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ID genes mediate tumor reinitiation during breast cancer lung metastasis
Abstract
The establishment of distant metastases depends on the capacity of small numbers of cancer cells to regenerate a tumor after entering a target tissue. The mechanisms that confer this capacity remain to be defined. Here we identify a role for the transcriptional inhibitors of differentiation Id1 and Id3 as selective mediators of lung metastatic colonization in the triple negative [TN, i.e., lacking expression of estrogen receptor and progesterone receptor, and lacking Her2 (human epidermal growth factor receptor 2) amplification] subgroup of human breast cancer. Although broad expression of Id1 has recently been documented in tumors of the rare metaplastic subtype, here we report that rare Id1-expressing cells are also present in the more common TN subset of human breast tumors but not in other subtypes. We also provide evidence that Id1 expression is enriched in clinically obtained hormone receptor negative lung metastases. Functional studies demonstrate that Id1 and its closely related family member Id3 are required for tumor initiating functions, both in the context of primary tumor formation and during metastatic colonization of the lung microenvironment. In vivo characterization of lung metastatic progression reveals that Id1 and Id3 facilitate sustained proliferation during the early stages of metastatic colonization, subsequent to extravasation into the lung parenchyma. These results shed light on the proliferative mechanisms that initiate metastatic colonization, and they implicate Id1 and Id3 as mediators of this malignant function in the TN subgroup of breast cancers.
• proliferation
• stem cells
• triple negative
... The inhibitor of DNA binding and differentiation (ID) family of proteins are transcription regulators also reported to support breast cancer metastasis (Chen et al. 2012;Gupta et al. 2007). ID3 is part of the ID family of proteins that are expressed by embryonic and somatic stem cells; and contribute to stemness by enhancing proliferation and inhibiting differentiation. ...
... NRF-induced cancer stem cells from MCF-10A are referred to as BCSCs NRF1 or BCSCs NRF1+E2 if induced by treatment with estrogen. Transcription regulator ID3 is known to support the invasive growth of breast cancer cells and tumor initiating capacity of breast cancer metastases (Chen et al. 2012;Gupta et al. 2007). ID proteins have also been demonstrated to be secreted angiogenic transcription factors in arthritis, but whether endothelial ID3 may influence the growth of BCSCs is not known (Isozaki et al. 2014). ...
... ID proteins have been reported to facilitate breast cancer metastatic colonization of the lung and brain (Gupta et al. 2007;Nair et al. 2014). To validate our in vitro findings, we determined whether ESCs guided BCSCs in vivo and whether this migration was exacerbated by exposure to PCB153. ...
Treatment options for brain metastatic breast cancer are limited because the molecular mechanism for how breast cancer cells infiltrate the brain is not fully understood. For breast tumors to metastasize to the brain first, cells need to detach from the primary tumor, enter in the blood circulation, survive within the microvascular niche, and then cross the blood–brain barrier (BBB) to colonize into the brain. It is critical to understand how breast cancer cells transmigrate through the BBB to prevent brain metastasis. Nuclear respiratory factor 1 (NRF1) transcription factor has been reported to be highly active in several human cancers and its aberrant expression facilitates in the acquisition of breast cancer stem cells (BCSCs). Inhibitor of differentiation protein 3 (ID3), a transcription regulating protein, induces pluripotent endothelial stem cells (ESCs). Herein, we investigated if NRF1-induced BCSCs could cross a BBB model and guiding of BCSCs by ID3-induced ESCs across the BBB. BCSCs and ESCs were subjected to functional gain/loss experiments to determine if NRF1/ID3 contributed to lineage-specific BCSCs organ entry. First, we tested whether NRF1 promoted migration of breast cancer using a BBB model consisting of BCSCs or MDA-MB231 cells, brain endothelial cell layer, and astrocytes. NRF1 overexpression increased the propensity for BCSCs and NRF1-induced MDA-MB231 cells to adhere to brain endothelial cells and migrate across a human BBB model. Increased adhesion of NRF1-induced BCSCs to ESCsID3 was detected. NRF1-induced BCSCs crossed through the BBB model and this was promoted by ESCsID3. We also showed that environmental relevant exposure to PCBs (PCB153 and PCB77) produced differential effects. Treatment with PCB153 showed increased growth of NRF1-induced BCSCs tumor spheroids and increased in vivo migration of ESCsID3. Exosomal ID3 released from endothelial cells also supported the growth of NRF1-induced BCSCs and provide the basis for paracrine effects by ESCsID3 associated with breast tumors. Xenograft experiments showed that ID3 overexpressing brain ESCs not only supported the growth of BCSC tumor spheroids but guided them to the neural crest in zebrafish. These findings show for the first time a novel role for ID3 and NRF1 by which ESCsID3 help guide BCSCsNRF1 to distant metastatic sites where they most likely facilitate the colonization, survival, and proliferation of BCSCs. This knowledge is important for pre-clinical testing of NRF1/ID3 modifying agents to prevent the spread of breast cancer to the brain.
... ID proteins are expressed in a tissue-specific and stage-dependent manner and are required for the maintenance of self-renewal and multipotency of embryonic and many tissue stem cells (Liang et al., 2009;Hong et al., 2011;Stankic et al., 2013;Aloia et al., 2015). Previous studies have reported a functional redundancy among the four members of the mammalian Id family, in particular Id1 and Id3 (referred to collectively here as Id), and their overlapping expression patterns during normal development and cancer (Lyden et al., 1999;Gupta et al., 2007;Anido et al., 2010;O'Brien et al., 2012;Niola et al., 2013). Id2 and Id4 were not investigated in this work as they are found to have independent functions from Id1 and Id3. ...
... A number of studies have implied a significant role for ID1 and ID3 in breast cancer progression and metastasis (Gupta et al., 2007). We have previously demonstrated that Id1 cooperates with activated Ras signaling and promotes mammary tumor initiation and metastasis in vivo by supporting long-term self-renewal and proliferative capacity (Swarbrick et al., 2008). ...
... The Id1 shRNA sequence which targets 5 ′ -GGGACCTGCAGCTGGAGCTGAA-3 ′ has been validated earlier (Gao et al., 2008). The Id3 sequence was adopted from Gupta et al. (2007) and targets the sequence 5 ′ -ATGGATGAGCTTCGATCTTAA-3 ′ . shRNA directed against EGFP was used as the control. ...
Breast cancers display phenotypic and functional heterogeneity and several lines of evidence support the existence of cancer stem cells (CSCs) in certain breast cancers, a minor population of cells capable of tumor initiation and metastatic dissemination. Identifying factors that regulate the CSC phenotype is therefore important for developing strategies to treat metastatic disease. The Inhibitor of Differentiation Protein 1 (Id1) and its closely related family member Inhibitor of Differentiation 3 (Id3) (collectively termed Id) are expressed by a diversity of stem cells and are required for metastatic dissemination in experimental models of breast cancer. In this study, we show that ID1 is expressed in rare neoplastic cells within ER-negative breast cancers. To address the function of Id1 expressing cells within tumors, we developed independent murine models of Triple Negative Breast Cancer (TNBC) in which a genetic reporter permitted the prospective isolation of Id1⁺ cells. Id1⁺ cells are enriched for self-renewal in tumorsphere assays in vitro and for tumor initiation in vivo. Conversely, depletion of Id1 and Id3 in the 4T1 murine model of TNBC demonstrates that Id1/3 are required for cell proliferation and self-renewal in vitro, as well as primary tumor growth and metastatic colonization of the lung in vivo. Using combined bioinformatic analysis, we have defined a novel mechanism of Id protein function via negative regulation of the Roundabout Axon Guidance Receptor Homolog 1 (Robo1) leading to activation of a Myc transcriptional programme.
... ID1 and ID3 belong to the family of basic-helix-loop-helix (bHLH) transcription factors; however, they lack DNA binding domains 27 . They inhibit differentiation and sustain proliferation of tumor cells by binding to other bHLH transcription factors, ETS proteins and RB in a dominant-negative manner and preventing them from binding to DNA 27,28 . While a few indirect ID1/ID3 targets have been identified, these are mostly associated with proliferation (downstream of RB) and highly context-dependent 27 . ...
... While a few indirect ID1/ID3 targets have been identified, these are mostly associated with proliferation (downstream of RB) and highly context-dependent 27 . ID1 and ID3 have previously been characterized as part of a common murine and human lung metastatic signature in triple negative breast cancer cells 28,29 and extensively validated as regulators of metastasis 27,28,30 . ID1 and ID3 have also been implicated in anchorage-independent growth in soft agar in small cell lung cancer 31 ; however, they have not previously been studied in ILC. ...
... While a few indirect ID1/ID3 targets have been identified, these are mostly associated with proliferation (downstream of RB) and highly context-dependent 27 . ID1 and ID3 have previously been characterized as part of a common murine and human lung metastatic signature in triple negative breast cancer cells 28,29 and extensively validated as regulators of metastasis 27,28,30 . ID1 and ID3 have also been implicated in anchorage-independent growth in soft agar in small cell lung cancer 31 ; however, they have not previously been studied in ILC. ...
Invasive lobular carcinoma (ILC) is a histological subtype of breast cancer with distinct molecular and clinical features from the more common subtype invasive ductal carcinoma (IDC). ILC cells exhibit anchorage-independent growth in ultra-low attachment (ULA) suspension cultures, which is largely attributed to the loss of E-cadherin. In addition to anoikis resistance, herein we show that human ILC cell lines exhibit enhanced cell proliferation in ULA cultures as compared to IDC cells. Proteomic comparison of ILC and IDC cell lines identified induction of PI3K/Akt and p90-RSK pathways specifically in ULA culture in ILC cells. Further transcriptional profiling uncovered unique upregulation of the inhibitors of differentiation family transcription factors ID1 and ID3 in ILC ULA culture, the knockdown of which diminished the anchorage-independent growth of ILC cell lines through cell cycle arrest. We find that ID1 and ID3 expression is higher in human ILC tumors as compared to IDC, correlated with worse prognosis uniquely in patients with ILC and associated with upregulation of angiogenesis and matrisome-related genes. Altogether, our comprehensive study of anchorage independence in human ILC cell lines provides mechanistic insights and clinical implications for metastatic dissemination of ILC and implicates ID1 and ID3 as novel drivers and therapeutic targets for lobular breast cancer.
... However, their expression frequently becomes reactivated in cancer, providing cancer cells with stem cell-like traits [31,32]. In TNBC, ID1 and ID3 are requisite for self-renewal, metastasis, tumor re-initiation, and colonization, making them attractive targets for cancer stem cell therapy [30,[33][34][35]. ...
... To identify downstream target gene(s) that enhance stem-like features in breast cancer, we ranked ZNF148 ChIP-enrichment scores, and the expression fold changes of genes within the enriched GO terms (Fig. 5E) and identified Inhibitor of DNA binding 3 HLH protein (ID3) as a potential candidate. ID1 and ID3 proteins are functionally redundant and have been shown to promote breast cancer cell self-renewal, tumor cell dissemination, and metastatic colonization of the lung and tumor re-initiation [33][34][35]. Importantly, the ZNF148 occupancy was significantly enriched at the promoter and gene body of ID3 (Fig. 7A). A closer examination of the other family members of ID genes, ID1, ID2, and ID4 loci, all showed ZNF148 occupancy at the promoter and gene body, similar to ID3, albeit statistically less significant in MACS2 peak calling scores (Supplemental Fig. 7). ...
The MYC proto-oncogene (MYC) is one of the most frequently overexpressed genes in breast cancer that drives cancer stem cell-like traits, resulting in aggressive disease progression and poor prognosis. In this study, we identified zinc finger transcription factor 148 (ZNF148, also called Zfp148 and ZBP-89) as a direct target of MYC. ZNF148 suppressed cell proliferation and migration and was transcriptionally repressed by MYC in breast cancer. Depletion of ZNF148 by short hairpin RNA (shRNA) and CRISPR/Cas9 increased triple-negative breast cancer (TNBC) cell proliferation and migration. Global transcriptome and chromatin occupancy analyses of ZNF148 revealed a central role in inhibiting cancer cell de-differentiation and migration. Mechanistically, we identified the Inhibitor of DNA binding 1 and 3 (ID1, ID3), drivers of cancer stemness and plasticity, as previously uncharacterized targets of transcriptional repression by ZNF148. Silencing of ZNF148 increased the stemness and tumorigenicity in TNBC cells. These findings uncover a previously unknown tumor suppressor role for ZNF148, and a transcriptional regulatory circuitry encompassing MYC, ZNF148, and ID1/3 in driving cancer stem cell traits in aggressive breast cancer.
... High ID expression is associated with increased metastatic potential. For example, in breast cancer, high levels of ID1, ID3, and ID4 are present in the tumor cells of the highly metastatic, triple-negative subtype 45,46 . In metastatic tumor cells, ID1 and ID3 facilitate the mesenchymal-to-epithelial conversion required for macrometastatic disease progression, likely through antagonism of the bHLH protein Twist 47 . ...
... There was no significant difference in the number of GFP-positive cells between AGX51 and DMSO groups after either 24 or 48 h of treatment (Fig. 5f). These data are consistent with genetic models that found that Id1/Id3 inhibition does not prevent extravasation of metastasizing breast cancer cells, but rather the conversion of micro-to macro-metastatic lesions 45,47 . ...
ID proteins are helix-loop-helix (HLH) transcriptional regulators frequently overexpressed in cancer. ID proteins inhibit basic-HLH transcription factors often blocking differentiation and sustaining proliferation. A small-molecule, AGX51, targets ID proteins for degradation and impairs ocular neovascularization in mouse models. Here we show that AGX51 treatment of cancer cell lines impairs cell growth and viability that results from an increase in reactive oxygen species (ROS) production upon ID degradation. In mouse models, AGX51 treatment suppresses breast cancer colonization in the lung, regresses the growth of paclitaxel-resistant breast tumors when combined with paclitaxel and reduces tumor burden in sporadic colorectal neoplasia. Furthermore, in cells and mice, we fail to observe acquired resistance to AGX51 likely the result of the inability to mutate the binding pocket without loss of ID function and efficient degradation of the ID proteins. Thus, AGX51 is a first-in-class compound that antagonizes ID proteins, shows strong anti-tumor effects and may be further developed for the management of multiple cancers.
... High Id expression is associated with increased metastatic potential. For example, in breast cancer, high levels of ID1, ID3 and ID4 are present in the tumor cells of the highly metastatic, triple negative subtype (Gupta et al., 2007;Wen et al., 2012). In metastatic tumor cells, Id1 and Id3 facilitate the mesenchymal-to-epithelial conversion required for macro-metastatic disease progression, likely through antagonism of the bHLH protein Twist (Stankic et al., 2013). ...
... These data are consistent with genetic models that found that Id1/Id3 inhibition does not prevent extravasation of metastasizing breast cancer cells, but rather the conversion of microto macro-metastatic lesions (Gupta et al., 2007;Stankic et al., 2013). ...
Id proteins are helix-loop-helix (HLH) transcriptional regulators frequently overexpressed in cancer. Id proteins inhibit basic HLH transcription factors through protein-protein interactions, often inhibiting differentiation and sustaining proliferation. We recently identified a small-molecule, AGX51, which targets Id proteins for degradation and impairs ocular neovascularization in mouse models. Here we show that AGX51 treatment of cancer cell lines impaired cell growth and viability that results from a dramatic increase in ROS production upon Id degradation. In mouse models, AGX51 treatment suppressed breast cancer colonization in the lung, regressed the growth of paclitaxel-resistant breast tumors when combined with paclitaxel and reduced tumor burden in a model of sporadic colorectal neoplasia. Furthermore, in cells and mice, we failed to observe acquired resistance to AGX51 likely the result of the immutability of the binding pocket and efficient degradation of the Id proteins. Thus, AGX51 is a first-in-class compound that antagonizes Id proteins, shows strong anti-tumor effects and may be further developed for the management of multiple cancers.
... In addition, in breast cancer cells, TGF-β induced ANGPTL4 via the SMAD signaling pathway, and this cytokine could disrupt lung capillary walls and seed pulmonary metastases [219]. Because there are inherent differences in the microvasculature of these two tissues, lung metastasis requires robust extravasation functions provided by ANGPTl4 and other factors and additional lung colonizing functions achieved by ID1/ID3 [220]. Therefore, the vasculature disruptive mechanism provides a selective invasive advantage in the lung but not bone. ...
... In addition, in breast cancer cells, TGF-β induced 477 ANGPTL4 via the SMAD signaling pathway, and this cytokine could disrupt lung capillary walls 478 and seed pulmonary metastases [219]. Because there are inherent differences in the microvasculature 479 of these two tissues, lung metastasis requires robust extravasation functions provided by ANGPTl4 480 and other factors and additional lung colonizing functions achieved by ID1/ID3 [220]. Therefore, the 481 vasculature disruptive mechanism provides a selective invasive advantage in the lung but not bone. ...
Transforming growth factor β (TGF-β) is a secreted cytokine that regulates cell proliferation, migration, and the differentiation of a plethora of different cell types. Consistent with these findings, TGF-β plays a key role in controlling embryogenic development, inflammation, and tissue repair, as well as in maintaining adult tissue homeostasis. TGF-β elicits a broad range of context-dependent cellular responses, and consequently, alterations in TGF-β signaling have been implicated in many diseases, including cancer. During the early stages of tumorigenesis, TGF-β acts as a tumor suppressor by inducing cytostasis and the apoptosis of normal and premalignant cells. However, at later stages, when cancer cells have acquired oncogenic mutations and/or have lost tumor suppressor gene function, cells are resistant to TGF-β-induced growth arrest, and TGF-β functions as a tumor promotor by stimulating tumor cells to undergo the so-called epithelial-mesenchymal transition (EMT). The latter leads to metastasis and chemotherapy resistance. TGF-β further supports cancer growth and progression by activating tumor angiogenesis and cancer-associated fibroblasts and enabling the tumor to evade inhibitory immune responses. In this review, we will consider the role of TGF-β signaling in cell cycle arrest, apoptosis, EMT and cancer cell metastasis. In particular, we will highlight recent insights into the multistep and dynamically controlled process of TGF-β-induced EMT and the functions of miRNAs and long noncoding RNAs in this process. Finally, we will discuss how these new mechanistic insights might be exploited to develop novel therapeutic interventions.
... Cancer cells use TNT to communicate with other cancer cells and stromal cells in the tumor microenvironment, such as mesenchymal stem cells, fibroblasts, macrophages, and neurons. 71 Membrane myoferlin, together with LST1, is involved in the induction and deformation of the plasma membrane and its fusion to form TNT. 55,69,72 Myoferlin expression is increased in HCC. [73][74][75][76][77][78][79] Myoferlin as an oncogene protein is involved in various HCC progression processes including proliferation and invasion, metastasis, and EMT. ...
Nowadays, hepatocellular carcinoma (HCC) is the second leading cause of cancer deaths, and identifying the effective factors in causing this disease can play an important role in its prevention and treatment. Tumors provide effective agents for invasion and metastasis to other organs by establishing appropriate communication between cancer cells and the microenvironment. Epithelial-to-mesenchymal transition (EMT) can be mentioned as one of the effective phenomena in tumor invasion and metastasis. Several factors are involved in inducing this phenomenon in the tumor microenvironment, which helps the tumor survive and migrate to other places. It can be effective to identify these factors in the use of appropriate treatment strategies and greater patient survival. This study investigated the molecular differences between tumor border cells and tumor core cells or internal tumor cells in HCC for specific EMT genes. Expression of NOTCH1, ID1, and LST1 genes showed a significant increase at the HCC tumor border. Targeting these genes can be considered as a useful therapeutic strategy to prevent distant metastasis in HCC patients.
... Patients with breast cancer who had lymph node metastases typically contained a high expression level of ID1 and low levels of KLF17 [44]. It was also shown that inhibiting ID1 and ID3 did not affect breast cancer cells' capacity to migrate to the lung [45]. ...
Background
Inhibitor of DNA Binding 2 (ID2) plays a crucial role in tumor cell proliferation, invasion, metastasis, and stemness. Aberrant ID2 expression is associated with poor prognosis in various cancers. However, the specific function of ID2 in thyroid cancer remain unclear.
Method
The TCGA database were utilized to explore the clinical relevance of ID2 in cancer. GO, KEGG, and TIMER were employed to predict the potential roles of ID2 in cancer. Functional analysis, including CCK-8, colony formation, transwell, wound healing, and sphere formation experiments, were conducted to determine the biological functions of ID2 in human cancers. Western blot (WB), RT-qPCR, and immunohistochemical (IHC) analyses were used to investigate the relationship between ID2 and downstream targets.
Results
Our study revealed significant overexpression of ID2 in various malignant tumor cells. Knocking ID2 significantly inhibited cancer cell proliferation and invasion, while overexpressing ID2 enhanced these capabilities. Additionally, ID2 mediates resistance of cancer cells to protein kinase B (or Akt) inhibitions. Further WB and IHC experiments indicated that ID2 promotes the phosphorylation activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, thereby upregulating the expression of downstream proliferation, epithelial-mesenchymal transition (EMT), and stemness-related markers.
Conclusion
We found that ID2 significantly promotes thyroid cancer cell proliferation, migration, EMT, and stemness through the PI3K/Akt pathway. Moreover, ID2 plays a crucial role in regulating cancer immune responses. It may serve as a potential biomarker for enhancing the efficacy of chemotherapy, targeted therapy, and immunotherapy against cancer.
... Another protein that regulates cellular responses to TGF-β is ID1. ID1 modulates activities of transcription factors that are often upregulated in solid cancers and thus has effects on differentiation, senescence, and apoptosis [60,61]. In mice, overexpression of ID1 causes the development of adenomas [62] and, when Ras is also overexpressed, metastatic breast cancer [63]. ...
... The ID gene family has important roles in development and their deregulated expression is connected to poor prognosis. Increased expression of ID1 has been associated with both primary tumor formation and lung colonization [47] presumably via mesenchymal -epithelial transition essential for the latter process [48]. Earlier studies demonstrated that MDA-MB-231 are genetically heterogeneous and in vitro selection for more aggressive populations with altered genome structure have been reported [49]. ...
Promyelocytic leukemia protein (PML) modulates diverse cell functions that contribute to both tumor-suppressor and pro-oncogenic effects, depending on the cellular context. We show here that PML knockdown (KD) in MDA-MB-231, but not MCF7, breast cancer cells, prolonged stem-cell-like survival, and increased cell proliferation and migration, which is in line with gene-enrichment results from their RNA sequencing analysis. Of note, increased migration was accompanied by higher levels of the epithelial-mesenchymal transition (EMT) regulator Twist-related protein 2 (TWIST2). We showed here that PML binds to TWIST2 via its basic helix-loop-helix (bHLH) region and functionally interferes with suppression of the epithelial target of TWIST2, CD24. In addition, PML ablation in MDA-MB-231 cells led to higher protein levels of hypoxia-inducible factor 1-alpha (HIF1a), resulting in a higher cell hypoxic response. Functionally, PML directly suppressed induction of the HIF1a target gene vascular endothelial growth factor A (VEGFa). In line with these results, tumor xenografts of MDA-MB-231 PML-KD cells had enhanced aggressive properties, including higher microvessel density, faster local growth and higher metastatic ability, with a preference for lung. Collectively, PML suppresses the cancer aggressive behavior by multiple mechanisms that impede both the HIF-hypoxia-angiogenic and EMT pathways.
... ID proteins, also known as inhibitors of DNA-binding proteins, antagonized the global activity of the bHLH family members by repressing their DNA-binding functions107,139 . During BC lung metastasis, ID1 and ID3 are required for tumor re-initiation throughout extravasation into the lung parenchyma in vivo140 . In glioma, TGF stimulation helps to maintain the glioma-initiating cells (GICs) so-called glioma stem cells that have the ability to initiate tumors seeding. ...
Epithelial to mesenchymal transition (EMT) is well-known mechanism which is associated with poor prognosis and tumor escape from immune escape surveillance in cancer. In addition to the transcriptional regulation of PD-L1 in breast cancer (BC) undergoing EMT, here we show that CMTM6 is overexpressed in EMT-activated BC cells as compared with epithelial cells. CMTM6 was recently consider as a key regulator for the stabilization and recycling of PD-L1 at the cell surface in a broad range of cancer types. We, therefore silenced CMTM6 and observed a partial but significant downregulation of surface PD-L1 in BC undergoing EMT. Further investigations demonstrate that other members of the CMTM family contribute to PD-L1 regulation. In addition to CMTM6, CMTM3 and CMTM7 were also found to be upregulated in EMT-induced BC cells. However only CMTM7 silencing modulate the SNAI-induced PD-L1 surface expression as compared with control cells. Dual CMTM6 and CMTM7 silencing significantly reduced for up to 50% the expression of the SNAI-dependent PD-L1 surface expression in mesenchymal tumor cells.Supplementary results showed that hypoxia, a common feature observed in the tumor microenvironment, positively regulated CMTM6 gene expression in several cancer cell lines. Moreover, preliminary data show that CMTM7, but not CMTM6 is transcriptionally regulated by SNAI1, but not ZEB1 in EMT-activated BC cells. Our data also suggest that targeting CMTM6 and/or CMTM7 were able to increase T cell survival by caspase3/7 measurement activity, to the same degree as PD-1/PD-L1 blockade in vitro.Overall, our results provided additional highlight EMT as new inducers of the CMTM family expression in BC as well as a comprehensive overview on PD-L1 regulation during EMT and its impact on T-cell mediated cell death via CMTM6 and CMTM7 expression.
... The ID gene family has important roles in development and their deregulated expression is connected to poor prognosis. Increased expression of ID1 has been associated with both primary tumor formation and lung colonization [41] presumably via mesenchymal -epithelial transition essential for the latter process [42] . The role and interaction of PML with EMT factors seems to be complex. ...
Promyelocytic Leukemia Protein (PML) is the core protein of nuclear bodies (NBs) that regulate a large number of cellular processes, including, context dependent, tumor-suppressor and pro-oncogenic effects. PML knockdown (KD) in breast cancer lines MDA-MB-231, but not MCF7, cells showed higher cell proliferation, increased migration properties and prolonged stem cell-like survival in line with gene enrichment results from RNA sequencing analysis. MDA-MB-231 PML KD cells showed an increase of hypoxic and mesenchymal characteristics manifested by higher HIF1a and the EMT-TWIST2 protein levels respectively. Mechanistically, PML loss caused an increase of HIF1a and TWIST2 RNA levels. Interestingly, TWIST2 binds to PML. Moreover, PML directly opposed the action of HIF1a and TWIST2 on VEGFa and CD24 reporters, respectively. Tumor xenografts of MDA-MB-231 PML KD cells showed a higher micro vessel content, grew faster and had higher metastatic ability with a preference for lung. Thus, PML opposes the aggressive cancer phenotype by multiple mechanisms that antagonize the HIF-hypoxia-angiogenic and EMT axis.
... Additionally, ID proteins are necessary for angiogenesis and vascularization of tumors [9]. In breast cancer, high expression levels of ID proteins are associated with cancer proliferation and metastasis [10][11][12]. Moreover, overexpression of ID3 has been observed in cases of esophageal squamous cell carcinoma [13] and cervical cancer [14]. ...
Simple Summary
Ovarian cancer is one of the most fatal gynecological malignancies in women. Even though primary treatment might result in complete remission, approximately 60–80% patients with advanced-stage ovarian cancer experience a relapse. To improve their survival outcomes, several novel agents have been proposed; however, the majority of these have demonstrated limited efficacy in the treatment of ovarian cancer. Interestingly, an imbalance in the basic helix-loop-helix inhibitor of DNA binding (bHLH/ID) protein networks has been associated with oncogenesis, but the exact expression patterns of bHLH/ID in ovarian cancer are not yet known. We hypothesized that such an imbalance in bHLH/ID activity may be integral to ovarian cancer pathogenesis, and modulating the bHLH/ID balance might be a promising approach to treat ovarian cancer.
Abstract
The E2A and inhibitor of DNA binding (ID) proteins are transcription factors involved in cell cycle regulation and cellular differentiation. Imbalance of ID/E2A activity is associated with oncogenesis in various tumors, but their expression patterns and prognostic values are still unknown. We evaluated ID and E2A expression in ovarian cancer cells, and assessed the possibility of reprogramming ovarian cellular homeostasis by restoring the ID/E2A axis. We analyzed copy number alterations, mutations, methylations, and mRNA expressions of ID 1–4 and E2A using The Cancer Genome Atlas data of 570 ovarian serous cystadenocarcinoma patients. Incidentally, 97.2% cases exhibited gain of ID 1–4 or loss of E2A. Predominantly, ID 1–4 were hypomethylated, while E2A was hypermethylated. Immunohistochemical analysis revealed that ID-3 and ID-4 expressions were high while E2A expression was low in cancerous ovarian tissues. Correlation analysis of ID and E2A levels with survival outcomes of ovarian cancer patients indicated that patients with high ID-3 levels had poor overall survival. We also determined the effect of E2A induction on ovarian cancer cell growth in vitro and in vivo using SKOV-3/Luc cells transduced with tamoxifen-inducible E47, a splice variant of E2A. Interestingly, E47 induced SKOV-3 cell death in vitro and inhibited tumor growth in SKOV-3 implanted mice. Therefore, restoring ID/E2A balance is a promising approach for treating ovarian cancer.
... Recent translational studies and genomic sequencing have shown several links to dormancy in solid tumors [124][125][126][127]. In particular, in breast and prostatic cancer, signature genes such as NR2F1, SHARP1, BMP7high and COCOlow seem to be related with dormancy and reactivation in specific metastatic sites [128]. ...
Simple Summary
The world of small molecules in solid tumors as cancer stem cells (CSCs), circulating tumor cells (CTCs) and cancer-associated fibroblasts (CAFs) continues to be under-debated, but not of minor interest in recent decades. One of the main problems in regard to cancer is the development of tumor recurrence, even in the early stages, in addition to drug resistance and, consequently, ineffective or an incomplete response against the tumor. The findings behind this resistance are probably justified by the presence of small molecules such as CSCs, CTCs and CAFs connected with the tumor microenvironment, which may influence the aggressiveness and the metastatic process. The mechanisms, connections, and molecular pathways behind them are still unknown. Our review would like to represent an important step forward to highlight the roles of these molecules and the possible connections among them.
Abstract
The importance of defining new molecules to fight cancer is of significant interest to the scientific community. In particular, it has been shown that cancer stem cells (CSCs) are a small subpopulation of cells within tumors with capabilities of self-renewal, differentiation, and tumorigenicity; on the other side, circulating tumor cells (CTCs) seem to split away from the primary tumor and appear in the circulatory system as singular units or clusters. It is becoming more and more important to discover new biomarkers related to these populations of cells in combination to define the network among them and the tumor microenvironment. In particular, cancer-associated fibroblasts (CAFs) are a key component of the tumor microenvironment with different functions, including matrix deposition and remodeling, extensive reciprocal signaling interactions with cancer cells and crosstalk with immunity. The settings of new markers and the definition of the molecular connections may present new avenues, not only for fighting cancer but also for the definition of more tailored therapies.
... Regarding LKB1-SIKs pathway, it was discovered that it plays an important role in driving metastasis. The dysregulation of LKB1-SIK-CREB axis induces the expression of the inhibitor of DNA binding 1 (ID1) (156), an oncogene already studied and demonstrated to be essential for lung colonization by breast cancer (157). In NSCLC, this gene controls cell-cell and cell-ECM interaction, cytoskeleton and other aspects involved in anchorage-independent growth and colonization promoting in this way metastasis. ...
LKB1 (liver kinase B1) is a master regulator of several processes such as metabolism, proliferation, cell polarity and immunity. About one third of non-small cell lung cancers (NSCLCs) present LKB1 alterations, which almost invariably lead to protein loss, resulting in the absence of a potential druggable target. In addition, LKB1-null tumors are very aggressive and resistant to chemotherapy, targeted therapies and immune checkpoint inhibitors (ICIs). In this review, we report and comment strategies that exploit peculiar co-vulnerabilities to effectively treat this subgroup of NSCLCs. LKB1 loss leads to an enhanced metabolic avidity, and treatments inducing metabolic stress were successful in inhibiting tumor growth in several preclinical models. Biguanides, by compromising mitochondria and reducing systemic glucose availability, and the glutaminase inhibitor telaglenastat (CB-839), inhibiting glutamate production and reducing carbon intermediates essential for TCA cycle progression, have provided the most interesting results and entered different clinical trials enrolling also LKB1-null NSCLC patients. Nutrient deprivation has been investigated as an alternative therapeutic intervention, giving rise to interesting results exploitable to design specific dietetic regimens able to counteract cancer progression. Other strategies aimed at targeting LKB1-null NSCLCs exploit its pivotal role in modulating cell proliferation and cell invasion. Several inhibitors of LKB1 downstream proteins, such as mTOR, MEK, ERK and SRK/FAK, resulted specifically active on LKB1-mutated preclinical models and, being molecules already in clinical experimentation, could be soon proposed as a specific therapy for these patients. In particular, the rational use in combination of these inhibitors represents a very promising strategy to prevent the activation of collateral pathways and possibly avoid the potential emergence of resistance to these drugs. LKB1-null phenotype has been correlated to ICIs resistance but several studies have already proposed the mechanisms involved and potential interventions. Interestingly, emerging data highlighted that LKB1 alterations represent positive determinants to the new KRAS specific inhibitors response in KRAS co-mutated NSCLCs. In conclusion, the absence of the target did not block the development of treatments able to hit LKB1-mutated NSCLCs acting on several fronts. This will give patients a concrete chance to finally benefit from an effective therapy.
... Notably, within the activin B signature, three members of the family of inhibitors of DNA binding and cell differentiation (ID) proteins (ID1, ID2 and ID3) were among the top induced genes (Fig. 4d). These proteins are recognized to promote stem cell self-renewal and multipotency, and were previously shown to facilitate metastatic colonization by breast cancer cells 26,27 . ...
Disseminated cancer cells frequently lodge near vasculature in secondary organs. However, our understanding of the cellular crosstalk invoked at perivascular sites is still rudimentary. Here, we identify intercellular machinery governing formation of a pro-metastatic vascular niche during breast cancer colonization in the lung. We show that specific secreted factors, induced in metastasis-associated endothelial cells (ECs), promote metastasis in mice by enhancing stem cell properties and the viability of cancer cells. Perivascular macrophages, activated via tenascin C (TNC) stimulation of Toll-like receptor 4 (TLR4), were shown to be crucial in niche activation by secreting nitric oxide (NO) and tumor necrosis factor (TNF) to induce EC-mediated production of niche components. Notably, this mechanism was independent of vascular endothelial growth factor (VEGF), a key regulator of EC behavior and angiogenesis. However, targeting both macrophage-mediated vascular niche activation and VEGF-regulated angiogenesis resulted in added potency to curb lung metastasis in mice. Together, our findings provide mechanistic insights into the formation of vascular niches in metastasis. Hongu et al. find that perivascular macrophages stimulate activation of the pro-metastatic vascular niche via tenascin C stimulation of TLR4 and show that combined TLR4 and VEGF inhibition prevents TNC-mediated metastatic vascular activity.
... Conversely, in the absence of the EMT factors Snail and TGF-β, the Inhibitor Of DNA Binding 1 (ID1) factor can induce MET by antagonizing TWIST1 activity (Stankic et al. 2013). Interestingly, ID1 was earlier identified as a member of a group of genes that enables lung cancer colonization (Gupta et al. 2007). Along with the ID1 role in HIF stabilization and angiogenesis (Kim et al. 2007), IDs emerge as key factors in both the early (angiogenic) and late (colonization metastatic) process. ...
The promyelocytic leukemia protein (PML) is the core organizer of the cognate nuclear bodies (PML-NBs). Through physical interaction or modification of diverse protein clients, PML-NBs regulate a multitude of - often antithetical- biological processes such as antiviral and stress response, inhibition of cell proliferation and autophagy, and promotion of apoptosis or senescence. Although PML was originally recognized as a tumor-suppressive factor, more recent studies revealed a “double faced” agent role for PML. Indeed PML displayed tumor cell pro-survival and pro-migratory functions via inhibition of migration suppressing molecules or promotion of transforming growth factor beta (TGF-β) mediated Epithelial-Mesenchymal Transition (EMT) that may promote cancer cell dissemination. In this line, PML was found to correlate with poor patient prognosis in distinct tumor contexts. Furthermore in the last decade, a number of publications have implicated PML in the physiology of normal or cancer stem cells (CSCs). Promyelocytic leukemia protein is activating fatty acid oxidation (FAO), a metabolic mechanism required for the asymmetric divisions and maintenance of hematopoietic stem cells (HSCs). In embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), PML is required for maintenance of the naïve and acquisition of the induced pluripotency state respectively. Correspondingly, PML ablation causes significant morphological, gene expression and lineage choice changes. In this review, we focus on the mechanisms orchestrated by PML and PML-NBs in cancer and healthy stem cells from cell physiology to the regulation of chromatin dynamics.
... Inhibitor of DNA binding 1 (ID1) is an oncogenic protein, promoting cancer survival, proliferation, angiogenesis, and metastasis (Lyden et al., 1999;Perk et al., 2005;Gupta et al., 2007;Lasorella et al., 2014). ID1 protein has a helix-loop-helix domain (HLH), which negatively regulates the activity of HLH transcription factors (TF) by decreasing the TF/DNA-binding (Sun et al., 1991;Alani et al., 2001). ...
Hypoxia is a universal pathological feature of solid tumors. Hypoxic tumor cells acquire metastatic and lethal phenotypes primarily through the activities of hypoxia-inducible factor 1 alpha (HIF1α). Therefore, HIF1α is considered as a promising therapeutic target. However, HIF inhibitors have not proven to be effective in clinical testing. The underlying mechanism is unclear. We report that oncogenic protein ID1 is upregulated in hypoxia by HIF1α shRNA or pharmacological inhibitors. In turn, ID1 supports tumor growth in hypoxia in vitro and in xenografts in vivo, conferring adaptive survival response and resistance. Mechanistically, ID1 proteins interfere HIF1-mediated gene transcription activation, thus ID1 protein degradation is accelerated by HIF1α-dependent mechanisms in hypoxia. Inhibitions of HIF1α rescues ID1, which compensates the loss of HIF1α by the upregulation of GLS2 and glutamine metabolism, thereby switching the metabolic dependency of HIF1α -inhibited cells from glucose to glutamine.
... In contrast, Wnt1-driven tumors exhibit features in common with human basal subtype that falls within the subgroup of TN (triple negative tumors) (lack ER and PR expression and Her2 gene amplification. Id-1 and Id-3 expression is seen in tumor cells of Wnt-1 driven tumors but not in Neu-induced tumors [145]. ...
Exquisite regulation of telomere length is essential for the preservation of the lifetime function and self-renewal of stem cells. However, multiple oncogenic pathways converge on induction of telomere attrition or telomerase overexpression and these events can by themselves trigger malignant transformation. Activation of NFκB, the outcome of telomere complex damage, is present in leukemia stem cells but absent in normal stem cells and can activate DOT1L which has been linked to MLL-fusion leukemias. Tumors that arise from cells of early and late developmental stages appear to follow two different oncogenic routes in which the role of telomere and telomerase signaling might be differentially involved. In contrast, direct malignant transformation of stem cells appears to be extremely rare. This suggests an inherent resistance of stem cells to cancer transformation which could be linked to a stem cell’specific mechanism of telomere maintenance. However, tumor protection of normal stem cells could also be conferred by cell extrinsic mechanisms.
... Thus, the reduced SNAI2/SNAI1 ratio correlates with the decreased levels of proliferation markers and indicates an upregulated EMT response by SNAI1, supported by an upregulation of FOSL1 and MUC1 levels. Surprisingly, ID1 level that is shown by the previous study to be implicated in breast cancer metastasis (Gupta et al., 2007;Gumireddy et al., 2014) was noted to be downregulated. A previous study has shown ID1 to inhibit TWIST-mediated EMT to promote mesenchymal-epithelial transition at metastatic sites where SNAI1 expression is low and underscores that ID1 does not affect SNAI1-mediated EMT at the primary tumor site (Stankic et al., 2013). ...
The field of 3D cell cultures is currently emerging, and material development is essential in striving toward mimicking the microenvironment of a native tissue. By using the response of reporter cells to a 3D environment, a comparison between materials can be assessed, allowing optimization of material composition and microenvironment. Of particular interest, the response can be different in a normoxic and hypoxic culturing conditions, which in turn may alter the conclusion regarding a successful recreation of the microenvironment. This study aimed at determining the role of such environments to the conclusion of a better resembling cell culture model to native tissue. Here, the breast cancer cell line MCF7 was cultured in normoxic and hypoxic conditions on patient-derived scaffolds and compared at mRNA and protein levels to cells cultured on 3D printed scaffolds, Matrigel, and conventional 2D plastics. Specifically, a wide range of mRNA targets (40), identified as being regulated upon hypoxia and traditional markers for cell traits (cancer stem cells, epithelial–mesenchymal transition, pluripotency, proliferation, and differentiation), were used together with a selection of corresponding protein targets. 3D cultured cells were vastly different to 2D cultured cells in gene expression and protein levels on the majority of the selected targets in both normoxic and hypoxic culturing conditions. By comparing Matrigel and 3DPS-cultured cells to cells cultured on patient-derived scffolds, differences were also noted along all categories of mRNA targets while specifically for the GLUT3 protein. Overall, cells cultured on patient-derived scaffolds closely resembled cells cultured on 3D printed scaffolds, contrasting 2D and Matrigel-cultured cells, regardless of a normoxic or hypoxic culturing condition. Thus, these data support the use of either a normoxic or hypoxic culturing condition in assays using native tissues as a blueprint to optimize material composition.
... Then, we focused on variants in 127 genes (Additional file 2: Table S7) that have been reported as metastasis-related [45][46][47]. We found that this patient had somatically acquired deletions and amplifications in 17 of these genes (Fig. 5B); 8 of the genomic lesions were present in the primary tumor, including the MTDH, ANGPTL4, and ID1 genes associated with lung metastasis [46,48]; and the IL11 and TGFB1 genes in which genomic alterations have been associated with bone metastasis [46,49]. ...
Background
Metastatic breast cancer is a deadly disease with a low 5-year survival rate. Tracking metastatic spread in living patients is difficult and thus poorly understood.
Methods
Via rapid autopsy, we have collected 30 tumor samples over 3 timepoints and across 8 organs from a triple-negative metastatic breast cancer patient. The large number of sites sampled, together with deep whole-genome sequencing and advanced computational analysis, allowed us to comprehensively reconstruct the tumor’s evolution at subclonal resolution.
Results
The most unique, previously unreported aspect of the tumor’s evolution that we observed in this patient was the presence of “subclone incubators,” defined as metastatic sites where substantial tumor evolution occurs before colonization of additional sites and organs by subclones that initially evolved at the incubator site. Overall, we identified four discrete waves of metastatic expansions, each of which resulted in a number of new, genetically similar metastasis sites that also enriched for particular organs (e.g., abdominal vs bone and brain). The lung played a critical role in facilitating metastatic spread in this patient: the lung was the first site of metastatic escape from the primary breast lesion, subclones at this site were likely the source of all four subsequent metastatic waves, and multiple sites in the lung acted as subclone incubators. Finally, functional annotation revealed that many known drivers or metastasis-promoting tumor mutations in this patient were shared by some, but not all metastatic sites, highlighting the need for more comprehensive surveys of a patient’s metastases for effective clinical intervention.
Conclusions
Our analysis revealed the presence of substantial tumor evolution at metastatic incubator sites in a patient, with potentially important clinical implications. Our study demonstrated that sampling of a large number of metastatic sites affords unprecedented detail for studying metastatic evolution.
... Then we focused on mutations in 128 genes (Supplementary Table 7) that have been reported as metastasis-related [28][29][30]. We found that this patient had somatically acquired deletions and amplifications in 17 of these genes (Fig. 5B); eight of the genomic lesions were present in the primary tumor, including the MTDH, ANGPTL4, and ID1 genes associated with lung metastasis [29,31]; and the IL11 and TGFB1 genes in which genomic alterations have been associated with bone metastasis [29,32]. ...
Background: Metastatic breast cancer is a deadly disease with a low 5-year survival rate. Tracking metastatic spread in living patients is difficult, and thus poorly understood.
Results: Via rapid autopsy, we have collected 30 tumor samples over 3 timepoints and across 8 organs from a triple-negative metastatic breast cancer patient. The large number of sites sampled, together with deep whole genome sequencing and advanced computational analysis, allowed us to comprehensively reconstruct the tumor's evolution at subclonal resolution. The most unique, previously not reported aspect of the tumor's evolution we observed in this patient was the presence of "subclone incubators", i.e. already metastatic sites where substantial tumor evolution occurred before colonization of additional sites and organs by subclones that evolved at the incubator site. Overall, we identified four discrete waves of metastatic expansions, each of which resulted in a number of new, genetically similar metastasis sites that also enriched for particular organs (e.g. abdominal vs bone and brain). The lung played a critical role in facilitating metastatic spread in this patient: the lung was the first site of metastatic escape from the primary breast lesion; subclones at this site were the source of all four subsequent metastatic waves; and multiple sites in the lung acted as subclone incubators. Finally, functional annotation revealed that many known driver or metastasis-promoting tumor mutations in this patient were shared by some, but not all metastatic sites, highlighting the need for more comprehensive surveys of a patient's metastases for effective clinical intervention.
Conclusions: Our analysis revealed the presence of substantial tumor evolution at metastatic incubator sites, with potentially important clinical implications. Our study demonstrated that sampling of a large number of metastatic sites affords unprecedented detail for studying metastatic evolution.
... They act as dominant negative inhibitors of basic helixloop-helix (bHLH) transcription factors, by heterodimerising with them and preventing them from binding to DNA [51]. Expression of Id1 and Id3 has been implicated in tumor initiation and metastatic growth [52][53][54]. Accordingly, their expression correlates with poor prognosis for many types of cancer [55], including melanoma [56]. ...
Cancer heterogeneity is a result of genetic mutations within the cancer cells. Their proliferation is not only driven by autocrine functions but also under the influence of cancer microenvironment, which consists of normal stromal cells such as infiltrating immune cells, cancer-associated fibroblasts, endothelial cells, pericytes, vascular and lymphatic channels. The relationship between cancer cells and cancer microenvironment is a critical one and we are just on the verge to understand it on a molecular level. Cancer microenvironment may serve as a selective force to modulate cancer cells to allow them to evolve into more aggressive clones with ability to invade the lymphatic or vascular channels to spread to regional lymph nodes and distant sites. It is important to understand these steps of cancer evolution within the cancer microenvironment towards invasion so that therapeutic strategies can be developed to control or stop these processes.
... Within the signature, three members of the family of inhibitors of DNA binding and cell differentiation (ID) proteins (ID1, ID2 and ID3) were among the top induced genes (Figure 4b). These proteins are recognized to promote stem cell self-renewal and multipotency and were shown to facilitate metastatic colonization by breast cancer cells 15,16 . To analyze the phenotype of activin B-stimulated cancer cells, we stratified gene expression datasets from samples of breast cancer patients (Metabric or samples of lung metastases) based on expression levels of the activin B response signature (Figure 4d). ...
When cancers progress to metastasis, disseminated cancer cells frequently lodge near vasculature in secondary organs. However, our understanding of the cellular crosstalk evoked at perivascular sites is still rudimentary. In this study, we identify intercellular machinery governing formation of a pro-metastatic vascular niche during breast cancer colonization in lungs. We show that four secreted factors, INHBB, SCGB3A1, OPG and LAMA1, induced in metastasis-associated endothelial cells (ECs), are essential components of the vascular niche and promote metastasis in mice by enhancing stem cell properties and survival ability of cancer cells. Notably, blocking VEGF, a key regulator of EC behavior, dramatically suppressed EC proliferation, whereas no impact was observed on the expression of the four vascular niche factors in lung ECs. However, perivascular macrophages, activated via the TNC-TLR4 axis, were shown to be crucial for EC-mediated production of niche components. Together, our findings provide mechanistic insights into the formation of vascular niches in metastasis.
... TGF-β, which controls the expression of the inhibitor of differentiation family of proteins (ID), has been reported to promote metastasis and regulate the dormant state of cancer cells. In this line, increased ID1 and ID3 expression in basal or triple-negative breast tumors reactivates and sustains cancer cell proliferation in the lung during metastatic colonization [150,151]. On the other hand, Coco, a secreted antagonist of TGF-β ligands, has been reported to facilitate escape of dormancy in disseminated breast cancer cells by blocking BMP signaling, thus inducing lung-specific colonization [152]. Additionally, disseminated prostate cancer cells in the bone marrow have been shown to escape their dormant state by downregulating TGF-β2 expression and activating its downstream target myosin light chain kinase (MLCK) [153]. ...
Progression from localized to metastatic disease requires cancer cells spreading to distant organs through the bloodstream. Only a small proportion of these circulating tumor cells (CTCs) survives dissemination due to anoikis, shear forces and elimination by the immune system. However, all metastases originate from CTCs capable of surviving and extravasating into distant tissue to re-initiate a tumor. Metastasis initiation is not always immediate as disseminated tumor cells (DTCs) may enter a non-dividing state of cell dormancy. Cancer dormancy is a reversible condition that can be maintained for many years without being clinically detectable. Subsequently, late disease relapses are thought to be due to cancer cells ultimately escaping from dormant state. Cancer dormancy is usually associated with minimal residual disease (MRD), where DTCs persist after intended curative therapy. Thus, MRD is commonly regarded as an indicator of poor prognosis in all cancers. In this review, we examine the current understanding of MRD and immunity during cancer progression to metastasis and discuss clinical perspectives for oncology.
... Atf3 (8.735-fold up-regulated) promoted skin cancer cell proliferation and enhanced skin keratinocyte carcinogenesis by inhibiting p53 expression and activating Stat3 (1.605-fold up-regulated) phosphorylation [39,40]. Id1 (63.185-fold up-regulated) is an attractive therapeutic target in cancer, since it is associated with tumor angiogenesis and metastasis [41,42]. Subverting Id1 activation could prevent tumor progression, which is critical in melanoma [43]. ...
Aims
We systematically characterized metastatic murine B16-F10 melanoma, a sub-line derived from murine melanoma B16-F1 cells.
Materials and methods
RNA-sequencing and network analyses (Ingenuity Pathway Analysis) were performed to identify novel potential metastasis mechanisms. Chromosomal aberrations were identified by multicolor fluorescence in situ hybridization (mFISH) using all 21 murine whole chromosome painting probes.
Key findings
Numerous genes were overexpressed in B16-F10 cells, some of which have been already described as being metastasis-linked. Nr5a1/sf1, a known prognostic marker for adrenal tumors, was 177-fold upregulated in B16-F10 cells compared to B16-F1 cells. Hoxb8 was 75-fold upregulated, which was previously associated with gastric cancer progression and metastasis. Ptk7, which is linked with tumorigenesis and metastasis of esophageal squamous carcinoma, was 67-fold upregulated. B16-F10 cells acquired additional chromosomal aberrations compared to B16-F1 cells, including dic(4)(pter->qter:qter->pter), +dic(6;15), +der(10)t(10;?1;16).
Significance
In addition to well-known metastatic genes, numerous novel genes and genomic aberrations were identified, which may serve as targets for treatment in the future. Transcriptomic and genetic analyses in B16-F10 cells unraveled a range of novel metastasis mechanisms, which may also have important implications for future treatment strategies.
... Even through autopsy, the primary origin of CUPs is uncertain (Myung et al., 2001;Petrushev et al., 2011). Because of limited treatment plan for CUP patients, the treatment efficacy is often unpredictable, and those patients usually have poor prognosis (Sun and Zhang, 2006;Gupta et al., 2007;Carmeliet and Jain, 2011;Petrakis et al., 2013). The immunohistochemistry assay is usually considered to be a diagnostic method for CUP patients. ...
Some carcinomas show that one or more metastatic sites appear with unknown origins. The identification of primary or metastatic tumor tissues is crucial for physicians to develop precise treatment plans for patients. With unknown primary origin sites, it is challenging to design specific plans for patients. Usually, those patients receive broad-spectrum chemotherapy, while still having poor prognosis though. Machine learning has been widely used and already achieved significant advantages in clinical practices. In this study, we classify and predict a large number of tumor samples with uncertain origins by applying the random forest and Naive Bayesian algorithms. We use the precision, recall, and other measurements to evaluate the performance of our approach. The results have showed that the prediction accuracy of this method was 90.4 for 7,713 samples. The accuracy was 80% for 20 metastatic tumors samples. In addition, the 10-fold cross-validation is used to evaluate the accuracy of classification, which reaches 91%.
... The main biological properties of ID proteins are inhibition of differentiation and conservation of the self-renewal capability and multipotency of stem cells 11 . ID proteins are overexpressed in several human carcinomas [11][12] . More specifically, Id-1 protein expression is directly involved in cancer initiation and/or progression in different types of human malignancies including cervical 9, 13-15 . ...
Epstein–Barr virus (EBV) has been recently shown to be co-present with high-risk human papillomaviruses (HPVs) in human cervical cancer; thus, these oncoviruses play an important role in the initiation and/or progression of this cancer. Accordingly, our group has recently viewed the presence and genotyping distribution of high-risk HPVs in cervical cancer in Syrian women; our data pointed out that HPVs are present in 95.45% of our samples. Herein, we aim to explore the co-prevalence of EBV and high-risk HPVs in 44 cervical cancer tissues from Syrian women using polymerase chain reaction (PCR), immunohistochemistry (IHC) and tissue microarray (TMA) analyses. We found that EBV and high-risk HPVs are co-present in 15/44 (34%) of the samples. Additionally, we report that the co-expression of LMP1 and E6 genes of EBV and high-risk HPVs, respectively, is associated with poorly differentiated squamous cell carcinomas phenotype; this is accompanied by a strong and diffused Id-1 overexpression, which is an important regulator of cell invasion and metastasis. These data imply that EBV and HPVs are co-present in cervical cancer in the Middle East area including Syria and their co-presence is associated with a more aggressive cancer phenotype. Future investigations are needed to elucidate the exact role of EBV and HPVs cooperation in cervical carcinogenesis.
... The BMP complex phosphorylates Smad-1/5, which translocates to the nucleus and regulates transcriptional events including the inhibitor of differentiation proteins (Id1, Id2, and Id3) [12][13][14]. Id1 has tumorigenic effects regulating self-renewal and migration of cancer cells [15][16][17][18][19]. Recent studies suggest that the BMP signaling cascade mediates cell survival independent of its regulation of Smad-1/5 transcription. ...
Background: Bone morphogenetic protein (BMP) is an evolutionarily conserved morphogen that is reactivated in lung carcinomas. BMP receptor inhibitors promote cell death of lung carcinomas by mechanisms not fully elucidated. The studies here reveal novel mechanisms by which the “survivin” inhibitor Ym155 in combination with the BMP receptor inhibitor JL5 synergistically induces death of lung cancer cells.
Methods: This study examines the mechanism by which Ym155 in combination with JL5 downregulates BMP signaling and induces cell death of non-small cell lung carcinoma (NSCLC) cell lines. Validation experiments were performed on five passage 0 primary NSCLC cell lines.
Results: We found that Ym155, which is reported to be a survivin inhibitor, potently inhibits BMP signaling by causing BMPR2 mislocalization into the cytoplasm and its decreased expression. The combination of Ym155 and the BMP receptor inhibitor JL5 synergistically causes the downregulation of BMP Smad-1/5 dependent and independent signaling and the induction of cell death of lung cancer cell lines and primary lung tumors. Cell death involves the nuclear translocation of apoptosis inducing factor (AIF) from the mitochondria to the nucleus. This causes DNA double stranded breaks independent of caspase activation, which occur only when JL5 and Ym155 are used in combination. Knockdown of BMPR2 together with Ym155 also induced AIF localization to the nucleus.
Conclusions: These studies suggest that inhibition of BMPR2 together with Ym155 can induce AIF caspase-independent cell death. AIF caspase-independent cell is an evolutionarily conserved cell death pathway that has never been targeted to induce cell death in cancer cells. These studies provide mechanistic insight of how to target AIF caspase-independent cell death using BMP inhibitors.
... Similar studies have demonstrated that exposure of breast tumor cells to chemotherapy in vitro followed by their transplantation into immune compromised mice (termed ex vivo) also results in an increase in BTIC frequency [7]. The resistance of BTIC to chemotherapy may explain why some breast cancer patients experience a remission after treatment followed by an oftenfatal tumor recurrence [8]. Importantly, non-tumorigenic tumor cells can be experimentally induced to become BTIC by initiating an epithelial-to-mesenchymal transition (EMT) [9]. ...
Background:
Breast tumor initiating cells (BTIC) are stem-like cells that initiate and sustain tumor growth, and drive disease recurrence. Identifying therapies targeting BTIC has been hindered due primarily to their scarcity in tumors. We previously reported that BTIC frequency ranges between 15% and 50% in multiple mammary tumors of 3 different transgenic mouse models of breast cancer and that this frequency is maintained in tumor cell populations cultured in serum-free, chemically defined media as non-adherent tumorspheres. The latter enabled high-throughput screening of small molecules for their capacity to affect BTIC survival. Antagonists of several serotonin receptors (5-HTRs) were among the hit compounds. The most potent compound we identified, SB-699551, selectively binds to 5-HT5A, a Gαi/o protein coupled receptor (GPCR).
Methods:
We evaluated the activity of structurally unrelated selective 5-HT5A antagonists using multiple orthogonal assays of BTIC frequency. Thereafter we used a phosphoproteomic approach to uncover the mechanism of action of SB-699551. To validate the molecular target of the antagonists, we used the CRISPR-Cas9 gene editing technology to conditionally knockout HTR5A in a breast tumor cell line.
Results:
We found that selective antagonists of 5-HT5A reduced the frequency of tumorsphere initiating cells residing in breast tumor cell lines and those of patient-derived xenografts (PDXs) that we established. The most potent compound among those tested, SB-699551, reduced the frequency of BTIC in ex vivo assays and acted in concert with chemotherapy to shrink human breast tumor xenografts in vivo. Our phosphoproteomic experiments established that exposure of breast tumor cells to SB-699551 elicited signaling changes in the canonical Gαi/o-coupled pathway and the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) axis. Moreover, conditional mutation of the HTR5A gene resulted in the loss of tumorsphere initiating cells and BTIC thus mimicking the effect of SB-699551.
Conclusions:
Our data provide genetic, pharmacological and phosphoproteomic evidence consistent with the on-target activity of SB-699551. The use of such agents in combination with cytotoxic chemotherapy provides a novel therapeutic approach to treat breast cancer.
... The BMP complex phosphorylates Smad-1/5, which translocates to the nucleus and regulates transcriptional events including the inhibitor of differentiation proteins (Id1, Id2, and Id3) [12][13][14]. Id1 has tumorigenic effects regulating self-renewal and migration of cancer cells [15][16][17][18][19]. Recent studies suggest that the BMP signaling cascade mediates cell survival independent of its regulation of Smad-1/5 transcription. ...
Background: Bone morphogenetic protein (BMP) is an evolutionarily conserved morphogen that is reactivated in lung carcinomas. BMP receptor inhibitors promote cell death of lung carcinomas by mechanisms not fully elucidated. The studies here reveal novel mechanisms by which the “survivin” inhibitor Ym155 in combination with the BMP receptor inhibitor JL5 synergistically induces death of lung cancer cells.
Methods: This study examines the mechanism by which Ym155 in combination with JL5 downregulates BMP signaling and induces cell death of non-small cell lung carcinoma (NSCLC) cell lines. Validation experiments were performed on five passage 0 primary NSCLC cell lines.
Results: We found that Ym155, which is reported to be a survivin inhibitor, potently inhibits BMP signaling by causing BMPR2 mislocalization into the cytoplasm and its decreased expression. The combination of Ym155 and the BMP receptor inhibitor JL5 synergistically causes the downregulation of BMP Smad-1/5 dependent and independent signaling and the induction of cell death of lung cancer cell lines and primary lung tumors. Cell death involves the nuclear translocation of apoptosis inducing factor (AIF) from the mitochondria to the nucleus. This causes DNA double stranded breaks independent of caspase activation, which occur only when JL5 and Ym155 are used in combination. Knockdown of BMPR2 together with Ym155 also induced AIF localization to the nucleus.
Conclusions: These studies suggest that inhibition of BMPR2 together with Ym155 can induce AIF caspase-independent cell death. AIF caspase-independent cell is an evolutionarily conserved cell death pathway that has never been targeted to induce cell death in cancer cells. These studies provide mechanistic insight of how to target AIF caspase-independent cell death using BMP inhibitors.
... The main biological properties of Id proteins are inhibition of differentiation and conservation of the self-renewal capability and multipotency of stem cells (11). Id proteins are overexpressed in several human carcinomas (11,12). More speci fically, Id-1 protein expression is directly involved in cancer initiation and/or progression in different types of human malignancies including cervical (9,(13)(14)(15). ...
Epstein-Barr virus (EBV) has been recently shown to be co-present with high-risk human papillomaviruses (HPVs) in human cervical cancer; thus, these oncoviruses play an important role in the initiation and/or progression of this cancer. Accordingly, our group has recently viewed the presence and genotyping distribution of high-risk HPVs in cervical cancer in Syrian women; our data pointed out that HPVs are present in 42/44 samples (95%). Herein, we aim to explore the co-prevalence of EBV and high-risk HPVs in 44 cervical cancer tissues from Syrian women using polymerase chain reaction, immunohistochemistry, and tissue microarray analyses. We found that EBV and high-risk HPVs are co-present in 15/44 (34%) of the samples. However, none of the samples was exclusively EBV-positive. Additionally, we report that the co-expression of LMP1 and E6 genes of EBV and high-risk HPVs, respectively, is associated with poorly differentiated squamous cell carcinomas phenotype; this is accompanied by a strong and diffuse overexpression of Id-1 (93% positivity), which is an important regulator of cell invasion and metastasis. These data imply that EBV and HPVs are co-present in cervical cancer samples in the Middle East area including Syria and their co-presence is associated with a more aggressive cancer phenotype. Future investigations are needed to elucidate the exact role of EBV and HPVs cooperation in cervical carcinogenesis.
... Intriguingly, study also showed that Id1 could down-regulate the ability of PC3 cells to form osteolytic lesions in vivo, although in the same study it was observed that knockdown of Id1 in PC3 cells inhibited the proliferation of cancer cells in vitro [67]. While evidence also suggests that Id1 is a key factor in promoting cancer metastasis to lungs [68], the discrepancy of microenvironment in different sites may be the cause. Moreover, Id1 was showed mediating chemosensitivity enhancement. ...
The inhibitor of DNA binding (Id) proteins are regulators of cell cycle and cell differentiation. Of all Id family proteins, Id1 is mostly linked to tumorigenesis, cellular senescence as well as cell proliferation and survival. Id1 is a stem cell-like gene more than a classical oncogene. Id1 is overexpressed in numerous types of cancers and exerts its promotion effect to these tumors through different pathways. Briefly, Id1 was found significantly correlated with EMT-related proteins, K-Ras signaling, EGFR signaling, BMP signaling, PI3K/Akt signaling, WNT and SHH signaling, c-Myc signaling, STAT3 signaling, RK1/2 MAPK/Egr1 pathway and TGF-β pathway, etc. Id1 has potent effect on facilitating tumorous angiogenesis and metastasis. Moreover, high expression of Id1 plays a facilitating role in the development of drug resistance, including chemoresistance, radiation resistance and resistance to drugs targeting angiogenesis. However, controversial results were also obtained. Overall, Id1 represent a promising target of anti-tumor therapeutics based on its potent promotion effect to cancer. Numerous drugs were found exerting their anti-tumor function through Id1-related signaling pathways, such as fucoidan, berberine, tetramethylpyrazine, crizotinib, cannabidiol and vinblastine.
... GAPDH served as a loading control. are enriched for genes of the LMS [22,23]. A defining trait of LM cells is their stem-like phenotype. ...
Lack of insight into the identity of the cells that initiate metastasis hampers the development of antimetastatic therapies. Only a tiny fraction of tumor cells termed metastasis-initiating cells (MICs) are able to successfully seed metastases, causing recurrence and therapeutic resistance. Using metastasis models, we describe a subpopulation of MIC derivates from lung metastases that do not have proliferation advantages, express high levels of the PDGF receptors and EMT/stemness-related genes, and are unique in their ability to initiate metastasis. PDGF factors specifically boost the metastatic potential of MIC populations in a PDGFR-dependent manner. However, PDGFR inhibition preferentially suppresses lung metastases, but does not reduce the primary tumor burden. Thus, we found that PDGFR inhibition blocks AKT activation, whereas SGK1, which shares high-similarity kinase domain and overlap substrates with AKT overexpression remains active in MICs. SGK1 and PDGF signaling act in concert to promote metastatic formation, and SGK1 inhibition confers vulnerability to PDGFR inhibitors, also eliciting a powerful antitumor effect. In vivo, SGK1 inhibitors sensitize xenograft tumors to PDGFR-targeted therapies by reducing primary tumor growth and lung metastasis. Consequently, dual inhibition of PDGFR and SGK1 exhibited strong antitumor activities in established breast cancer cell lines in vitro and in vivo. Therefore, this approach not only provides insight into MIC transformation but also aids the design of improved therapeutic strategies for advanced breast cancer.
... These are transcription factors implicated in promoting proliferation and migration as well as inhibiting cell differentiation in many types of cancer [34]. The expression of ID1 and ID3 in cell clusters of basal or triple-negative subtype breast tumors is required to reactivate and sustain proliferation of cancer cells in the lungs during metastatic colonization [35,36]. Coco, a secreted antagonist of TGF-β ligands, has also been shown to facilitate the escape of dormancy in disseminated cells in the lungs by blocking lung-derived paracrine bone morphogenetic protein (BMP) signaling. ...
Metastasis, a multistep process during which tumor cells disseminate to secondary organs, represents the main cause of death for cancer patients. Metastatic dormancy is a late stage during cancer progression, following extravasation of cells at a secondary site, where the metastatic cells stop proliferating but survive in a quiescent state. When the microenvironmental conditions are favorable, they re-initiate proliferation and colonize, sometimes years after treatment of the primary tumor. This phenomenon represents a major clinical obstacle in cancer patient care. In this review, we describe the current knowledge regarding the genetic or epigenetic mechanisms that are activated by cancer cells that either sustain tumor dormancy or promote escape from this inactive state. In addition, we focus on the role of the microenvironment with emphasis on the effects of extracellular matrix proteins and in factors implicated in regulating dormancy during colonization to the lungs, brain, and bone. Finally, we describe the opportunities and efforts being made for the development of novel therapeutic strategies to combat metastatic cancer, by targeting the dormancy stage.
Introduction
Lung adenocarcinoma (LUAD) is a common pathological type of lung cancer. The presence of lymph node metastasis plays a crucial role in determining the overall treatment approach and long‐term prognosis for early LUAD, therefore accurate prediction of lymph node metastasis is essential to guide treatment decisions and ultimately improve patient outcomes.
Methods
We performed transcriptome sequencing on T1 LUAD patients with positive or negative lymph node metastases and combined this data with The Cancer Genome Atlas Program cohort to identify potential risk molecules at the tissue level. Subsequently, by detecting the expression of these risk molecules by real‐time quantitative PCR in serum samples, we developed a model to predict the risk of lymph node metastasis from a training cohort of 96 patients and a validation cohort of 158 patients.
Results
Through transcriptome sequencing analysis of tissue samples, we identified 11 RNA (miR‐412, miR‐219, miR‐371, FOXC1, ID1, MMP13, COL11A1, PODXL2, CXCL13, SPOCK1 and MECOM) associated with positive lymph node metastases in T1 LUAD. As the expression of FOXC1 and COL11A1 was not detected in serum, we constructed a predictive model that accurately identifies patients with positive lymph node metastases using the remaining nine RNA molecules in the serum of T1 LUAD patients. In the training set, the model achieved an area under the curve (AUC) of 0.89, and in the validation set, the AUC was 0.91.
Conclusions
We have established a new risk prediction model using serum samples from T1 LUAD patients, enabling noninvasive identification of those with positive lymph node metastases.
Cancer cells adapt to varying stress conditions to survive through plasticity. Stem cells exhibit a high degree of plasticity, allowing them to generate more stem cells or differentiate them into specialized cell types to contribute to tissue development, growth, and repair. Cancer cells can also exhibit plasticity and acquire properties that enhance their survival. TGF-β is an unrivaled growth factor exploited by cancer cells to gain plasticity. TGF-β-mediated signaling enables carcinoma cells to alter their epithelial and mesenchymal properties through epithelial-mesenchymal plasticity (EMP). However, TGF-β is a multifunctional cytokine; thus, the signaling by TGF-β can be detrimental or beneficial to cancer cells depending on the cellular context. Those cells that overcome the anti-tumor effect of TGF-β can induce epithelial-mesenchymal transition (EMT) to gain EMP benefits. EMP allows cancer cells to alter their cell properties and the tumor immune microenvironment (TIME), facilitating their survival. Due to the significant roles of TGF-β and EMP in carcinoma progression, it is essential to understand how TGF-β enables EMP and how cancer cells exploit this plasticity. This understanding will guide the development of effective TGF-β-targeting therapies that eliminate cancer cell plasticity.
Cholangiocarcinoma (CCA) is a common malignant tumor of the biliary tract that carries a high burden of morbidity and a poor prognosis. Due to the lack of precise diagnostic methods, many patients are often diagnosed at advanced stages of the disease. The current treatment options available are of varying efficacy, underscoring the urgency for the discovery of more effective biomarkers for early diagnosis and improved treatment. Recently, single-cell sequencing (SCS) technology has gained popularity in cancer research. This technology has the ability to analyze tumor tissues at the single-cell level, thus providing insights into the genomics and epigenetics of tumor cells. It also serves as a practical approach to study the mechanisms of cancer progression and to explore therapeutic strategies. In this review, we aim to assess the heterogeneity of CCA using single-cell sequencing technology, with the ultimate goal of identifying possible biomarkers and potential treatment targets.
Incomplete tumor ablation and subsequent tumor metastasis usually occur during photothermal anti-tumor processes. The combination of photothermal and immunotherapy has proven to be a promising method to conquer technical challenges. Inhibiting the programmed death ligand-1 (PD-L1)/programmed cell death protein 1 (PD-1) immune pathway represents one of the most successful immunotherapy strategies. Whereas, the PD-L1 expression level significantly differs, leading to a relatively low response rate to the immune checkpoint blockade (ICB) approaches. Therefore, improving the expression level of PD-L1 becomes one potential method to enhance the response rate. Herein, NIH 3T3 cells were educated to steadily express PD-1 protein. Furthermore, the synthesized molybdenum nitride was then coated with PD-1 protein-modified cytomembrane, which endows it with immune checkpoint blocking capability. Moreover, under the irradiation of near-infrared light, the local mild heat released from the molybdenum nitride causes the apoptosis of tumor cells. More importantly, the elevated temperature simultaneously helps elevate the expression level of PD-L1, further enhancing the response rate of ICB. Finally, the PD-1 cytomembrane coatings interact with the upregulated PD-L1, leading to the activation of the immune system. In summary, we confirmed that the PD-1 protein-coated molybdenum nitride could synergistically ablate tumors and avoid metastasis.
Treatment options for brain metastatic breast cancer is limited because the molecular mechanism for how breast cancer cells infiltrate the brain is not fully understood. In order for breast tumors to metastasize to the brain first cells need to detach from the primary tumor, enter in the blood circulation, survive within the microvascular niche, and then cross the blood brain barrier (BBB) to colonize into the brain. It is critical to understand how breast cancer cells transmigrate through the BBB to prevent brain metastasis. Nuclear respiratory factor 1 (NRF1) transcription factor has been reported to be highly active in several human cancers and its aberrant expression facilitates in the acquisition of breast cancer stem cells (BCSCs). Inhibitor of differentiation protein 3 (ID3), a transcription regulating protein, induces pluripotent endothelial stem cells (ESCs). Herein we investigated if NRF1-induced BCSCs could cross a BBB model and guiding of BCSCs by ID3-induced ESCs across the BBB. BCSCs and ESCs were subjected to functional gain/loss experiments to determine if NRF1/ID3 contributed to lineage specific BCSCs organ entry. First, we tested whether NRF1 promoted migration of breast cancer using a BBB model consisting of BCSCs or MDA-MB231 cells, brain endothelial cell layer, and astrocytes. NRF1 overexpression increased the propensity for BCSCs and NRF1-induced MDA-MB231 cells to adhere to brain endothelial cells and migrate across a human BBB model. Increased adhesion of NRF1 induced BCSCs to ESCs ID3 was detected. NRF1 induced BCSCs crossed through the BBB model and this was promoted by ESCs ID3 . We also showed that environmental relevant exposure to PCBs (PCB153 and PCB77) produced differential effects. Treatment with PCB153 showed increased growth of NRF1 induced BCSCs tumor spheroids and increased in vivo migration of ESCs ID3 . Exosomal ID3 released from endothelial cells also supported the growth of NRF1 induced BCSCs and provide the basis for paracrine effects by ESCs ID3 associated with breast tumors. Xenograft experiments showed that ID3 overexpressing brain ESCs not only supported the growth of BCSC tumorospheroids but guided them to the neural crest in zebrafish. These findings show for the first time a novel role for ID3 and NRF1 by which ESCs ID3 help guide BCSCs NRF1 to distant metastatic sites where they most likely facilitate the colonization, survival, and proliferation of BCSCs. This knowledge is important for pre-clinical testing of NRF1/ID3 modifying agents to prevent the spread of breast cancer to the brain.
Triple Negative Breast Cancer (TNBC) is an aggressive breast cancer subtype with limited treatment options for chemo-resistant disease. Evidence from preclinical models and patient circulating tumor cells indicate that both the androgen receptor (AR) and TGFb pathways support anchorage independent survival, or anoikis resistance, to TNBC. Inhibition or knockdown of AR causes reduced TNBC growth in soft agar, invasion, mammosphere formation, and tumorigenicity. The transforming growth factor β (TGFβ) pathway, known to be important for epithelial to mesenchymal transition (EMT), is also heavily involved in anoikis resistance and is active in TNBC circulating tumor cells (CTCs). While there is evidence for a strong link between TGFβ and AR in prostate cancer, the collaboration between these two pathways in anoikis resistance and the potential for a combined targeting strategy was only recently studied in TNBC. Gene expression profiling combined with reverse phase protein array of TNBC cells in anchorage independent culture conditions revealed increased components of the TGFβ signaling pathway at the level of ligands, receptors and phosphorylated, nuclear Smad 3 localization and activity combined with AR activity and a positive feedback loop between these two pathways. These data are substantiated by the finding that when both TGFβ pathway components and AR are highly expressed in human breast cancer publicly available data sets, a poor outcome is predicted. These findings warrant further investigations into whether combined inhibition of the AR and TGFβ pathways may decrease metastatic recurrence rate and mortality from AR positive TNBC, which responds less well to chemotherapeutic approaches.
I have recently theorized that several similarities exist between the tumor process and embryo development. Starting from an initial cancer stem cell (CSC0), similar to an embryonic stem cell (ESC), after implantation in a niche, primary self-renewing CSCs (CSC1s) would arise, which then generate secondary proliferating CSCs (CSC2s). From these epithelial CSCs, tertiary mesenchymal CSCs (CSC3s) would arise, which, under favorable stereotrophic conditions, by asymmetric proliferation, would generate cancer progenitor cells (CPCs) and then cancer differentiated cells (CDCs), thus giving a defined cell heterogeneity and hierarchy. CSC1s–CSC2s–CSC3s–CPCs–CDCs would constitute a defined “tumor growth module,” able to generate new tumor modules, forming a spherical avascular mass, similar to a tumor sphere. Further growth in situ of this initial tumor would require implantation in the host and vascularization through the overexpression of some aspecific checkpoint molecules, such as CD44, ID, LIF, HSP70, and HLA-G. To expand and spread in the host tissues, this vascularized tumor would then carry on a real growth strategy based on other specific checkpoint factors, such as those contained in the extracellular vesicles (EVs), namely, microRNAs, messenger RNAs, long non-coding RNAs, and integrins. These EV components would be crucial in tumor progression because they can mediate intercellular communications in the surrounding microenvironment and systemically, dictating to recipient cells a new tumor-enslaved phenotype, thus determining pre-metastatic conditions. Moreover, by their induction properties, the EV contents could also frustrate in time the effects of cytolytic tumor therapies, where EVs released by killed CSCs might enter other cancer and non-cancer cells, thus giving chemoresistance, non-CSC/CSC transition (recurrence), and metastasis. Thus, antitumor cytotoxic treatments, “shielded” from the EV-specific checkpoints by suitable adjuvant agents, simultaneously targeting the aforesaid aspecific checkpoints should be necessary for dismantling the hierarchic tumor structure, avoiding recurrence and preventing metastasis.
Owing to increased awareness of the importance of mammogram and advances in surgical technology, survival rate of patients with primary breast cancer has dramatically increased. Despite all these advances in breast cancer treatment, there are no currently available treatments for this disease once it metastasizes to distant organs including bones, lungs, brain, and liver. This is mainly attributed to the complexity of metastatic process. Recent advances in technology enabled cancer biologists to dissect each step of the metastatic process, and this led to discovery of major players and molecules in this process. In this section, we will discuss recent discovery and advances in the field of breast cancer metastasis research.
Oncogenic transformation has been the major cause of global mortality since decades. Despite established therapeutic regimes, majority of cancer patients either present with tumor relapse, refractory disease or therapeutic resistance. Numerous drug candidates are being explored to tap the key reason being poor tumor remission rates, from novel chemotherapy agents to immunotherapy to exploring natural compound derivatives with effective anti-cancer potential. One of these natural product metabolites, emodin has present with significant potential to target tumor oncogenic processes: induction of apoptosis and cell cycle arrest, tumor angiogenesis, and metastasis to chemoresistance in malignant cells. Based on the present scientific excerpts on safety and effectiveness of emodin in targeting hallmarks of tumor progression, emodin is being promisingly explored using nanotechnology platforms for long-term sustained treatment and management of cancer patients. In this review, we summarize the up-to-date scientific literature supporting the anti-neoplastic potential of emodin. We also provide an insight into toxicity and safety profile of emodin and how emodin has emerged as an effective therapeutic alternative in synergism with established conventional chemotherapeutic regimes for management and treatment of tumor progression.
In this chapter, we provide a comprehensive review of the pathogenesis, features, diagnostic evaluation, management, survival, and complications from lung metastasis. Lung metastasis may arise due to a combination of dietary influences, genetic and immunologic changes using normal cellular processes to assist in metastatic spread. Computed tomography with or without positron emission tomography is most frequently used to detect lung metastases since MRI is less sensitive than these modalities. Patients with isolated or oligometastatic disease can be considered for definitive management, including radiofrequency ablation (RFA), stereotactic radiation therapy, and surgical resection with non-surgical interventions typically offered for unresectable tumors or poor surgical candidates. Primary tumor types that commonly have lung metastasis include colon carcinoma, renal cell carcinoma, sarcoma, melanoma, head and neck squamous cell carcinoma, germ cell tumors, breast cancer, and giant cell tumor of bone, and survival after treatment is highly dependent on the primary site. Common complications of metastatic disease include malignant pleural effusions, managed with indwelling catheters or sclerotic agents, and bronchial obstructions, managed with endobronchial stenting, photodynamic therapy, and radiation therapy.
The tumorigenicity of cancer cells is highly influenced by the extracellular matrix (ECM) through mechanisms that are poorly understood. Here it is reported that a variety of 3D ECM microenvironments strongly induce expression of Id1 and Id3 in melanoma cells. Genetic ablation of Id1/Id3 impairs melanoma cell outgrowth in 3D Matrigel culture and inhibits melanoma initiation in vivo. Mechanistically, 3D ECM microenvironments hinder diffusion of endogenously produced bone morphogenetic proteins, thereby fostering autocrine signaling and Id1/Id3 expression. A compound screen identifies new coumarin derivatives that potently inhibit both Id1/Id3 expression and melanoma initiation in vivo. Together, the findings reveal a novel mechanism through which the ECM increases tumorigenicity, identify Id1/Id3 as melanoma‐relevant therapeutic targets, and characterize inhibitors of Id1/Id3 expression with therapeutic potential.
Metastasis is the main culprit of the great majority of cancer-related deaths. However, the complicated process of the invasion-metastasis cascade remains the least understood aspect of cancer biology. Telomerase plays a pivotal role in bypassing cellular senescence and sustaining the cancer progression by maintaining telomere homeostasis and genomic integrity. Telomerase reverse transcriptase (TERT) exerts a series of fundamental functions that are independent of its enzymatic cellular activity, including proliferation, inflammation, epithelia-mesenchymal transition (EMT), angiogenesis, DNA repair, and gene expression. Accumulating evidence indicates that TERT may facilitate most steps of the invasion-metastasis cascade. In this review, we summarize important advances that have revealed some of the mechanisms by which TERT facilitates tumor metastasis, providing an update on the non-canonical functions of telomerase beyond telomere maintaining.
Mammary epithelial cells constitutively expressing Id-1 protein are unable to differentiate, acquire the ability to proliferate, and invade the extracellular matrix. In addition, Id-1 is aberrantly over-expressed in aggressive and metastatic breast cancer cells, as well as in human breast tumor biopsies from infiltrating carcinomas, suggesting Id-1 might be an important regulator of breast cancer progression. We show that human metastatic breast cancer cells become significantly less invasive in vitro and less metastatic in vivo when Id-1 is down-regulated by stable transduction with antisense Id-1. Expression of the matrix metalloproteinase MT1-MMP is decreased in proportion to the decrease in Id-1 protein levels, representing a potential mechanism for the reduction of invasiveness. Further, to more accurately recapitulate the biology of and potential therapeutic approaches to tumor metastasis, we targeted Id-1 expression systemically in tumor-bearing mice by using a nonviral approach. We demonstrate significant reduction of both Id-1 and MT1-MMP expressions as well as the metastatic spread of 4T1 breast cancer cells in syngeneic BALB/c mice. In conclusion, our studies have identified Id-1 as a critical regulator of breast cancer progression and suggest the feasibility of developing novel therapeutic approaches to target Id-1 expression to reduce breast cancer metastasis in humans.
On the subject of acute myeloid leukemia (AML), there is little consensus about the target cell within the hematopoietic stem cell hierarchy that is susceptible to leukemic transformation, or about the mechanism that underlies the phenotypic, genotypic and clinical heterogeneity. Here we demonstrate that the cell capable of initiating human AML in non-obese diabetic mice with severe combined immunodeficiency disease (NOD/SCID mice) - termed the SCID leukemia-initiating cell, or SL-IC - possesses the differentiative and proliferative capacities and the potential for self-renewal expected of a leukemic stem cell. The SL-ICs from all subtypes of AML analyzed, regardless of the heterogeneity in maturation characteristics of the leukemic blasts, were exclusively CD34++ CD38-, similar to the cell-surface phenotype of normal SCID-repopulating cells, suggesting that normal primitive cells, rather than committed progenitor cells, are the target for leukemic transformation. The SL-ICs were able to differentiate in vivo into leukemic blasts, indicating that the leukemic clone is organized as a hierarchy.
Amplification of the Neu (ErbB-2 or HER-2) receptor tyrosine kinase occurs in 20 to 30% of human mammary carcinomas, correlating with a poor clinical prognosis. We have previously demonstrated that four (Y1144 Y1201, Y1227 and Y1253) of the five known Neu autophosphorylation sites can independently mediate transforming signals. The transforming potential of two of these mutants correlates with their capacity to recruit Grb2 directly to Y1144 (YB) or indirectly through Shc to Y1227 (YD). Here, we demonstrate that these transformation-competent neu mutants activate extracellular signal-regulated kinases and stimulate Ets-2-dependent transcription. Although the transforming potential of three of these mutants (YB, YD, and YE) was susceptible to inhibition by Rap1A, a genetic antagonist of Ras, the transforming potential of YC was resistant to inhibition by Rap1A. To further address the significance of these ErbB-2-coupled signaling molecules in induction of mammary cancers, transgenic mice expressing mutant Neu receptors lacking the known autophosphorylation sites (NYPD) or those coupled directly to either Grb2 (YB) or Shc (YD) adapter molecules were derived. In contrast to the NYPD strains, which developed focal mammary tumors after a long latency period with low penetrance, all female mice derived from YB and YD strains rapidly developed mammary tumors. Although female mice from several independent YB or YD lines developed mammary tumors, the YB strains developed lung metastases at substantially higher rates than the YD strains. These observations argue that Grb2 and Shc play important and distinct roles in ErbB-2/Neu-induced mammary tumorigenesis and metastasis.
Breast cancer is a genetically and clinically heterogeneous disease, and the contributions of different target cells and different oncogenic mutations to this heterogeneity are not well understood. Here we report that mammary tumors induced by components of the Wnt signaling pathway contain heterogeneous cell types and express early developmental markers, in contrast to tumors induced by other signaling elements. Expression of the Wnt-1 protooncogene in mammary glands of transgenic mice expands a population of epithelial cells expressing progenitor cell markers, keratin 6 and Sca-1; subsequent tumors express these markers and contain luminal epithelial and myoepithelial tumor cells that share a secondary mutation, loss of Pten, implying that they arose from a common progenitor. Mammary tumors arising in transgenic mice expressing beta-catenin and c-Myc, downstream components of the canonical Wnt signaling pathway, also contain a significant proportion of myoepithelial cells and cells expressing keratin 6. Progenitor cell markers and myoepithelial cells, however, are lacking in mammary tumors from transgenic mice expressing Neu, H-Ras, or polyoma middle T antigen. These results suggest that mammary stem cells and/or progenitors to mammary luminal epithelial and myoepithelial cells may be the targets for oncogenesis by Wnt-1 signaling elements. Thus, the developmental heterogeneity of different breast cancers is in part a consequence of differential effects of oncogenes on distinct cell types in the breast.
Why are tumours heterogeneous, in terms of cell phenotype and proliferative potential, even in cases in which all cells are derived from a single clone? Ongoing mutagenesis can partially explain this heterogeneity, but it also seems that some tumours arise from small populations of 'cancer stem cells' that give rise to phenotypically diverse cancer cells, with less proliferative potential. These cancer stem cells are likely to arise from mutations that dysregulate normal stem-cell self-renewal. Using this information, it might be possible to devise more effective therapies.
By means of in vivo selection, transcriptomic analysis, functional verification and clinical validation, here we identify a set of genes that marks and mediates breast cancer metastasis to the lungs. Some of these genes serve dual functions, providing growth advantages both in the primary tumour and in the lung microenvironment. Others contribute to aggressive growth selectively in the lung. Many encode extracellular proteins and are of previously unknown relevance to cancer metastasis.
Previous work has shown that cyclin D1 expression is required for neu- and ras-induced, but not wnt- or c-myc-induced, breast tumorigenesis in mice. Although cyclin D1 binds and activates cyclin-dependent kinase 4 (Cdk4), thereby mediating activation of a program of E2F-dependent gene expression, it has been suggested that the oncogenic activities of cyclin D1 are independent of Cdk4. To determine whether Cdk4 expression is required for breast tumorigenesis in mice, we have generated compound mice ectopically expressing the neu or wnt oncogenes in the mammary glands of wild-type and Cdk4-/- mice. Our results show that Cdk4 expression is required for efficient neu-induced tumorigenesis but is dispensable for wnt-induced breast tumorigenesis. In contrast to results previously observed in the mammary glands of cyclin D1-/- virgin females, our results show defects in mammary gland development in Cdk4-/- virgin females, suggesting differences in compensatory mechanisms in the absence of either subunit of the cyclin D1/Cdk4 complex. These results suggest that drugs targeted to inhibit Cdk4 activities could be developed to specifically treat certain breast tumors as Cdk4 is not essential for viability.
Microarray profiling of invasive breast carcinomas has identified five distinct subtypes of tumors (luminal A, luminal B, normal breast-like, HER2 overexpressing, and basal-like) that are associated with different clinical outcomes. The basal-like subtype is associated with poor clinical outcomes and is the subtype observed in BRCA1-related breast cancers. The aim of this study was to characterize the histologic and immunophenotypic properties of breast basal-like carcinomas that were first positively identified using DNA microarray analysis. Detailed histologic review was performed on 56 tumors with known microarray profiles (23 basal-like, 23 luminal, and 12 HER2+). Immunohistochemistry for estrogen receptor (ER), HER2, EGFR, smooth muscle actin (SMA), p63, CD10, cytokeratin 5/6, cytokeratin 8/18, and vimentin was performed on 18 basal-like, 16 luminal, and 12 HER2+ tumors. The basal-like tumors were grade 3 ductal/NOS (21/23) or metaplastic (2/23) carcinomas that frequently showed geographic necrosis (17/23), a pushing border of invasion (14/23), and a stromal lymphocytic response (13/23). Most basal-like tumors showed immunoreactivity for vimentin (17/18), luminal cytokeratin 8/18 (15/18), EGFR (13/18), and cytokeratin 5/6 (11/18), while positivity for the myoepithelial markers SMA (4/18), p63 (4/18) and CD10 (2/18) was infrequent. All basal-like tumors tested were ER- and HER2-. Morphologic features significantly associated with the basal-like subtype included markedly elevated mitotic count (P<0.0001), geographic tumor necrosis (P=0.0003), pushing margin of invasion (P=0.0001), and stromal lymphocytic response (P=0.01). The most consistent immunophenotype seen in the basal-like tumors was negativity for ER and HER2, and positivity for vimentin, EGFR, cytokeratin 8/18, and cytokeratin 5/6. The infrequent expression of myoepithelial markers in basal-like carcinomas does not support a direct myoepithelial cell derivation of these tumors. These findings should further assist in the identification of basal-like carcinomas in clinical specimens, facilitating treatment and epidemiologic studies of this tumor subtype.
Elucidation of the cellular and molecular mechanisms that maintain mammary epithelial tissue integrity is of broad interest and paramount to the design of more effective treatments for breast cancer. Evidence from both in vitro and in vivo experiments suggests that mammary cell differentiation is a hierarchical process originating in an uncommitted stem cell with self-renewal potential. However, analysis of the properties and regulation of mammary stem cells has been limited by a lack of methods for their prospective isolation. Here we report the use of multi-parameter cell sorting and limiting dilution transplant analysis to demonstrate the purification of a rare subset of adult mouse mammary cells that are able individually to regenerate an entire mammary gland within 6 weeks in vivo while simultaneously executing up to ten symmetrical self-renewal divisions. These mammary stem cells are phenotypically distinct from and give rise to mammary epithelial progenitor cells that produce adherent colonies in vitro. The mammary stem cells are also a rapidly cycling population in the normal adult and have molecular features indicative of a basal position in the mammary epithelium.
The contribution of bone marrow–derived circulating endothelial progenitor cells (CEPs) to tumor angiogenesis has been controversial,
primarily because of their low numbers in blood vessels of untreated tumors. We show that treatment of tumor-bearing mice
with vascular disrupting agents (VDAs) leads to an acute mobilization of CEPs, which home to the viable tumor rim that characteristically
remains after such therapy. Disruption of this CEP spike by antiangiogenic drugs or by genetic manipulation resulted in marked
reductions in tumor rim size and blood flow as well as enhanced VDA antitumor activity. These findings also provide a mechanistic
rationale for the enhanced efficacy of VDAs when combined with antiangiogenic drugs.
The association between large tumor size and metastatic risk in a majority of clinical cancers has led to questions as to whether these observations are causally related or whether one is simply a marker for the other. This is partly due to an uncertainty about how metastasis-promoting gene expression changes can arise in primary tumors. We investigated this question through the analysis of a previously defined “lung metastasis gene-expression signature” (LMS) that mediates experimental breast cancer metastasis selectively to the lung and is expressed by primary human breast cancer with a high risk for developing lung metastasis. Experimentally, we demonstrate that the LMS promotes primary tumor growth that enriches for LMS⁺ cells, and it allows for intravasation after reaching a critical tumor size. Clinically, this corresponds to LMS⁺ tumors being larger at diagnosis compared with LMS⁻ tumors and to a marked rise in the incidence of metastasis after LMS⁺ tumors reach 2 cm. Patients with LMS-expressing primary tumors selectively fail in the lung compared with the bone or other visceral sites and have a worse overall survival. The mechanistic linkage between metastasis gene expression, accelerated tumor growth, and likelihood of metastatic recurrence provided by the LMS may help to explain observations of prognostic gene signatures in primary cancer and how tumor growth can both lead to metastasis and be a marker for cells destined to metastasize.
• cancer
• genomics
• oncogenesis
Metastasis entails numerous biological functions that collectively enable cancerous cells from a primary site to disseminate and overtake distant organs. Using genetic and pharmacological approaches, we show that the epidermal growth factor receptor ligand epiregulin, the cyclooxygenase COX2, and the matrix metalloproteinases 1 and 2, when expressed in human breast cancer cells, collectively facilitate the assembly of new tumour blood vessels, the release of tumour cells into the circulation, and the breaching of lung capillaries by circulating tumour cells to seed pulmonary metastasis. These findings reveal how aggressive primary tumorigenic functions can be mechanistically coupled to greater lung metastatic potential, and how such biological activities may be therapeutically targeted with specific drug combinations.
Although numerous mouse models of breast carcinomas have been developed, we do not know the extent to which any faithfully represent clinically significant human phenotypes. To address this need, we characterized mammary tumor gene expression profiles from 13 different murine models using DNA microarrays and compared the resulting data to those from human breast tumors.
Unsupervised hierarchical clustering analysis showed that six models (TgWAP-Myc, TgMMTV-Neu, TgMMTV-PyMT, TgWAP-Int3, TgWAP-Tag, and TgC3(1)-Tag) yielded tumors with distinctive and homogeneous expression patterns within each strain. However, in each of four other models (TgWAP-T121, TgMMTV-Wnt1, Brca1Co/Co;TgMMTV-Cre;p53+/- and DMBA-induced), tumors with a variety of histologies and expression profiles developed. In many models, similarities to human breast tumors were recognized, including proliferation and human breast tumor subtype signatures. Significantly, tumors of several models displayed characteristics of human basal-like breast tumors, including two models with induced Brca1 deficiencies. Tumors of other murine models shared features and trended towards significance of gene enrichment with human luminal tumors; however, these murine tumors lacked expression of estrogen receptor (ER) and ER-regulated genes. TgMMTV-Neu tumors did not have a significant gene overlap with the human HER2+/ER- subtype and were more similar to human luminal tumors.
Many of the defining characteristics of human subtypes were conserved among the mouse models. Although no single mouse model recapitulated all the expression features of a given human subtype, these shared expression features provide a common framework for an improved integration of murine mammary tumor models with human breast tumors.
Ectopic activation of the Wnt signaling pathway is highly oncogenic for many human tissues. Here, we show that ectopic Wnt signaling increases the effective stem cell activity in mouse mammary glands in vivo. Furthermore, Wnt effectors induce the accumulation of mouse mammary epithelial progenitors (assayed by Hoechst dye exclusion, a surrogate stem cell marker, side population cells) both in vivo and in vitro. The longevity of stem cells makes them good candidate tumor precursors, and we propose that Wnt-induced progenitor amplification is likely to be key to tumor initiation. In support of this notion, mammary glands from a tumor-resistant strain of mice (carrying a null mutation in syndecan-1) contain fewer side population cells. When this strain is crossed to mice that overexpress effectors of the beta-catenin/T cell factor Wnt pathway, the amplification of progenitors is reduced, together with all subsequent events of tumor development. We propose that the growth dynamic of the stem cell fraction is a major determinant of tumor susceptibility.
To study phenotype-genotype correlations, ErbB/Ras pathway tumors (transgenic for ErbB2, c-Neu, mutants of c-Neu, polyomavirus middle T antigene (PyV-mT), Ras, and bi-transgenic for ErbB2/Neu with ErbB3 and with progesterone receptor) from four different institutions were histopathologically compared with Wnt pathway tumors [transgenes Wnt1, Wnt10b, dominant-negative glycogen synthase kinase 3-β, β-Catenin, and spontaneous mutants of adenomatous polyposis coli gene (Apc)]. ErbB/Ras pathway tumors tend to form solid nodules consisting of poorly differentiated cells with abundant cytoplasm. ErbB/Ras pathway tumors also have scanty stroma and lack myoepithelial or squamous differentiation. In contrast, Wnt pathway tumors exhibit myoepithelial, acinar, or glandular differentiation, and, frequently, combinations of these. Squamous metaplasia is frequent and may include transdifferentiation to epidermal and pilar structures. Most Wnt pathway tumors form caricatures of elongated, branched ductules, and have well-developed stroma, inflammatory infiltrates, and pushing margins. Tumors transgenic for interacting genes such as protein kinase CK2α (casein kinase IIα), and the fibroblast growth factors (Fgf) Int2/Fgf3 or keratinocyte growth factor (Kgf/Fgf7) also have the Wnt pathway phenotype. Because the tumors from the ErbB/Ras and the Wnt pathway are so distinct and can be readily identified using routine hematoxylin and eosin sections, we suggest that pathway pathology is applicable in both basic and clinical cancer research.
Id proteins may control cell differentiation by interfering with DNA binding of transcription factors. Here we show that targeted disruption of the dominant negative helix–loop–helix proteins Id1 and Id3 in mice results in premature withdrawal of neuroblasts from the cell cycle and expression of neural-specific differentiation markers. The Id1–Id3 double knockout mice also display vascular malformations in the forebrain and an absence of branching and sprouting of blood vessels into the neuroectoderm. As angiogenesis both in the brain and in tumours requires invasion of avascular tissue by endothelial cells, we examined the Id knockout mice for their ability to support the growth of tumour xenografts. Three different tumours failed to grow and/or metastasize in Id1+/-Id3-/- mice, and any tumour growth present showed poor vascularization and extensive necrosis. Thus, the Id genes are required to maintain the timing of neuronal differentiation in the embryo and invasiveness of the vasculature. Because the Id genes are expressed at very low levels in adults, they make attractive new targets for anti-angiogenic drug design.
Transcriptional activation of the int-1 gene by proviral insertion mutations is thought to be a key step in mammary tumor induction by the mouse mammary tumor virus (MMTV). To test this hypothesis, we have constructed an int-1 allele resembling those found in virus-induced tumors, with an MMTV LTR placed 5' to the int-1 gene in the opposite transcriptional orientation. Transgenic mice harboring this allele express int-1 RNA at high levels in mammary and salivary glands of male and female mice and in male reproductive organs. The mammary glands of males and virgin females are grossly hyperplastic compared with those of nontrasgenic littermates. Mammary and (less frequently) salivary adenocarcinomas occur in these animals at rates indicating that transcriptional activation of int-1 and associated hyperplasia are initiating events in multistep carcinogenesis.
Mammary epithelial cells undergo changes in growth, invasion, and differentiation throughout much of adulthood, and most strikingly during pregnancy, lactation, and involution. Although the pathways of milk protein expression are being elucidated, little is known, at a molecular level, about control of mammary epithelial cell phenotypes during normal tissue morphogenesis and evolution of aggressive breast cancer. We developed a murine mammary epithelial cell line, SCp2, that arrests growth and functionally differentiates in response to a basement membrane and lactogenic hormones. In these cells, expression of Id-1, an inhibitor of basic helix-loop-helix transcription factors, declines prior to differentiation, and constitutive Id-1 expression blocks differentiation. Here, we show that SCp2 cells that constitutively express Id-1 slowly invade the basement membrane but remain anchorage dependent for growth and do not form tumors in nude mice. Cells expressing Id-1 secreted a approximately 120-kDa gelatinase. From inhibitor studies, this gelatinase appeared to be a metalloproteinase, and it was the only metalloproteinase detectable in conditioned medium from these cells. A nontoxic inhibitor diminished the activity of this metalloproteinase in vitro and repressed the invasive phenotype of Id-1-expressing cells in culture. The implications of these findings for normal mammary-gland development and human breast cancer were investigated. A gelatinase of approximately 120 kDa was expressed by the mammary gland during involution, a time when Id-1 expression is high and there is extensive tissue remodeling. Moreover, high levels of Id-1 expression and the activity of a approximately 120-kDa gelatinase correlated with a less-differentiated and more-aggressive phenotype in human breast cancer cells. We suggest that Id-1 controls invasion by normal and neoplastic mammary epithelial cells, primarily through induction of a approximately 120-kDa gelatinase. This Id-1-regulated invasive phenotype could contribute to involution of the mammary gland and possibly to the development of invasive breast cancer.
Breast cancer is the most common malignancy among women. Most of these cancers overexpress cyclin D1, a component of the core cell-cycle machinery. We previously generated mice lacking cyclin D1 using gene targeting. Here we report that these cyclin D1-deficient mice are resistant to breast cancers induced by the neu and ras oncogenes. However, animals lacking cyclin D1 remain fully sensitive to other oncogenic pathways of the mammary epithelium, such as those driven by c-myc or Wnt-1. Our analyses revealed that, in mammary epithelial cells, the Neu-Ras pathway is connected to the cell-cycle machinery by cyclin D1, explaining the absolute dependency on cyclin D1 for malignant transformation in this tissue. Our results suggest that an anti-cyclin D1 therapy might be highly specific in treating human breast cancers with activated Neu-Ras pathways.
To study phenotype-genotype correlations, ErbB/Ras pathway tumors (transgenic for ErbB2, c-Neu, mutants of c-Neu, polyomavirus middle T antigene (PyV-mT), Ras, and bi-transgenic for ErbB2/Neu with ErbB3 and with progesterone receptor) from four different institutions were histopathologically compared with Wnt pathway tumors [transgenes Wnt1, Wnt10b, dominant-negative glycogen synthase kinase 3-beta, beta-Catenin, and spontaneous mutants of adenomatous polyposis coli gene (Apc)]. ErbB/Ras pathway tumors tend to form solid nodules consisting of poorly differentiated cells with abundant cytoplasm. ErbB/Ras pathway tumors also have scanty stroma and lack myoepithelial or squamous differentiation. In contrast, Wnt pathway tumors exhibit myoepithelial, acinar, or glandular differentiation, and, frequently, combinations of these. Squamous metaplasia is frequent and may include transdifferentiation to epidermal and pilar structures. Most Wnt pathway tumors form caricatures of elongated, branched ductules, and have well-developed stroma, inflammatory infiltrates, and pushing margins. Tumors transgenic for interacting genes such as protein kinase CK2alpha (casein kinase IIalpha), and the fibroblast growth factors (Fgf) Int2/Fgf3 or keratinocyte growth factor (Kgf/Fgf7) also have the Wnt pathway phenotype. Because the tumors from the ErbB/Ras and the Wnt pathway are so distinct and can be readily identified using routine hematoxylin and eosin sections, we suggest that pathway pathology is applicable in both basic and clinical cancer research.
Researchers have been studying metastasis for more than 100 years, and only recently have we gained insight into the mechanisms by which metastatic cells arise from primary tumours and the reasons that certain tumour types tend to metastasize to specific organs. Stephen Paget's 1889 proposal that metastasis depends on cross-talk between selected cancer cells (the 'seeds') and specific organ microenvironments (the 'soil') still holds forth today. It is now known that the potential of a tumour cell to metastasize depends on its interactions with the homeostatic factors that promote tumour-cell growth, survival, angiogenesis, invasion and metastasis. How has this field developed over the past century, and what major breakthroughs are most likely to lead to effective therapeutic approaches?
We investigated the molecular basis for osteolytic bone metastasis by selecting human breast cancer cell line subpopulations with elevated metastatic activity and functionally validating genes that are overexpressed in these cells. These genes act cooperatively to cause osteolytic metastasis, and most of them encode secreted and cell surface proteins. Two of these genes, interleukin-11 and CTGF, encode osteolytic and angiogenic factors whose expression is further increased by the prometastatic cytokine TGF beta. Overexpression of this bone metastasis gene set is superimposed on a poor-prognosis gene expression signature already present in the parental breast cancer population, suggesting that metastasis requires a set of functions beyond those underlying the emergence of the primary tumor.
The basic helix-loop-helix (bHLH) family of transcription factors functions in the coordinated regulation of gene expression, cell lineage commitment, and cell differentiation in most mammalian tissues. Helix-loop-helix Id (Inhibitor of DNA binding) proteins are distinct from bHLH transcription factors in that they lack the basic domain necessary for DNA binding. Id proteins thus function as dominant negative regulators of bHLH transcription factors. The inhibition of bHLH factor activity by forced constitutive expression of Id proteins is closely associated with the inhibition of differentiation in a number of different cell types, including mammary epithelial cells. Moreover, recent literature suggests important roles of HLH proteins in many normal and transformed tissues, including mammary gland. Therefore, future directions for prognosis or therapeutic treatments of breast cancer may be able to exploit bHLH and Id genes as useful molecular targets. The purpose of this review is to summarize the evidence implicating HLH proteins in the regulation of normal and transformed mammary epithelial cell phenotypes.
The intestinal epithelium follows the paradigms of stem cell biology established for other self-renewing tissues. With a unique topology, it constitutes a two-dimensional structure folded into valleys and hills: the proliferative crypts and the differentiated villi. Its unprecedented self-renewal rate appears reflected in a high susceptibility to malignant transformation. The molecular mechanisms that control homeostatic self-renewal and those that underlie colorectal cancer are remarkably symmetrical. Here, we discuss the biology of the intestinal epithelium, emphasizing the roles played by Wnt, bone morphogenic protein, and Notch signaling cascades in epithelial self-renewal and cancer.
Over the past few decades, biologists have identified key molecular signatures associated with a wide range of human cancers. Recently, animal models have been particularly useful in establishing whether such signatures have functional relevance; the overexpression of pro-oncogenic or loss of anti-oncogenic factors have been evaluated for their effects on various tumour models. The aim of this review is to analyze the potential role of the inhibitor of DNA binding (Id) proteins in cancer and examine whether deregulated Id activity is tumorigenic and contributes to hallmarks of malignancy, such as loss of differentiation (anaplasia), unrestricted proliferation and neoangiogenesis.
Gene-expression-profiling studies of primary breast tumors performed by different laboratories have resulted in the identification of a number of distinct prognostic profiles, or gene sets, with little overlap in terms of gene identity.
To compare the predictions derived from these gene sets for individual samples, we obtained a single data set of 295 samples and applied five gene-expression-based models: intrinsic subtypes, 70-gene profile, wound response, recurrence score, and the two-gene ratio (for patients who had been treated with tamoxifen).
We found that most models had high rates of concordance in their outcome predictions for the individual samples. In particular, almost all tumors identified as having an intrinsic subtype of basal-like, HER2-positive and estrogen-receptor-negative, or luminal B (associated with a poor prognosis) were also classified as having a poor 70-gene profile, activated wound response, and high recurrence score. The 70-gene and recurrence-score models, which are beginning to be used in the clinical setting, showed 77 to 81 percent agreement in outcome classification.
Even though different gene sets were used for prognostication in patients with breast cancer, four of the five tested showed significant agreement in the outcome predictions for individual patients and are probably tracking a common set of biologic phenotypes.
Id proteins are a class of dominant-negative antagonists of helix-loop-helix transcription factors and have been shown to control differentiation of a variety of cell types in diverse organisms. Although the importance of Id1 in tumor endothelial cells is well established, the expression and role of the Id1 protein in human cancer cells is controversial. To explore this issue, we developed and characterized a highly specific rabbit monoclonal antibody against Id1 to assess its expression in human breast, prostate, and bladder malignancies. Our results show that in usual types of human mammary carcinomas, the Id1 protein is expressed exclusively in the endothelium. Interestingly, we detected nuclear expression of the Id1 protein in the tumor cells in 10 of 45 cases of poorly differentiated and highly aggressive carcinoma with metaplastic morphology. Similarly, only 1 of 30 prostate cancer samples showed Id1-positive tumor cells, whereas in almost all, endothelial cells showed high Id1 expression. Intriguingly, whereas normal prostate glands do not show any Id1 protein expression, basal layer cells of benign prostate glands in proximity to tumors expressed high levels of the Id1 protein. In contrast to the lack of Id1 expression in the usual types of mammary and prostate cancers, the majority of transitional cell bladder tumors showed Id1 protein expression in both tumor and endothelial cells. These results suggest that further refinement of Id1 expression patterns in a variety of tumor types will be necessary to identify and study the functional roles played by Id1 in human neoplastic processes.
How tumors spread and kill their host organism remains an enigma, but not for lack of attention. For more than a century, cancer biologists have postulated that metastasis results from the interplay of wandering tumor cells with permissive target tissues. Yet, decades of scrutiny into the molecular bases of cancer have largely focused on what causes oncogenic transformation and the incipient emergence of tumors. By comparison, the study of how tumor cells take steps toward metastasis (that is, by altering their microenvironment, entering the circulation, and colonizing a distant organ) has received less attention. Progressively, however, the idea has emerged that tumors are more than just a mass of transformed cells. A renewed focus on the problem of metastasis is now apparent, and for good reason—metastasis remains the cause of 90% of deaths from solid tumors.
Triple-negative breast cancer (estrogen receptor-negative, progesterone receptor-negative, and HER2-negative) is a high risk breast cancer that lacks the benefit of specific therapy that targets these proteins.
In this study, the authors examined a large and well characterized series of invasive breast carcinoma (n = 1944) with a long-term clinical follow-up (median, 56 months) by using tissue microarray. The series were also stained with concurrent immunohistochemical prognostic panels (estrogen receptor, progesterone receptor, HER-2, androgen receptor, epidermal growth factor receptor (EGFR), P-cadherin, E-cadherin, and basal (CK5/6, CK14), and p53), to characterize this specific subgroup of breast cancer and to identify prognostic markers that can identify tumors with more aggressive behavior.
Of informative cases, 16.3% were of the triple-negative phenotype. The majority of these tumors were grade 3, ductal/no-specific-type carcinomas. There were positive associations with larger size, pushing margins, poorer Nottingham Prognostic Index, development of recurrence and distant metastasis, and poorer outcome. In addition, associations were found with loss of expression of androgen receptor and E-cadherin, and positive expression of basal cytokeratins (basal phenotype), P-cadherin, p53, and EGFR. In all tumors, tumor size, lymph node stage, and androgen receptor were the most useful prognostic markers. In the lymph node-positive subgroup, both size and androgen receptor retained their prognostic significance. However, in the lymph node-negative tumors, basal phenotype was the sole prognostic marker identified in this subgroup. Other parameters including age, histological grade, tumor size, vascular invasion or other biomarkers included in the current study were not significant.
The authors concluded that assessment of androgen receptor and basal phenotype, in addition to the established pathologic variables, mainly lymph node status and tumor size, can be used to select high-risk and low-risk patients at the time of primary surgery and can provide valuable information on treatment options in these triple-negative tumors.
The advent of technology for the analysis of human tissue samples for the activity of the entire genome initiated a quest for the molecular profiling of tumors. The aim of this quest was a better system for classifying cancers, a clarification of the origin of these diseases, a more accurate prognostic capacity than was previously available, and an improved ability to choose among possible therapies. Progress in the molecular profiling of solid tumors began with the identification of sets of genes whose expression can be used to classify breast cancers into five so-called intrinsic subtypes.1 Soon thereafter, two independent comparisons . . .
Cells with distinct phenotypes including stem-cell-like properties have been proposed to exist in normal human mammary epithelium and breast carcinomas, but their detailed molecular characteristics and clinical significance are unclear. We determined gene expression and genetic profiles of cells purified from cancerous and normal breast tissue using markers previously associated with stem-cell-like properties. CD24+ and CD44+ cells from individual tumors were clonally related but not always identical. CD44+ cell-specific genes included many known stem-cell markers and correlated with decreased patient survival. The TGF-beta pathway was specifically active in CD44+ cancer cells, where its inhibition induced a more epithelial phenotype. Our data suggest prognostic relevance of CD44+ cells and therapeutic targeting of distinct tumor cell populations.
Metastasis can be viewed as an evolutionary process, culminating in the prevalence of rare tumour cells that overcame stringent physiological barriers as they separated from their original environment and developmental fate. This phenomenon brings into focus long-standing questions about the stage at which cancer cells acquire metastatic abilities, the relationship of metastatic cells to their tumour of origin, the basis for metastatic tissue tropism, the nature of metastasis predisposition factors and, importantly, the identity of genes that mediate these processes. With knowledge cemented in decades of research into tumour-initiating events, current experimental and conceptual models are beginning to address the genetic basis for cancer colonization of distant organs.
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