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CDK7 Inhibition Affects the Proliferation of Ovarian Cancer Cells (A) Lysates from different ovarian cancer cell lines, either untreated or treated with DMSO, the PI3K inhibitor LY294002, or the CDK7 inhibitor THZ1 for 4 hr were subjected to immunoblotting with the indicated antibodies. Vinculin was used as loading control. The experiment was repeated three times with analogous results. (legend continued on next page)
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Our understanding of the molecular determinants of cancer is still inadequate because of cancer heterogeneity. Here, using epithelial ovarian cancer (EOC) as a model system, we analyzed a minute amount of patient-derived epithelial cells from either healthy or cancerous tissues by single-shot mass-spectrometry-based phosphoproteomics. Using a multi...
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... cells were compared to cells treated with THZ1, a specific inhibitor of CDK7 that covalently binds to a conserved cysteine in the kinase domain of CDK7 ( Kwiatkowski et al., 2014). The mitogen-activated protein kinase kinase (MEK) inhibitor U0126 ( Duncia et al., 1998) was used as control ( Figure S6A). The specific effects of THZ1 and U0126 treatment on the phosphorylation of POLR2A and extracellular signal-regulated kinases (ERK), respectively, were confirmed by WB analysis ( Figure 5B). ...
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... specific effects of THZ1 and U0126 treatment on the phosphorylation of POLR2A and extracellular signal-regulated kinases (ERK), respectively, were confirmed by WB analysis ( Figure 5B). We quantified 13,194 phosphorylation sites using label-free quantitation (Table S6) and observed high reproducibility between biological replicates with Pearson correlation coefficients above 0.75 ( Figure S6B). The quality of this phosphoproteomics dataset was comparable to that observed in primary cells, as assessed by high reproducibility of the independent biological replicates (Figures S6B-S6F). ...
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... CDK7 has recently emerged as a prominent target for treating cancer (Cao and Shilatifard, 2014;Christensen et al., 2014), we tested EOC sensitivity to THZ1 using five EOC cell lines (OVCAR3, SKOV3, HEYA8, COV362, and COV318). THZ1 treatment reduced the activation of POLR2A after 4-hr treatment as well as cell proliferation after 72-hr treatment in all cell lines (Fig- ures 6A and 6B). The inhibition of phosphatidylinositol 3-kinase (PI3K), one of the few genes known to be mutated in ovarian cancer (Cancer Genome Atlas Research Network, 2011), with LY294002 was used as a control. ...
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... inhibition of phosphatidylinositol 3-kinase (PI3K), one of the few genes known to be mutated in ovarian cancer (Cancer Genome Atlas Research Network, 2011), with LY294002 was used as a control. Finally, THZ1 treatment did not affect the proliferation of HeLa, A549, or MCF7 cancer cells ( Figure 6C), which are of cervix, lung, and breast cancer origin, respectively, implying that CDK7 inhibition is specific for EOC. To verify the causal link between CDK7 activation and POLR2A phosphorylation, we transfected OVCAR3, COV318, or COV362 cells with two different small interfering RNA (siRNA) sequences against CDK7. ...
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... verify the causal link between CDK7 activation and POLR2A phosphorylation, we transfected OVCAR3, COV318, or COV362 cells with two different small interfering RNA (siRNA) sequences against CDK7. Ablation of CDK7 reduced dramatically the phosphorylation of POLR2A in all the cell lines ( Figure 6D). Moreover, CDK7 knockdown also resulted in the inhibition of cell proliferation for all three cell lines ( Figure 6E), thus supporting the role of CDK7 in EOC cell proliferation. ...
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... of CDK7 reduced dramatically the phosphorylation of POLR2A in all the cell lines ( Figure 6D). Moreover, CDK7 knockdown also resulted in the inhibition of cell proliferation for all three cell lines ( Figure 6E), thus supporting the role of CDK7 in EOC cell proliferation. ...
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... results might open a novel therapeutic window for the treatment of EOC, in line with recent studies reporting that blocking CDK7 with THZ1, a covalent inhibitor of CDK7 ( Kwiatkowski et al., 2014), specifically killed triple-negative breast cancer cells ( Wang et al., 2015). Perhaps CDK7-dependent phosphorylation of POLR2A ( Figure 6) is responsible for cancer cell proliferation, in line with recent data linking POLR2A activation with colon cancer ( Liu et al., 2015). For example, the CDK7 inhibitor THZ1, which does not interfere with CDK7 phosphorylation ( Kwiatkowski et al., 2014), can be combined with the blockade of transcriptional regulators (Asghar et al., 2015;Gonda and Ramsay, 2015). ...
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Citations
... The purity of primary cell culture was consistently over 95%. Tissue isolation and culture conditions of primary cells were performed as described previously [40]. ...
... Lentiviral vectors were generated by transient co-transfection of the packaging cell line HEK293T, purchased from ATCC and cultured as described previously [40], with 10 μg of the following anti-FOXM1 shRNA from Genecopoeia: sh-C (5'-TAATACGACTCACTATAGGG-3'; HSH096566-LVRU6GP-c); sh-E (5'-TAATACGACTCACTATAGGG-3'; HSH096566-LVRU6GPe) or with the scrambled control (CSHCTR001-LVRU6GP, Genecopoeia), and the following packaging vectors: PMD2G (3 μg), Rre (5 μg), and REV (2.5 μg), using the calcium phosphate precipitation method. The supernatant from HEK293T, containing the virus particles, was supplemented with 8 μg/ml of polybrene and used to transduce recipient cells. ...
In ovarian tumors, the omental microenvironment profoundly influences the behavior of cancer cells and sustains the acquisition of stem-like traits, with major impacts on tumor aggressiveness and relapse. Here, we leverage a patient-derived platform of organotypic cultures to study the crosstalk between the tumor microenvironment and ovarian cancer stem cells. We discovered that the pro-tumorigenic transcription factor FOXM1 is specifically induced by the microenvironment in ovarian cancer stem cells, through activation of FAK/YAP signaling. The microenvironment-induced FOXM1 sustains stemness, and its inactivation reduces cancer stem cells survival in the omental niche and enhances their response to the PARP inhibitor Olaparib. By unveiling the novel role of FOXM1 in ovarian cancer stemness, our findings highlight patient-derived organotypic co-cultures as a powerful tool to capture clinically relevant mechanisms of the microenvironment/cancer stem cells crosstalk, contributing to the identification of tumor vulnerabilities.
... On top of this, the analysis of phosphorylated proteins (phosphoproteomics) is capable of defining protein activation dynamics that modify the functional properties of cells (e.g. phosphorylation patterns during mitotic states) 23,24 . With these points in mind, we employed a combined proteomic and phosphoproteomic approach to define protein and phosphorylation patterns in ESR1 mutant models under estrogen deprivation (mimicking AI treatment) and estrogen stimulation conditions. ...
Three quarters of all breast cancers express the estrogen receptor (ER, ESR1 gene), which promotes tumor growth and constitutes a direct target for endocrine therapies. ESR1 mutations have been implicated in therapy resistance in metastatic breast cancer, in particular to aromatase inhibitors. ESR1 mutations promote constitutive ER activity and affect other signaling pathways, allowing cancer cells to proliferate by employing mechanisms within and without direct regulation by the ER. Although subjected to extensive genetic and transcriptomic analyses, understanding of protein alterations remains poorly investigated. Towards this, we employed an integrated mass spectrometry based proteomic approach to profile the protein and phosphoprotein differences in breast cancer cell lines expressing the frequent Y537N and Y537S ER mutations. Global proteome analysis revealed enrichment of mitotic and immune signaling pathways in ER mutant cells, while phosphoprotein analysis evidenced enriched activity of proliferation associated kinases, in particular CDKs and mTOR. Integration of protein expression and phosphorylation data revealed pathway-dependent discrepancies (motility vs proliferation) that were observed at varying degrees across mutant and wt ER cells. Additionally, protein expression and phosphorylation patterns, while under different regulation, still recapitulated the estrogen-independent phenotype of ER mutant cells. Our study is the first proteome-centric characterization of ESR1 mutant models, out of which we confirm estrogen independence of ER mutants and reveal the enrichment of immune signaling pathways at the proteomic level.
... The purity of primary cell culture was consistently over 95%. Tissue isolation and culture conditions of primary cells were performed as described previously [16]. ...
... Alpha-tubulin and Lamin were used as loading controls for cytoplasmic and nuclear fractions, respectively. patient-derived primary cultures, an approach that has recently revealed novel and unexpected players not only in OC per se but also in the OCSC subset [16,17,49,50]. Along this line, primary cultures unveiled MGP as a hallmark of OCSC and provided the rationale for subsequent functional studies. ...
Ovarian cancer (OC) displays the highest mortality among gynecological tumors, mainly due to early peritoneal dissemination, the high frequency of tumor relapse following primary debulking, and the development of chemoresistance. All these events are thought to be initiated and sustained by a subpopulation of neoplastic cells, termed ovarian cancer stem cells (OCSC), that are endowed with self-renewing and tumor-initiating properties. This implies that interfering with OCSC function should offer novel therapeutic perspectives to defeat OC progression. To this aim, a better understanding of the molecular and functional makeup of OCSC in clinically relevant model systems is essential. We have profiled the transcriptome of OCSC vs. their bulk cell counterpart from a panel of patient-derived OC cell cultures. This revealed that Matrix Gla Protein (MGP), classically known as a calcification-preventing factor in cartilage and blood vessels, is markedly enriched in OCSC. Functional assays showed that MGP confers several stemness-associated traits to OC cells, including a transcriptional reprogramming. Patient-derived organotypic cultures pointed to the peritoneal microenvironment as a major inducer of MGP expression in OC cells. Furthermore, MGP was found to be necessary and sufficient for tumor initiation in OC mouse models, by shortening tumor latency and increasing dramatically the frequency of tumor-initiating cells. Mechanistically, MGP-driven OC stemness was mediated by the stimulation of Hedgehog signaling, in particular through the induction of the Hedgehog effector GLI1, thus highlighting a novel MGP/Hedgehog pathway axis in OCSC. Finally, MGP expression was found to correlate with poor prognosis in OC patients, and was increased in tumor tissue after chemotherapy, supporting the clinical relevance of our findings. Thus, MGP is a novel driver in OCSC pathophysiology, with a major role in stemness and in tumor initiation.
... Recently, advanced proteomic technologies applied in primary ovarian cancers have reported (a) biomarkers for differential diagnosis of histotypes [15,16]; (b) patient stratification for precision therapy [17][18][19][20][21]; ...
High-grade serous ovarian carcinoma (HGSOC) is the most common subtype of ovarian cancer with 5-year survival rates below 40%. Neoadjuvant chemotherapy (NACT) followed by interval debulking surgery (IDS) is recommended for patients with advanced-stage HGSOC unsuitable for primary debulking surgery (PDS). However, about 40% of patients receiving this treatment exhibited chemoresistance of uncertain molecular mechanisms and predictability. Here, we built a high-quality ovarian-tissue-specific spectral library containing 130,735 peptides and 10,696 proteins on Orbitrap instruments. Compared to a published DIA pan-human spectral library (DPHL), this spectral library provides 10% more ovary-specific and 3% more ovary-enriched proteins. This library was then applied to analyze data-independent acquisition (DIA) data of tissue samples from an HGSOC cohort treated with NACT, leading to 10,070 quantified proteins, which is 9.73% more than that with DPHL. We further established a six-protein classifier by parallel reaction monitoring (PRM) to effectively predict the resistance to additional chemotherapy after IDS (Log-rank test, p = 0.002).The classifier was validated with 57 patients from an independent clinical center (p = 0.014). Thus, we have developed an ovary-specific spectral library for targeted proteome analysis, and propose a six-protein classifier that could potentially predicts chemoresistance in HGSOC patients after NACT-IDS treatment.
... These cultures, however, share some limitations with immortalized cell lines, such as the loss of the native tissue architecture and the lack of a tumor microenvironment [13]. On the other hand, the presence of a single cell type, namely, tumor cells, renders primary cultures amenable for the application of several omics-based analyses of the epithelial compartment of the tumor, which could help in identifying relevant signatures of this disease [18,19]. For example, Francavilla and colleagues analyzed the phosphoproteomics profile of ex vivo primary OC cells compared to normal tissue and this approach unveiled the presence of a cancer-specific signature (i.e., the phosphorylation of the cyclin-dependent kinase CDK-7 and the activation of its target, RNA polymerase II) that was validated in tumor specimens by immunohistochemistry, demonstrating the ability of primary cells to recapitulate in vitro the activation of signaling pathways relevant for OC proliferation [18]. ...
... On the other hand, the presence of a single cell type, namely, tumor cells, renders primary cultures amenable for the application of several omics-based analyses of the epithelial compartment of the tumor, which could help in identifying relevant signatures of this disease [18,19]. For example, Francavilla and colleagues analyzed the phosphoproteomics profile of ex vivo primary OC cells compared to normal tissue and this approach unveiled the presence of a cancer-specific signature (i.e., the phosphorylation of the cyclin-dependent kinase CDK-7 and the activation of its target, RNA polymerase II) that was validated in tumor specimens by immunohistochemistry, demonstrating the ability of primary cells to recapitulate in vitro the activation of signaling pathways relevant for OC proliferation [18]. ...
Simple Summary
Ovarian cancer (OC) is a highly lethal neoplasm with a poor rate of response to current treatment. A prerequisite to designing and validating innovative and more efficacious therapies is a deeper understanding of the biological mechanisms that underlie OC progression and chemoresistance. Such an objective, in turn, requires experimental models that mimic the disease as faithfully as possible. In this context, great help comes from the use of patient-derived material, which is key to establishing clinically relevant models. This review summarizes the different categories of in vitro patient-derived OC models and outlines their ability to represent specific aspects of OC biology and provide new tools for personalizing the treatment of such a devastating disease.
Abstract
Epithelial ovarian cancer (OC) is the most lethal gynecological malignancy worldwide due to a late diagnosis caused by the lack of specific symptoms and rapid dissemination into the peritoneal cavity. The standard of care for OC treatment is surgical cytoreduction followed by platinum-based chemotherapy. While a response to this frontline treatment is common, most patients undergo relapse within 2 years and frequently develop a chemoresistant disease that has become unresponsive to standard treatments. Moreover, also due to the lack of actionable mutations, very few alternative therapeutic strategies have been designed as yet for the treatment of recurrent OC. This dismal clinical perspective raises the need for pre-clinical models that faithfully recapitulate the original disease and therefore offer suitable tools to design novel therapeutic approaches. In this regard, patient-derived models are endowed with high translational relevance, as they can better capture specific aspects of OC such as (i) the high inter- and intra-tumor heterogeneity, (ii) the role of cancer stem cells (a small subset of tumor cells endowed with tumor-initiating ability, which can sustain tumor spreading, recurrence and chemoresistance), and (iii) the involvement of the tumor microenvironment, which interacts with tumor cells and modulates their behavior. This review describes the different in vitro patient-derived models that have been developed in recent years in the field of OC research, focusing on their ability to recapitulate specific features of this disease. We also discuss the possibilities of leveraging such models as personalized platforms to design new therapeutic approaches and guide clinical decisions.
... The purity of primary cell culture was consistently over 95%. Tissue isolation and culture conditions of primary cells were performed as described previously [16]. ...
... Alpha-tubulin and Lamin were used as loading controls for cytoplasmic and nuclear fractions, respectively. patient-derived primary cultures, an approach that has recently revealed novel and unexpected players not only in OC per se but also in the OCSC subset [16,17,49,50]. Along this line, primary cultures unveiled MGP as a hallmark of OCSC and provided the rationale for subsequent functional studies. ...
Ovarian cancer (OC) displays the highest mortality among gynecological tumors, mainly due to early peritoneal dissemination, the high frequency of tumor relapse following primary debulking and the development of chemoresistance. All these events are thought to be initiated and sustained by a subpopulation of neoplastic cells, termed ovarian cancer stem cells (OCSC), that are endowed with self-renewing and tumor-initiating properties. This implies that interfering with OCSC function should offer novel therapeutic perspectives to defeat OC progression. To this aim, a better understanding of the molecular and functional makeup of OCSC in clinically relevant model systems is essential. We have profiled the transcriptome of OCSC vs. their bulk cell counterpart from a panel of patient-derived OC cell cultures. This revealed that Matrix Gla Protein (MGP), classically known as a calcification-preventing factor in cartilage and blood vessels, is markedly enriched in OCSC. Functional assays showed that MGP confers several stemness-associated traits to OC cells, including a transcriptional reprogramming. Patient-derived organotypic cultures pointed to the peritoneal microenvironment as a major inducer of MGP expression in OC cells. Furthermore, MGP was found to be necessary and sufficient for tumor initiation in OC mouse models, by shortening tumor latency and increasing dramatically the frequency of tumor-initiating cells. Mechanistically, MGP-driven OC stemness was mediated by the stimulation of Hedgehog signaling, in particular through the induction of the Hedgehog effector GLI1, thus highlighting a novel MGP/Hedgehog pathway axis in OCSC. Finally, MGP expression was found to correlate with poor prognosis in OC patients, and was increased in tumor tissue after chemotherapy, supporting the clinical relevance of our findings.
Thus, MGP is a novel driver in OCSC pathophysiology, with a major role in stemness and in tumor initiation.
... We did not observe a significant effect on bulk abundance of Ser5P in cardiomyocytes, which may reflect redundant regulation of this phosphoform by other Pol II CTD kinases [14][15][16][17] . The IC 50 of THZ1 in these cardiomyocyte assays was 5-10 nM, which reflects a higher sensitivity to THZ1 than what has been observed in studies of growth inhibition of several cancer cell types [5][6][7][8][9][18][19][20][21][22][23][24][25][26] (Supplementary Fig. 1A). Under baseline conditions, THZ1 showed no significant effect on NRVM size, Nppa/Nppb expression, or cell death (Fig. 1B-D and Supplementary Fig. 1B). ...
Heart failure with reduced ejection fraction (HFrEF) is associated with high mortality, highlighting an urgent need for new therapeutic strategies. As stress-activated cardiac signaling cascades converge on the nucleus to drive maladaptive gene programs, interdicting pathological transcription is a conceptually attractive approach for HFrEF therapy. Here, we demonstrate that CDK7/12/13 are critical regulators of transcription activation in the heart that can be pharmacologically inhibited to improve HFrEF. CDK7/12/13 inhibition using the first-in-class inhibitor THZ1 or RNAi blocks stress-induced transcription and pathologic hypertrophy in cultured rodent cardiomyocytes. THZ1 potently attenuates adverse cardiac remodeling and HFrEF pathogenesis in mice and blocks cardinal features of disease in human iPSC-derived cardiomyocytes. THZ1 suppresses Pol II enrichment at stress-transactivated cardiac genes and inhibits a specific pathologic gene program in the failing mouse heart. These data identify CDK7/12/13 as druggable regulators of cardiac gene transactivation during disease-related stress, suggesting that HFrEF features a critical dependency on transcription that can be therapeutically exploited.
... Phosphorylation provides a promising strategy for the understanding of molecular determinants of OC. Francavilla et al. used this technique to show that cell proliferation is controlled by cyclin-dependent kinase 7 (CDK7), through an examination of epithelial cells from OC and healthy patients [55]. ...
The ability to identify ovarian cancer (OC) at its earliest stages remains a challenge. The patients present an advanced stage at diagnosis. This heterogeneous disease has distinguishable etiology and molecular biology. Next-generation sequencing changed clinical diagnostic testing, allowing assessment of multiple genes, simultaneously, in a faster and cheaper manner than sequential single gene analysis. Technologies of proteomics, such as mass spectrometry (MS) and protein array analysis, have advanced the dissection of the underlying molecular signaling events and the proteomic characterization of OC. Proteomics analysis of OC, as well as their adaptive responses to therapy, can uncover new therapeutic choices, which can reduce the emergence of drug resistance and potentially improve patient outcomes. There is an urgent need to better understand how the genomic and epigenomic heterogeneity intrinsic to OC is reflected at the protein level, and how this information could potentially lead to prolonged survival.
... Mass spectrometry-based proteomics enables large-scale analyses of protein expression and their posttranslational modifications (PTM) 8 . Although many studies have analyzed protein changes in ovarian cancer cell lines for the investigation of signaling events, cellular perturbations and response to therapies, few have analyzed them directly in ovarian cancer tissues [9][10][11][12] . These studies rely on the characterization of the proteome and phosphorylation changes, with none having integrated changes occurring at the level of ubiquitination. ...
Despite recent advances in the management of BRCA1 mutated high-grade serous ovarian cancer (HGSC), the physiology of these tumors remains poorly understood. Here we provide a comprehensive molecular understanding of the signaling processes that drive HGSC pathogenesis with the addition of valuable ubiquitination profiling, and their dependency on BRCA1 mutation-state directly in patient-derived tissues. Using a multilayered proteomic approach, we show the tight coordination between the ubiquitination and phosphorylation regulatory layers and their role in key cellular processes related to BRCA1-dependent HGSC pathogenesis. In addition, we identify key bridging proteins, kinase activity, and post-translational modifications responsible for molding distinct cancer phenotypes, thus providing new opportunities for therapeutic intervention, and ultimately advance towards a more personalized patient care.
... The isolation and culture of primary cells were performed as previously described. 14 Briefly, immediately after paracentesis, the ascitic fluid was centrifuged to obtain a pellet containing cell aggregates and single cells. Red blood cells were eliminated by two 5minute treatments with ACK (Ack Cell Lysing Buffer, Lonza) at room temperature. ...
High‐grade serous ovarian carcinoma (HGSOC) is a highly aggressive and intractable neoplasm, mainly because of its rapid dissemination into the abdominal cavity, a process that is favored by tumor‐associated peritoneal ascites. The precise molecular alterations involved in HGSOC onset and progression remain largely unknown due to the high biological and genetic heterogeneity of this tumor. We established a set of different tumor samples (termed the As11‐set) derived from a single HGSOC patient, consisting of peritoneal ascites, primary tumor cells, ovarian cancer stem cells (OCSC) and serially propagated tumor xenografts. The As11‐set was subjected to an integrated RNA‐seq and DNA‐seq analysis which unveiled molecular alterations that marked the different types of samples. Our profiling strategy yielded a panel of signatures relevant in HGSOC and in OCSC biology. When such signatures were used to interrogate the TCGA dataset from HGSOC patients, they exhibited prognostic and predictive power. The molecular alterations also identified potential vulnerabilities associated with OCSC, which were then tested functionally in stemness‐related assays. As a proof of concept, we defined PI3K signaling as a novel druggable target in OCSC.
