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Immunohistochemistry of thymus and thymic tumours. Normal mouse thymus at 6 weeks (A) and 18 weeks (B) showing expression of T-cell marker (CD3) and different cytokeratin markers specific to thymic medullar epithelial cells (CK5) and cortical epithelial cells (CK8). Immunohistochemistry showing thymic epithelial carcinoma (C), T-cell lymphoma (D) and lung metastasis of T-cell lymphoma (F). doi:10.1371/journal.pone.0040302.g002
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The Deleted in liver cancer one (Dlc1) tumor suppressor gene encodes a RhoGTPase activating protein (RhoGAP). The Dlc1 gene has multiple transcriptional isoforms and we have previously established a mouse strain containing a gene trap (gt) insertion, which specifically reduces the expression of the 6.1 kb isoform (isoform 2). This gene trapped alle...
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... necroscopy, some of these mice also showed neoplastic lesions in other organs including kidney, liver, lung, testis and ovary but, the majority of mice showed thymic tumours ( Figure 1A, Table 1). The majority of the lung tumours were metastatic thymic lymphoma cells in origin ( Figure 2E, Table 2). To determine overall survival, Kaplan-Meier analysis of the AdCMV-cre injected mice was carried out. ...
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... understand the nature of the thymic cancers, we performed immunohistochemistry using a T-cell specific marker CD3 and thymic epithelial cell specific markers, cytokeratin 5 (CK5) and cytokeratin 8 (CK8/Troma) [18]. In the normal young mice (6weeks old) the T lymphocytes were found evenly distributed in the both cortex and medulla regions of the thymus, whereas the medullar epithelial cells were strongly CK5 and cortical epithelial cells were strongly CK8 positive ( Figure 2A). However, in the adult mice the T lymphocytes were predominantly shifted towards the central medulla region and the cortical epithelial cells were strongly CK8 positive with isolated patches of CK5 positive cells ( Figure 2B). ...
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... the normal young mice (6weeks old) the T lymphocytes were found evenly distributed in the both cortex and medulla regions of the thymus, whereas the medullar epithelial cells were strongly CK5 and cortical epithelial cells were strongly CK8 positive ( Figure 2A). However, in the adult mice the T lymphocytes were predominantly shifted towards the central medulla region and the cortical epithelial cells were strongly CK8 positive with isolated patches of CK5 positive cells ( Figure 2B). Using these markers we have found a significantly higher number of thymic lymphomas 80% (8/10) in the K + mice versus 68.7% (11/16) in the KD mice (Figure 1B & 2D, Table 3). ...
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... also revealed that most of the thymic lymphoma tumours had metastasized to multiple foci in the lung. The majority of the lung metastases were of T-cell lymphoma in origin ( Figure 2E, Table 2). Quantification of the lung metastatic lesions of each cohort shows significantly higher frequency of metastases in KD mice compared with the K + mice ( Figure 3B). ...
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... established 4 thymic lymphoma (TL) and 3 thymic epithelial carcinoma (TEC) cell lines ( Figure 4A) from the primary tumours to study the expression of Dlc1 protein. These tumours were either CD3+ T-cells or CK 5 or CK 8 positive epithelial cells (Figure 2C &D). The TL and TEC cell lines spontaneously immortalized and were grown up to 20 passages without any decrease in growth potential. ...
Citations
... We overexpressed GFP-M1 in oncogenic K-RasG12D transformed ovarian cancer-derived mouse tumor cells (OC-033) developed by Sabbir et al. (2012) and studied OXPHOS architecture and function. 2D BN-PAGE/SDS-PAGE analysis using total cellular lysates from GFP-Chrm1 expressed OC-033 cells revealed that Chrm1 formed 2 major protein complexes at ≥1200 and -1000 kDa, respectively ( Figure 4A, red and blue dotted rectangles, respectively). ...
Introduction: Cholinergic Receptor Muscarinic 1 (CHRM1) is a G protein-coupled acetylcholine (ACh) receptor predominantly expressed in the cerebral cortex. In a retrospective postmortem brain tissues-based study, we demonstrated that severely (≥50% decrease) reduced CHRM1 proteins in the temporal cortex of Alzheimer’s patients significantly correlated with poor patient outcomes. The G protein-mediated CHRM1 signal transduction cannot sufficiently explain the mechanistic link between cortical CHRM1 loss and the appearance of hallmark Alzheimer’s pathophysiologies, particularly mitochondrial structural and functional abnormalities. Therefore, the objective of this study was to analyze the molecular, ultrastructural, and functional properties of cortical mitochondria using CHRM1 knockout (Chrm1-/-) and wild-type mice to identify mitochondrial abnormalities.
Methods: Isolated and enriched cortical mitochondrial fractions derived from wild-type and Chrm1-/- mice were assessed for respiratory deficits (oxygen consumption) following the addition of different substrates. The supramolecular assembly of mitochondrial oxidative phosphorylation (OXPHOS)-associated protein complexes (complex I-V) and cortical mitochondrial ultrastructure were investigated by blue native polyacrylamide gel electrophoresis and transmission electron microscopy (TEM), respectively. A cocktail of antibodies, specific to Ndufb8, Sdhb, Uqcrc2, Mtco1, and Atp5a proteins representing different subunits of complexes I-V, respectively was used to characterize different OXPHOS-associated protein complexes.
Results: Loss of Chrm1 led to a significant reduction in cortical mitochondrial respiration (oxygen consumption) concomitantly associated with reduced oligomerization of ATP synthase (complex V) and supramolecular assembly of complexes I-IV (Respirasome). Overexpression of Chrm1 in transformed cells (lacking native Chrm1) significantly increased complex V oligomerization and respirasome assembly leading to enhanced respiration. TEM analysis revealed that Chrm1 loss led to mitochondrial ultrastructural defects and alteration in the tinctorial properties of cortical neurons causing a significant increase in the abundance of dark cortical neurons (Chrm1-/- 85% versus wild-type 2%).
Discussion: Our findings indicate a hitherto unknown effect of Chrm1 deletion in cortical neurons affecting mitochondrial function by altering multiple interdependent factors including ATP synthase oligomerization, respirasome assembly, and mitochondrial ultrastructure. The appearance of dark neurons in Chrm1-/- cortices implies potentially enhanced glutamatergic signaling in pyramidal neurons under Chrm1 loss condition. The findings provide novel mechanistic insights into Chrm1 loss with the appearance of mitochondrial pathophysiological deficits in Alzheimer’s disease.
... Similar to humans [7], at least four murine splice/protein variants have been identified [8]. Gene trapped Dlc1 gt/+ mice harbour an insertion between exon 1 and 2 of the 6.1 kb transcript and exhibit developmental defects and disease-prone phenotypes [8,24,25]. However, Dlc1 gt/+ mice do not per se progress to cancer, suggesting that cooperative damage cues or oncogenic mutations are required for transformation. ...
Helicobacter (H.) pylori -induced gastritis is a risk factor for gastric cancer (GC). Deleted-in-liver-cancer-1 (DLC1/ARHGAP7) inhibits RHOA, a downstream mediator of virulence factor cytotoxin-A (CagA) signalling and driver of consensus-molecular-subtype-2 diffuse GC. DLC1 located to enterochromaffin-like and MIST1+ stem/chief cells in the stomach. DLC1+ cells were reduced in H. pylori gastritis and GC, and in mice infected with H. pylori . DLC1 positivity inversely correlated with tumour progression in patients. GC cells retained an N-terminal truncation variant DLC1v4 in contrast to full-length DLC1v1 in non-neoplastic tissues. H. pylori and CagA downregulated DLC1v1 / 4 promoter activities. DLC1v1/4 inhibited cell migration and counteracted CagA-driven stress phenotypes enforcing focal adhesion. CagA and DLC1 interacted via their N- and C-terminal domains, proposing that DLC1 protects against H. pylori by neutralising CagA. H. pylori -induced DLC1 loss is an early molecular event, which makes it a potential marker or target for subtype-aware cancer prevention or therapy.
... Masunaga et al., observed no methylation in the Phosphatase and Tensin homolog (PTEN) promoter region in TEN tumor cells [56]. Moreover, Deleted in liver cancer one (Dlc1) isoform 2 was described to undergo selective hypermethylation in oncogenic Kirsten rat sarcoma virus oncogene homolog (K-Ras) 2 induced TEN and significantly decrease the overall survival in Dlc1 gene trap mice [57]. Saito et al., showed that TC with Tet Methylcytosine Dioxygenase 2 (TET2) mutations had more hypermethylated genes and that hypermethylation correlated with down-regulation of gene expression [58]. ...
Thymic Epithelial Neoplasms (TENs) represent the most common tumors of the thymus gland. Epigenetic alterations are generally involved in initiation and progression of various cancer entities. However, little is known about the role of epigenetic modifications in TENs. In order to identify relevant studies, a literature review was conducted using the MEDLINE and LIVIVO databases. The search terms thymoma, thymic carcinoma, thymic epithelial neoplasm, epigenetics, DNA methylation, HDAC and miRNA were employed and we were able to identify forty studies focused on TENs and published between 1997 and 2021. Aberrant epigenetic alterations seem to be involved in the tumorigenesis of thymomas and thymic carcinomas, with numerous studies reporting on non-coding RNA clusters and altered gene methylation as possible biomarkers in different types of TENs. Interestingly, Histone Deacetylase Inhibitors have shown potent antitumor effects in clinical trials, thus possibly representing effective epigenetic therapeutic agents in TENs. Additional studies in larger patient cohorts are, nevertheless, needed to verify the clinical utility and safety of novel epigenetic agents in the treatment of patients with TENs.
... These findings indicate that DLC1 is present in human and mouse ECL cells. an increased RHOA activity, cytoskeletal changes and increased cell migration (Sabbir et al., 2012;Sabbir et al., 2010). Hence, these mice represent an appropriate model for the investigation of the role of DLC1 as a tumor suppressor and inhibitor of RHOA in ...
DLC1 is a tumor suppressor protein downregulated in gastric cancer. It is a negative regulator of RHOA, which is the major oncogenic driver mutation of human diffuse gastric cancer. Helicobacter infection leads to chronic gastric inflammation, which is a risk factor for the development of carcinoma. The Helicobacter toxin CagA activates numerous signaling pathways including RHOA. The role of DLC1 in Helicobacter-related gastric disease is unknown and was analyzed by this thesis. DLC1gt/+ mice showed increased gastric inflammatory infiltration. Involvement of DLC1 in the regulation of the immune response was confirmed by RT-qPCR analyses. Furthermore, DLC1 was shown to be localized to enterochromaffin-like cells. Quantitative gene expression analyses verified a crucial role of the tumor suppressor in homeostasis of gastric acid in vivo, thereby preventing the development of gastric cancer. This study further demonstrates an interaction between DLC1 and CagA. DLC1 was transcriptionally downregulated by CagA and the two proteins fulfilled antagonizing functions by complex formation. DLC1 counters the oncogenic signaling of CagA in vitro by promoting adhesion, suppressing proliferation and antagonizing CagA concerning the hypoxic stress response. DLC1 furthermore inhibited CagA-mediated G-protein-coupled RHOA activation. In vivo therapy of a preclinical model for human gastric cancer with an inhibitor of the RHO/ROCK-pathway efficiently reduced tumor growth. These findings propose inhibition of this pathway as a novel treatment strategy for human gastric cancer. In summary, this thesis postulates a protective role of DLC1 in initial steps of gastric disease by antagonizing CagA-mediated oncogenic signaling. Transcriptional downregulation of DLC1 by CagA promotes oncogenic effects and constitutes DLC1 as an early molecular marker for Helicobacter-related gastric disease. Due to the involvement of CagA and DLC1 in the regulation of RHOA, Helicobacter-related gastric disease can be assigned to diffuse genomically stable gastric cancer. This represents a new risk stratification for Helicobacter-infected gastric cancer patients. Suppression of tumor growth using an inhibitor of the RHOA downstream effector ROCK proposes DLC1 as a future druggable target in human gastric cancer.
... In this study, we have used a Dlc1 deficient ovarian tumour cell line (OC-033) for transfection experiments, which was derived from a heterozygous gene-trapped Dlc1 and K-RasG12D mouse tumour (Dlc1 gt/wt /LSL-KRas G12D/wt ) (Fig. S1) (Sabbir et al., 2012). In addition, we have also used a Neu (ErbB2) oncogene-transformed mammary cancer (BC-7-2) cell line, which constitutively expressed high levels Dlc1 protein (Fig. S1) (Dillon et al., 2009). ...
... Total cellular proteins were extracted as described (Sabbir et al., 2010). The cell lysate was pre-cleared and immunoprecipitated using anti-Dlc1 and anti-Myh9 antibodies as described (Sabbir et al., 2012). The western blots were immunoblotted with anti-GFP (ab-290), anti-Myh9 (Sc-98978, Santa Cruz Biotechnology (SCBT) Dallas, TX; ARP48072_P050, Aviva Systems Biology, San Diego, CA), anti-phospho-Myh9-Ser-1943 (CST-5026, New England Biolabs, Ipswich, MA), anti-Plectin (sc-33649, SCBT) and anti-Spectrin, antibodies (Sc-32931,SCBT) and visualized. ...
The Deleted in liver cancer 1 (Dlc1) gene codes for a Rho GTPase-activating protein that also acts as a tumour suppressor gene. Several studies have consistently found that overexpression leads to excessive cell elongation, cytoskeleton changes and subsequent cell death. However, none of these studies have been able to satisfactorily explain the Dlc1-induced cell morphological phenotypes and the function of the different Dlc1 isoforms. Therefore, we have studied the interacting proteins associated with the three major Dlc1 transcriptional isoforms using a mass spectrometric approach in Dlc1 overexpressing cells. We have found and validated novel interacting partners in constitutive Dlc1-expressing cells. Our study has shown that Dlc1 interacts with non-muscle myosin heavy chain II-A (Myh9), plectin and spectrin proteins in different multiprotein complexes. Overexpression of Dlc1 led to increased phosphorylation of Myh9 protein and activation of Rac1 GTPase. These data support a role for Dlc1 in induced cell elongation morphology and provide some molecular targets for further analysis of this phenotype.
... Rho GTPase has critical regulatory roles in thymus development, such as thymocyte proliferation and survival, and thymic egress [37,38]. Additionally, RhoGAP deletion is associated with thymic cancer [49]. CASP4 codes for caspase-4, an apoptosis-related cysteine peptidase which is also an activator of caspase-1 [39]. ...
Trisomy 21-driven transcriptional alterations in human thymus were characterized through gene coexpression network (GCN) and miRNA-target analyses. We used whole thymic tissue - obtained at heart surgery from Down syndrome (DS) and karyotipically normal subjects (CT) - and a network-based approach for GCN analysis that allows the identification of modular transcriptional repertoires (communities) and the interactions between all the system's constituents through community detection. Changes in the degree of connections observed for hierarchically important hubs/genes in CT and DS networks corresponded to community changes. Distinct communities of highly interconnected genes were topologically identified in these networks. The role of miRNAs in modulating the expression of highly connected genes in CT and DS was revealed through miRNA-target analysis. Trisomy 21 gene dysregulation in thymus may be depicted as the breakdown and altered reorganization of transcriptional modules. Leading networks acting in normal or disease states were identified. CT networks would depict the "canonical" way of thymus functioning. Conversely, DS networks represent a "non-canonical" way, i.e., thymic tissue adaptation under trisomy 21 genomic dysregulation. This adaptation is probably driven by epigenetic mechanisms acting at chromatin level and through the miRNA control of transcriptional programs involving the networks' high-hierarchy genes.
... The MEF-Vt and MEF-Cre cells displayed a similar increase in RasGTP (Fig. 1F), indicating that the stronger transformation of the MEF-Cre cells was not attributable to differences in RasGTP levels. After submission of this article, a study reported that the incidence of metastatic thymic tumors was increased in mice heterozygous for a genetrapped Dlc1 allele after Cre-mediated induction of oncogenic K-ras, compared with mice with 2 wild-type Dlc1 alleles (22). These results are analogous to our finding that reduced Dlc1 activity enhances the transforming ability of v-Ras. ...
The tumor suppressor gene deleted in liver cancer-1 (DLC1), which encodes a protein with strong RhoGAP (GTPase activating protein) activity and weak Cdc42GAP activity, is inactivated in various human malignancies. Following Dlc1 inactivation, mouse embryo fibroblasts (MEFs) with a conditional Dlc1 knockout allele reproducibly underwent neoplastic transformation. In addition to inactivation of Dlc1 and increased activity of Rho and Cdc42, transformation depended on the subsequent decreased expression of the Cdk4/6 inhibitors p15Ink4b and p16Ink4a together with increased expression and activation of Cdk4/6. The level of expression of these cell cycle regulatory genes was relevant to human tumors with low DLC1 expression. Analysis of publicly available annotated datasets of lung and colon cancer with gene expression microarray profiles indicated that, in pairwise comparisons, low DLC1 expression occurred frequently together (p<0.01) with downregulation of p15Ink4b or p16Ink4a or upregulation of CDK4 or CDK6. In addition, an unfavorable prognosis (p<0.05) was associated with low DLC1 and low p15Ink4b in lung cancer and colon cancer, low DLC1 and low p16Ink4a in lung cancer, low DLC1 and high CDK4 in lung cancer, and low DLC1 and high CDK6 in colon cancer. Thus, several genes and biochemical activities collaborate with the inactivation of DLC1 to give rise to cell transformation in MEFs, and the identified genes are relevant to human tumors with low DLC1 expression.
Hepatocellular carcinoma (HCC), a common primary liver cancer, is the third leading cause of death worldwide. DNA methylation changes are common in HCC and have been studied to be associated with hepatocarcinogenesis. In our study, we used the MassARRAY® EpiTYPER technology to investigate the methylation differences of deleted in liver cancer 1 (DLC1) (isoform 1 and 3) promoter between HCC tissues and corresponding adjacent noncancerous tissues and the association between methylation levels and clinicopathological features. In addition, the modified CRISPR-Cas9 system and the DNA methyltransferase inhibitor (DNMTi) were utilized to explore the functional correlation of epigenetic modifications and DLC1 gene regulation. The methylation levels of the DLC1 isoforms in HCC samples were found significantly lower than those in the adjacent noncancerous tissues (all p < 0.0001). Also, we found that the expression of DLC1 could be bidirectionally regulated by the modified CRISPR-Cas9 system and the DNMTi. Moreover, the hypomethylation of DLC1 in HCC samples was connected with the presence of satellite lesions (p = 0.0305) and incomplete tumor capsule (p = 0.0204). Receiver operator characteristic curve analysis demonstrated that the methylation levels of DLC1 could be applied to discriminate HCC patients (area under the curve = 0.728, p < 0.0001). The hypomethylation status was a key regulatory mechanism of DLC1 expression and might serve as a potential biomarker for HCC.
In advanced cancer, the RHOA GTPase is often active together with reduced expression of genes encoding Rho-specific GTPase-accelerating proteins (Rho-GAP), which negatively regulate RHOA and related GTPases. Here we used the The Cancer Genome Atlas dataset to examine 12 tumor types (including colon, breast, prostate, pancreas, lung adenocarcinoma, and squamous cell carcinoma) for the frequency of codon mutations of 10 Rho-GAP and experimentally tested biochemical and biological consequences for cancer-associated mutants that arose in the DLC1 tumor suppressor gene. DLC1 was the Rho-GAP gene mutated most frequently, with 5%–8% of tumors in five of the tumor types evaluated having DLC1 missense mutations. Furthermore, 20%–26% of the tumors in four of these five tumor types harbored missense mutations in at least one of the 10 Rho-GAPs. Experimental analysis of the DLC1 mutants indicated 7 of 9 mutants whose lesions were located in the Rho-GAP domain were deficient for Rho-GAP activity and for suppressing cell migration and anchorage-independent growth. Analysis of a DLC1 linker region mutant and a START domain mutant showed each was deficient for suppressing migration and growth in agar, but their Rho-GAP activity was similar to that of wild-type DLC1. Compared with the wild-type, the linker region mutant bound 14-3-3 proteins less efficiently, while the START domain mutant displayed reduced binding to Caveolin-1. Thus, mutation of Rho-GAP genes occurs frequently in some cancer types and the majority of cancer-associated DLC1 mutants evaluated were deficient biologically, with various mechanisms contributing to their reduced activity.
Significance
These findings indicate that point mutation of Rho-GAP genes is unexpectedly frequent in several cancer types, with DLC1 mutants exhibiting reduced function by various mechanisms.
Deleted in liver cancer‐1 (DLC1), a potential tumor suppressor, acts as a GTPase‐activating protein for Rho family members. In many human cancers, the DLC1 expression is frequently downregulated or inactivated, which allows cancer cells to proliferate and disseminate. In this review, we describe the characteristics and other members of the DLC1 family and delineate the signal pathways DLC1 involved in regulating cancer cell growth, colony formation, apoptosis, senescence, autophagy, migration and invasion. In addition, we explore the clinical data of DLC1 and the mechanisms that natural products upregulate the DLC1 expression to inhibit cancer. Despite these insights, many important unanswered questions remain about the exact mechanisms of DLC1‐mediated cancer suppression. Deleted in liver cancer‐1 (DLC1) is identified as a tumor suppressor. We introduce DLC1, other members of its family and the functions and signal pathways involving DLC1. We also describe the clinical data of DLC1 and the mechanism that natural products upregulate the DLC1 expression to inhibit cancer.
