Standardized and reproducible preclinical models that recapitulate the dynamics of prostate cance... more Standardized and reproducible preclinical models that recapitulate the dynamics of prostate cancer are urgently needed. We established a bank of transplantable patient-derived prostate cancer xenografts that capture the biologic and molecular heterogeneity currently confounding prognostication and therapy development. Xenografts preserved the histopathology, genome architecture, and global gene expression of donor tumors. Moreover, their aggressiveness matched patient observations, and their response to androgen withdrawal correlated with tumor subtype. The panel includes the first xenografts generated from needle biopsy tissue obtained at diagnosis. This advance was exploited to generate independent xenografts from different sites of a primary site, enabling functional dissection of tumor heterogeneity. Prolonged exposure of adenocarcinoma xenografts to androgen withdrawal led to castration-resistant prostate cancer, including the first-in-field model of complete transdifferentiation into lethal neuroendocrine prostate cancer. Further analysis of this model supports the hypothesis that neuroendocrine prostate cancer can evolve directly from adenocarcinoma via an adaptive response and yielded a set of genes potentially involved in neuroendocrine transdifferentiation. We predict that these next-generation models will be transformative for advancing mechanistic understanding of disease progression, response to therapy, and personalized oncology. Cancer Res; 74(4); 1272-83. Ó2013 AACR.
Low-grade serous ovarian carcinoma (LGSOC) is a rare tumor subtype with high case fatality rates ... more Low-grade serous ovarian carcinoma (LGSOC) is a rare tumor subtype with high case fatality rates in patients with metastatic disease. There is a pressing need to develop effective treatments using newly available preclinical models for therapeutic discovery and drug evaluation. Here, we use multiomics integration of wholeexome sequencing, RNA sequencing, and mass spectrometry-based proteomics on 14 LGSOC cell lines to elucidate novel biomarkers and therapeutic vulnerabilities. Comparison of LGSOC cell line data with LGSOC tumor data enabled predictive biomarker identification of MEK inhibitor (MEKi) efficacy, with KRAS mutations found exclusively in MEKi-sensitive cell lines and NRAS mutations found mostly in MEKi-resistant cell lines. Distinct patterns of Catalogue of Somatic Mutations in Cancer mutational signatures were identified in MEKi-sensitive and MEKi-resistant cell lines. Deletions of CDKN2A/B and MTAP genes were more frequent in cell lines than tumor samples and possibly represent key driver events in the absence of KRAS/NRAS/BRAF mutations. These LGSOC cell lines were representative models of the molecular aberrations found in LGSOC tumors. For prediction of in vitro MEKi efficacy, proteomic data provided better discrimination than gene expression data. Condensin, minichromosome maintenance, and replication factor C protein complexes were identified as potential treatment targets in MEKi-resistant cell lines. This study suggests that CDKN2A/B or MTAP deficiency may be exploited using synthetically lethal treatment strategies, highlighting the importance of using proteomic data as a tool for molecular drug prediction. Multiomics approaches are crucial to improving our understanding of the molecular underpinnings of LGSOC and applying this information to develop new therapies. Significance: These findings highlight the utility of global multiomics to characterize LGSOC cell lines as research models, to determine biomarkers of MEKi resistance, and to identify potential novel therapeutic targets.
Figure S1: Transplantable tumor lines retain the histolopathological characteristics of their par... more Figure S1: Transplantable tumor lines retain the histolopathological characteristics of their parent tumor. Xenografts LTL313H, LTL412 and LTL418 show similar tissue structure to the patient tumour from which they were originally derived (H&E stain; FFPE tissue). Immunohistochemical stains show that the protein expression of key markers (AR, PSA, PTEN, ERG) is also conserved. Note that LTL313H is derived from a biopsy sample the size of which is visible in the micrographs.
Background: Low-grade serous ovarian carcinoma (LGSOC) is a rare tumor subtype with high case fat... more Background: Low-grade serous ovarian carcinoma (LGSOC) is a rare tumor subtype with high case fatality rates. As such, there is a pressing need to develop more effective treatments using newly available preclinical models for therapeutic discovery and drug evaluation. Here, we use a multiomics approach to interrogate a collection of LGSOC patient-derived cell lines to elucidate novel biomarkers and therapeutic vulnerabilities. Methods: Fourteen LGSOC cell lines were interrogated using whole exome sequencing, RNA sequencing, and mass spectrometry-based proteomics. Somatic mutation, copy-number aberrations, gene and protein expression were analyzed and integrated using different computational approaches. LGSOC cell line data was compared to publicly available LGSOC tumor data (AACR GENIE cohort), and also used for predictive biomarker identification of MEK inhibitor (MEKi) efficacy. Protein interaction databases were evaluated to identify novel therapeutic targets. Results: KRAS mutations were exclusively found in MEKi-sensitive and NRAS mutations mostly in MEKiresistant cell lines. Analysis of COSMIC mutational signatures revealed distinct patterns of nucleotide substitution mutations in MEKi-sensitive and MEKi-resistant cell lines. Deletions of CDKN2A/B and MTAP genes (chromosome 9p21) were much more frequent in cell lines than tumor samples and possibly represent key driver events in the absence of KRAS/NRAS/BRAF mutations. For in-vitro MEKi efficacy prediction, proteomic data provided better discrimination than gene expression data. Condensin, MCM, and RFC protein complexes were identified as potential treatment targets in MEKi-resistant cell lines. Conclusions: Our LGSOC cell lines are representative models of the most common molecular aberrations found in LGSOC tumors. This study highlights the importance of using proteomic data in multiomics assessment of drug prediction and identification of potential therapeutic targets. CDKN2A/B and MTAP deficiency offer an opportunity to find synthetically lethal candidates for novel treatments. Multiomics approaches are crucial to improving our understanding of the molecular aberrations in LGSOC, establishing effective drug prediction programs and identifying novel therapeutic targets in LGSOC.
Background: Malignant Peritoneal Mesothelioma (PeM) is a rare and fatal cancer that originates fr... more Background: Malignant Peritoneal Mesothelioma (PeM) is a rare and fatal cancer that originates from the peritoneal lining of the abdomen. Standard treatment of PeM is limited to cytoreductive surgery and/or chemotherapy, and no effective targeted therapies for PeM exist. Some immune checkpoint inhibitor studies of mesothelioma have found positivity to be associated with a worse prognosis. Methods: To search for novel therapeutic targets for PeM, we performed a comprehensive integrative multiomics analysis of the genome, transcriptome, and proteome of 19 treatment-naïve PeM, and in particular we examined BAP1 mutation and copy-number status and its relationship to immune checkpoint inhibitor activation. Results: We found that PeM could be divided into tumors with an inflammatory tumor microenvironment and those without, and that this distinction correlated with haploinsufficiency of BAP1. To further investigate the role of BAP1, we used our recently developed cancer driver gene prioritization algorithm, HIT'nDRIVE, and observed that PeM with BAP1 haploinsufficiency form a distinct molecular subtype characterized by distinct gene expression patterns of chromatin remodeling, DNA repair pathways, and immune checkpoint receptor activation. We demonstrate that this subtype is correlated with an inflammatory tumor microenvironment and thus is a candidate for immune checkpoint blockade therapies. Conclusions: Our findings reveal BAP1 to be a potential, easily trackable prognostic and predictive biomarker for PeM immunotherapy that refines PeM disease classification. BAP1 stratification may improve drug response rates in ongoing phase-I and II clinical trials exploring the use of immune checkpoint blockade therapies in PeM in which BAP1 status is not considered. This integrated molecular characterization provides a comprehensive foundation for improved management of a subset of PeM patients.
Neuroendocrine prostate cancer (NEPC) is a highly aggressive subtype of prostate cancer (PCa) tha... more Neuroendocrine prostate cancer (NEPC) is a highly aggressive subtype of prostate cancer (PCa) that is becoming increasingly common in the clinic. While the vast majority of PCa presents as androgen-dependent adenocarcinoma, recent uses of increasingly potent therapeutics targeting the androgen receptor signaling axis has resulted in the promotion of NEPC transdifferentiation as a mechanism of treatment resistance. Unfortunately, there is currently no effective treatment option for NEPC. Altered cancer metabolism is now recognized as a hallmark of cancer and a crucial factor for promoting tumour growth and spread. In particular, altered glucose metabolism and the resultant acidification of the tumor microenvironment via increased lactic acid production has been shown to play an important role in multiple cancer-promoting processes, including tissue invasion/metastasis, angiogenesis, and suppression of local anticancer immunity. While increased glycolysis is not generally considered a phenomenon relevant to primary treatment-naive PCa, we have recently demonstrated its relavance to castration-resistant prostate cancer (CRPC) and thus suspect that it is also relevant to the more aggressive NEPC. Our laboratory has also developed a number of unique serially transplantable patient-derived xenograft (PDX) models of NEPC that are histologically highly similar to the donor tissues and retain important genetic and epigenetic features. In particular, we have developed the first spontaneous NEPC transdifferentiation model in the field (LTL331/331R). The gene expression profiles of these NEPC PDX models were compared to that of PCa adenocarcinoma PDX models. To determine whether certain metabolic pathway alterations were specific to NEPC, genes from a number of key metabolic pathways were compiled and overall pathway scores were generated using average expression z-scores. Furthermore, publically available gene expression data from NEPC patient tumors were used to validate our findings. From our analysis, we found that genes in the glycolysis pathway were signficiantlly upregulatied in both our PDX models and also in patient NEPC samples. Of particular interest is the upregulation of genes involved in the production and secretion of lactic acid, such as LDHA and MCT4. As such, our results suggest that elevated glycolysis and production of lactic acid could be a clinically important NEPC phenotype. Furthermore, the inhibition of glycolysis and particularly the inhibition of lactic acid secretion via MCT4 could be a potentially viable therapeutic strategy for NEPC. Citation Format: Stephen Y. Choi, Susan L. Ettinger, Dong Lin, Hui Xue, Robert H. Bell, Fan Mo, Michael Pollak, Colin C. Collins, Yuzhuo Wang. Elevated glycolytic gene signature in patient-derived neuroendocrine prostate cancer xenograft models and its clinical relevance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4420. doi:10.1158/1538-7445.AM2017-4420
Loss of tumour suppressor proteins, such as the retinoblastoma protein (Rb), results in tumour pr... more Loss of tumour suppressor proteins, such as the retinoblastoma protein (Rb), results in tumour progression and metastasis. Metastasis is facilitated by low oxygen availability within the tumour that is detected by hypoxia inducible factors (HIFs). The HIF1 complex, HIF1α and its dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT), is the master regulator of the hypoxic response. Previously, we demonstrated that Rb represses the transcriptional response to hypoxia by virtue of its association with HIF1. In this report, we further characterized the role of Rb in HIF1-regulated genetic programs by stably ablating Rb expression with retrovirally-introduced short hairpin RNA in LNCaP and 22rV1 human prostate cancer cells. DNA microarray analysis revealed that Rb regulates specific chromosomal gene clusters and loss of Rb in conjunction with hypoxia leads to dysregulation of HIF1-regulated genetic programs that promote cell invasion and neuroendocrine differentiation. Gene ontology analysis of the hypoxia-inducible genes sensitive to loss of Rb revealed that a significant portion of these genes are involved in neuroendocrine differentiation (NED), specifically ENO2, KISS1R and HTR5A. ENO2 is the bonafide marker of neuroendocrine differentiation and it's presence is a signature of late stage castrate resistant prostate cancer. Furthermore, we have functional evidence KISS1R is linked to intracellular calcium mobilization in 22RV1 cells. We have demonstrated that increased expression of HIF-regulated genes in response to loss of Rb activates Akt and ERK signaling pathways and promotes neuroendocrine differentiation and invasion. Inhibition of these signaling pathways significantly decreased actin polymerization in LNCaP cells. For the first time, we have established a direct link between hypoxic tumour environments, Rb inactivation and progression to late stage metastatic neuroendocrine prostate cancer. Understanding the molecular pathways responsible for progression of benign prostate tumours to metastasized and lethal forms will aid in the development of more effective prostate cancer therapies. Citation Format: Mark Labrecque, Mandeep Takhar, Rebecca J. Nason, Stephanie Santacruz, Kevin Tam, Shabnam Massah, Anne Haegert, Robert Bell, Manuel Altamirano-Dimas, Colin Collins, Frank Lee, Gratien Prefontaine, Michael Cox, Timothy Beischlag. Loss of retinoblastoma protein dysregulates HIF1-mediated genetic programs, and promotes tumor cell invasiveness and neuroendocrine differentiation in prostate cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2922.
The retinoblastoma protein (Rb) is capable of attenuating the hypoxic response in tumor cells. Th... more The retinoblastoma protein (Rb) is capable of attenuating the hypoxic response in tumor cells. This process is mediated by the hypoxia inducible factor 1α/2α (HIF1α/2α) and its dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT/ HIF1β). Rb modulates HIF activity by virtue of its association with the thyroid hormone receptor/retinoblastoma interacting protein 230 (TRIP230), an essential cofactor of the HIF1α/ARNT transcriptional complex. We used short hairpin RNA (shRNA) technology and microarray analysis to interrogate the Rb-negative and wild-type MCF7 cell transcriptomes and generated lists of genes that were either up- or downregulated in response to both loss of Rb and hypoxia. We found that loss of Rb enhances the expression of hypoxia-regulated genes involved in invasion and epithelial-to-mesenchymal transition and significantly decreases the expression of genes involved in cell anchoring and differentiation in MCF7 and MDA-MB-231 breast cancer cells. Genes were validated using both qRT-PCR and immuno-blot analysis. Additionally, gene ontology analysis revealed that AKT and ERK1/2 are downstream effectors of hypoxic gene programs that are sensitive to loss of Rb. Furthermore, these factors regulate the acquisition of a more invasive phenotype in breast cancer cells. Finally, we found that Rb knockdown in combination with pre-treatment of cells with hypoxia increased growth of tumor foci in the lungs after i.v. injection and increased the development of spontaneous metastases from orthotopically implanted breast tumor cells in female NOD-SCID mice. Primary tumors lacking Rb demonstrated enriched protein levels of genes identified in our arrays when compared to negative control tumors. These results show that Rb is a negative modulator of hypoxia-regulated genetic programs by virtue of its direct effects on the HIF-complex. Understanding the HIF complex and the molecular mechanisms controlling the progression from benign tumors to metastasized and lethal forms will allow us to develop more specific breast cancer therapies. Citation Format: Mandeep K. Takhar, Mark P. Labrecque, Kevin J. Tam, Anne Haegert, Robert H. Bell, Manuel Altamirano-Dimas, Colin C. Collins, Gratien G. Prefontaine, Michael E. Cox, Kevin L. Bennewith, Timothy V. Beischlag. The retinoblastoma protein regulates hypoxia-inducible factor-1α-mediated transcriptional programs, tumor cell invasiveness, tumor growth and metastasis in human breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2921.
migratoryand invasiveproperties tobecomemesenchymal stemcells.EMT has been implicated in the init... more migratoryand invasiveproperties tobecomemesenchymal stemcells.EMT has been implicated in the initiation of metastasis for cancer progression. METHODS: Exosomes were isolated by ultracentrifugation from T24 or UMUC3 invasive bladder cancer cell conditioned media. T24 exosomes were added to urothelial cells for 4, 6, 24 or 48 hours. RNA or protein was collected from the urothelial cells and qRT-PCR or western blotting was performed to measure expression of EMT markers. T24or UMUC3-derived exosomes were added to urothelial cells and plated in transwell inserts for migration and invasion assays. RESULTS: Urothelial cells treated with T24 or UMUC3 bladder cancer exosomes showed an increased expression in several mesenchymal markers, including a-SMA, S100A4, and Snail, as compared to PBS treated cells. Moreover, treatment of urothelial cells with T24or UMUC3-derived exosomes resulted in decreased expression of epithelial markers, including e-cadherin and b-catenin, as compared to the control, PBS treated cells. T24and UMUC3-derived exosomes also increased the migration and invasion of urothelial cells, and this was blocked by heparin pre-treatment. We further showed that exosomes isolated from patient urine and barbotage samples were able to induce the expression of several mesenchymal markers in recipient urothelial cells. CONCLUSIONS: In thisstudy,weestablished thatexosomes from invasive bladder cancer cells are able to induce EMT in recipient urothelial cells. We further showed that exosomes from invasive bladder cancer cells can promote migration and invasion of recipient urothelial cells. We also demonstrated that the effect on migration and invasion is mediated by heparin. Finally, we established that exosomes isolated from bladder cancer patients hada similar effect on the expression ofmesenchymalmarkers in urothelial cells as the exosomes isolated from bladder cancer cell lines.
Low-grade serous ovarian carcinoma (LGSOC) is a rare tumor subtype with high case fatality rates ... more Low-grade serous ovarian carcinoma (LGSOC) is a rare tumor subtype with high case fatality rates in patients with metastatic disease. There is a pressing need to develop effective treatments using newly available preclinical models for therapeutic discovery and drug evaluation. Here, we use multiomics integration of wholeexome sequencing, RNA sequencing, and mass spectrometry-based proteomics on 14 LGSOC cell lines to elucidate novel biomarkers and therapeutic vulnerabilities. Comparison of LGSOC cell line data with LGSOC tumor data enabled predictive biomarker identification of MEK inhibitor (MEKi) efficacy, with KRAS mutations found exclusively in MEKi-sensitive cell lines and NRAS mutations found mostly in MEKi-resistant cell lines. Distinct patterns of Catalogue of Somatic Mutations in Cancer mutational signatures were identified in MEKi-sensitive and MEKi-resistant cell lines. Deletions of CDKN2A/B and MTAP genes were more frequent in cell lines than tumor samples and possibly represent key driver events in the absence of KRAS/NRAS/BRAF mutations. These LGSOC cell lines were representative models of the molecular aberrations found in LGSOC tumors. For prediction of in vitro MEKi efficacy, proteomic data provided better discrimination than gene expression data. Condensin, minichromosome maintenance, and replication factor C protein complexes were identified as potential treatment targets in MEKi-resistant cell lines. This study suggests that CDKN2A/B or MTAP deficiency may be exploited using synthetically lethal treatment strategies, highlighting the importance of using proteomic data as a tool for molecular drug prediction. Multiomics approaches are crucial to improving our understanding of the molecular underpinnings of LGSOC and applying this information to develop new therapies. Significance: These findings highlight the utility of global multiomics to characterize LGSOC cell lines as research models, to determine biomarkers of MEKi resistance, and to identify potential novel therapeutic targets.
To avoid over-or under-treatment of primary prostate tumours, there is a critical need for molecu... more To avoid over-or under-treatment of primary prostate tumours, there is a critical need for molecular signatures to discriminate indolent from aggressive, lethal disease. Reprogrammed energy metabolism is an important hallmark of cancer, and abnormal metabolic characteristics of cancers have been implicated as potential diagnostic/ prognostic signatures. While genomic and transcriptomic heterogeneity of prostate cancer is well documented and associated with tumour progression, less is known about metabolic heterogeneity of the disease. Using a panel of high fidelity patientderived xenograft (PDX) models derived from hormone-naïve prostate cancer, we demonstrated heterogeneity of expression of genes involved in cellular energetics and macromolecular biosynthesis. Such heterogeneity was also observed in clinical, treatment-naïve prostate cancers by analyzing the transcriptome sequencing data. Importantly, a metabolic gene signature of increased one-carbon metabolism or decreased proline degradation was identified to be associated with significantly decreased biochemical disease-free patient survival. These results suggest that metabolic heterogeneity of hormone-naïve prostate cancer is of biological and clinical importance and motivate further studies to determine the heterogeneity in metabolic flux in the disease that may lead to identification of new signatures for tumour/ patient stratification and the development of new strategies and targets for therapy of prostate cancer.
Urologic Oncology-seminars and Original Investigations, Apr 1, 2015
To assess whether Bcl-2, an inhibitor of the apoptotic cascade, can predict response to neoadjuva... more To assess whether Bcl-2, an inhibitor of the apoptotic cascade, can predict response to neoadjuvant chemotherapy in patients with urothelial cancer of the bladder (UCB). Methods: Bcl-2 expression was analyzed in 2 different tissue microarrays (TMAs). One TMA was constructed of primary tumors and their corresponding lymph node (LN) metastases from 152 patients with chemotherapy-naive UCB treated by cystectomy and pelvic lymphadenectomy (chemotherapy-naive TMA cohort). The other TMA was constructed of tumor samples obtained from 55 patients with UCB before neoadjuvant chemotherapy (transurethral resection of the bladder cancer) and after cystectomy with pelvic lymphadenectomy (residual primary tumor [ypTþ], n ¼ 38); residual LN metastases [ypNþ], n ¼ 24) (prechemotherapy/postchemotherapy TMA cohort). Bcl-2 overexpression was defined as 10% or more cancer cells showing cytoplasmic immunoreactivity. Results: In both TMA cohorts, Bcl-2 overexpression was significantly (P o 0.05) more frequent in LN metastases than in primary tumors (chemotherapy-naive TMA group: 18/148 [12%] in primary tumors vs. 39/143 [27%] in metastases; postchemotherapy TMA: ypTþ 7/35 [20%] vs. ypNþ 11/19 [58%]). In the neoadjuvant setting, patients with Bcl-2 overexpression in transurethral resection of the bladder cancer specimens showed significantly (P ¼ 0.04) higher ypT stages and less regression in their cystectomy specimens than did the control group, and only one-eighth (13%) had complete tumor regression (ypT0 ypN0). In survival analyses, only histopathological parameters added significant prognostic information. Conclusions: Bcl-2 overexpression in chemotherapy-naive primary bladder cancer is related to poor chemotherapy response and might help to select likely nonresponders.
Standardized and reproducible preclinical models that recapitulate the dynamics of prostate cance... more Standardized and reproducible preclinical models that recapitulate the dynamics of prostate cancer are urgently needed. We established a bank of transplantable patient-derived prostate cancer xenografts that capture the biologic and molecular heterogeneity currently confounding prognostication and therapy development. Xenografts preserved the histopathology, genome architecture, and global gene expression of donor tumors. Moreover, their aggressiveness matched patient observations, and their response to androgen withdrawal correlated with tumor subtype. The panel includes the first xenografts generated from needle biopsy tissue obtained at diagnosis. This advance was exploited to generate independent xenografts from different sites of a primary site, enabling functional dissection of tumor heterogeneity. Prolonged exposure of adenocarcinoma xenografts to androgen withdrawal led to castration-resistant prostate cancer, including the first-in-field model of complete transdifferentiation into lethal neuroendocrine prostate cancer. Further analysis of this model supports the hypothesis that neuroendocrine prostate cancer can evolve directly from adenocarcinoma via an adaptive response and yielded a set of genes potentially involved in neuroendocrine transdifferentiation. We predict that these next-generation models will be transformative for advancing mechanistic understanding of disease progression, response to therapy, and personalized oncology. Cancer Res; 74(4); 1272-83. Ó2013 AACR.
Low-grade serous ovarian carcinoma (LGSOC) is a rare tumor subtype with high case fatality rates ... more Low-grade serous ovarian carcinoma (LGSOC) is a rare tumor subtype with high case fatality rates in patients with metastatic disease. There is a pressing need to develop effective treatments using newly available preclinical models for therapeutic discovery and drug evaluation. Here, we use multiomics integration of wholeexome sequencing, RNA sequencing, and mass spectrometry-based proteomics on 14 LGSOC cell lines to elucidate novel biomarkers and therapeutic vulnerabilities. Comparison of LGSOC cell line data with LGSOC tumor data enabled predictive biomarker identification of MEK inhibitor (MEKi) efficacy, with KRAS mutations found exclusively in MEKi-sensitive cell lines and NRAS mutations found mostly in MEKi-resistant cell lines. Distinct patterns of Catalogue of Somatic Mutations in Cancer mutational signatures were identified in MEKi-sensitive and MEKi-resistant cell lines. Deletions of CDKN2A/B and MTAP genes were more frequent in cell lines than tumor samples and possibly represent key driver events in the absence of KRAS/NRAS/BRAF mutations. These LGSOC cell lines were representative models of the molecular aberrations found in LGSOC tumors. For prediction of in vitro MEKi efficacy, proteomic data provided better discrimination than gene expression data. Condensin, minichromosome maintenance, and replication factor C protein complexes were identified as potential treatment targets in MEKi-resistant cell lines. This study suggests that CDKN2A/B or MTAP deficiency may be exploited using synthetically lethal treatment strategies, highlighting the importance of using proteomic data as a tool for molecular drug prediction. Multiomics approaches are crucial to improving our understanding of the molecular underpinnings of LGSOC and applying this information to develop new therapies. Significance: These findings highlight the utility of global multiomics to characterize LGSOC cell lines as research models, to determine biomarkers of MEKi resistance, and to identify potential novel therapeutic targets.
Figure S1: Transplantable tumor lines retain the histolopathological characteristics of their par... more Figure S1: Transplantable tumor lines retain the histolopathological characteristics of their parent tumor. Xenografts LTL313H, LTL412 and LTL418 show similar tissue structure to the patient tumour from which they were originally derived (H&E stain; FFPE tissue). Immunohistochemical stains show that the protein expression of key markers (AR, PSA, PTEN, ERG) is also conserved. Note that LTL313H is derived from a biopsy sample the size of which is visible in the micrographs.
Background: Low-grade serous ovarian carcinoma (LGSOC) is a rare tumor subtype with high case fat... more Background: Low-grade serous ovarian carcinoma (LGSOC) is a rare tumor subtype with high case fatality rates. As such, there is a pressing need to develop more effective treatments using newly available preclinical models for therapeutic discovery and drug evaluation. Here, we use a multiomics approach to interrogate a collection of LGSOC patient-derived cell lines to elucidate novel biomarkers and therapeutic vulnerabilities. Methods: Fourteen LGSOC cell lines were interrogated using whole exome sequencing, RNA sequencing, and mass spectrometry-based proteomics. Somatic mutation, copy-number aberrations, gene and protein expression were analyzed and integrated using different computational approaches. LGSOC cell line data was compared to publicly available LGSOC tumor data (AACR GENIE cohort), and also used for predictive biomarker identification of MEK inhibitor (MEKi) efficacy. Protein interaction databases were evaluated to identify novel therapeutic targets. Results: KRAS mutations were exclusively found in MEKi-sensitive and NRAS mutations mostly in MEKiresistant cell lines. Analysis of COSMIC mutational signatures revealed distinct patterns of nucleotide substitution mutations in MEKi-sensitive and MEKi-resistant cell lines. Deletions of CDKN2A/B and MTAP genes (chromosome 9p21) were much more frequent in cell lines than tumor samples and possibly represent key driver events in the absence of KRAS/NRAS/BRAF mutations. For in-vitro MEKi efficacy prediction, proteomic data provided better discrimination than gene expression data. Condensin, MCM, and RFC protein complexes were identified as potential treatment targets in MEKi-resistant cell lines. Conclusions: Our LGSOC cell lines are representative models of the most common molecular aberrations found in LGSOC tumors. This study highlights the importance of using proteomic data in multiomics assessment of drug prediction and identification of potential therapeutic targets. CDKN2A/B and MTAP deficiency offer an opportunity to find synthetically lethal candidates for novel treatments. Multiomics approaches are crucial to improving our understanding of the molecular aberrations in LGSOC, establishing effective drug prediction programs and identifying novel therapeutic targets in LGSOC.
Background: Malignant Peritoneal Mesothelioma (PeM) is a rare and fatal cancer that originates fr... more Background: Malignant Peritoneal Mesothelioma (PeM) is a rare and fatal cancer that originates from the peritoneal lining of the abdomen. Standard treatment of PeM is limited to cytoreductive surgery and/or chemotherapy, and no effective targeted therapies for PeM exist. Some immune checkpoint inhibitor studies of mesothelioma have found positivity to be associated with a worse prognosis. Methods: To search for novel therapeutic targets for PeM, we performed a comprehensive integrative multiomics analysis of the genome, transcriptome, and proteome of 19 treatment-naïve PeM, and in particular we examined BAP1 mutation and copy-number status and its relationship to immune checkpoint inhibitor activation. Results: We found that PeM could be divided into tumors with an inflammatory tumor microenvironment and those without, and that this distinction correlated with haploinsufficiency of BAP1. To further investigate the role of BAP1, we used our recently developed cancer driver gene prioritization algorithm, HIT'nDRIVE, and observed that PeM with BAP1 haploinsufficiency form a distinct molecular subtype characterized by distinct gene expression patterns of chromatin remodeling, DNA repair pathways, and immune checkpoint receptor activation. We demonstrate that this subtype is correlated with an inflammatory tumor microenvironment and thus is a candidate for immune checkpoint blockade therapies. Conclusions: Our findings reveal BAP1 to be a potential, easily trackable prognostic and predictive biomarker for PeM immunotherapy that refines PeM disease classification. BAP1 stratification may improve drug response rates in ongoing phase-I and II clinical trials exploring the use of immune checkpoint blockade therapies in PeM in which BAP1 status is not considered. This integrated molecular characterization provides a comprehensive foundation for improved management of a subset of PeM patients.
Neuroendocrine prostate cancer (NEPC) is a highly aggressive subtype of prostate cancer (PCa) tha... more Neuroendocrine prostate cancer (NEPC) is a highly aggressive subtype of prostate cancer (PCa) that is becoming increasingly common in the clinic. While the vast majority of PCa presents as androgen-dependent adenocarcinoma, recent uses of increasingly potent therapeutics targeting the androgen receptor signaling axis has resulted in the promotion of NEPC transdifferentiation as a mechanism of treatment resistance. Unfortunately, there is currently no effective treatment option for NEPC. Altered cancer metabolism is now recognized as a hallmark of cancer and a crucial factor for promoting tumour growth and spread. In particular, altered glucose metabolism and the resultant acidification of the tumor microenvironment via increased lactic acid production has been shown to play an important role in multiple cancer-promoting processes, including tissue invasion/metastasis, angiogenesis, and suppression of local anticancer immunity. While increased glycolysis is not generally considered a phenomenon relevant to primary treatment-naive PCa, we have recently demonstrated its relavance to castration-resistant prostate cancer (CRPC) and thus suspect that it is also relevant to the more aggressive NEPC. Our laboratory has also developed a number of unique serially transplantable patient-derived xenograft (PDX) models of NEPC that are histologically highly similar to the donor tissues and retain important genetic and epigenetic features. In particular, we have developed the first spontaneous NEPC transdifferentiation model in the field (LTL331/331R). The gene expression profiles of these NEPC PDX models were compared to that of PCa adenocarcinoma PDX models. To determine whether certain metabolic pathway alterations were specific to NEPC, genes from a number of key metabolic pathways were compiled and overall pathway scores were generated using average expression z-scores. Furthermore, publically available gene expression data from NEPC patient tumors were used to validate our findings. From our analysis, we found that genes in the glycolysis pathway were signficiantlly upregulatied in both our PDX models and also in patient NEPC samples. Of particular interest is the upregulation of genes involved in the production and secretion of lactic acid, such as LDHA and MCT4. As such, our results suggest that elevated glycolysis and production of lactic acid could be a clinically important NEPC phenotype. Furthermore, the inhibition of glycolysis and particularly the inhibition of lactic acid secretion via MCT4 could be a potentially viable therapeutic strategy for NEPC. Citation Format: Stephen Y. Choi, Susan L. Ettinger, Dong Lin, Hui Xue, Robert H. Bell, Fan Mo, Michael Pollak, Colin C. Collins, Yuzhuo Wang. Elevated glycolytic gene signature in patient-derived neuroendocrine prostate cancer xenograft models and its clinical relevance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4420. doi:10.1158/1538-7445.AM2017-4420
Loss of tumour suppressor proteins, such as the retinoblastoma protein (Rb), results in tumour pr... more Loss of tumour suppressor proteins, such as the retinoblastoma protein (Rb), results in tumour progression and metastasis. Metastasis is facilitated by low oxygen availability within the tumour that is detected by hypoxia inducible factors (HIFs). The HIF1 complex, HIF1α and its dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT), is the master regulator of the hypoxic response. Previously, we demonstrated that Rb represses the transcriptional response to hypoxia by virtue of its association with HIF1. In this report, we further characterized the role of Rb in HIF1-regulated genetic programs by stably ablating Rb expression with retrovirally-introduced short hairpin RNA in LNCaP and 22rV1 human prostate cancer cells. DNA microarray analysis revealed that Rb regulates specific chromosomal gene clusters and loss of Rb in conjunction with hypoxia leads to dysregulation of HIF1-regulated genetic programs that promote cell invasion and neuroendocrine differentiation. Gene ontology analysis of the hypoxia-inducible genes sensitive to loss of Rb revealed that a significant portion of these genes are involved in neuroendocrine differentiation (NED), specifically ENO2, KISS1R and HTR5A. ENO2 is the bonafide marker of neuroendocrine differentiation and it's presence is a signature of late stage castrate resistant prostate cancer. Furthermore, we have functional evidence KISS1R is linked to intracellular calcium mobilization in 22RV1 cells. We have demonstrated that increased expression of HIF-regulated genes in response to loss of Rb activates Akt and ERK signaling pathways and promotes neuroendocrine differentiation and invasion. Inhibition of these signaling pathways significantly decreased actin polymerization in LNCaP cells. For the first time, we have established a direct link between hypoxic tumour environments, Rb inactivation and progression to late stage metastatic neuroendocrine prostate cancer. Understanding the molecular pathways responsible for progression of benign prostate tumours to metastasized and lethal forms will aid in the development of more effective prostate cancer therapies. Citation Format: Mark Labrecque, Mandeep Takhar, Rebecca J. Nason, Stephanie Santacruz, Kevin Tam, Shabnam Massah, Anne Haegert, Robert Bell, Manuel Altamirano-Dimas, Colin Collins, Frank Lee, Gratien Prefontaine, Michael Cox, Timothy Beischlag. Loss of retinoblastoma protein dysregulates HIF1-mediated genetic programs, and promotes tumor cell invasiveness and neuroendocrine differentiation in prostate cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2922.
The retinoblastoma protein (Rb) is capable of attenuating the hypoxic response in tumor cells. Th... more The retinoblastoma protein (Rb) is capable of attenuating the hypoxic response in tumor cells. This process is mediated by the hypoxia inducible factor 1α/2α (HIF1α/2α) and its dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT/ HIF1β). Rb modulates HIF activity by virtue of its association with the thyroid hormone receptor/retinoblastoma interacting protein 230 (TRIP230), an essential cofactor of the HIF1α/ARNT transcriptional complex. We used short hairpin RNA (shRNA) technology and microarray analysis to interrogate the Rb-negative and wild-type MCF7 cell transcriptomes and generated lists of genes that were either up- or downregulated in response to both loss of Rb and hypoxia. We found that loss of Rb enhances the expression of hypoxia-regulated genes involved in invasion and epithelial-to-mesenchymal transition and significantly decreases the expression of genes involved in cell anchoring and differentiation in MCF7 and MDA-MB-231 breast cancer cells. Genes were validated using both qRT-PCR and immuno-blot analysis. Additionally, gene ontology analysis revealed that AKT and ERK1/2 are downstream effectors of hypoxic gene programs that are sensitive to loss of Rb. Furthermore, these factors regulate the acquisition of a more invasive phenotype in breast cancer cells. Finally, we found that Rb knockdown in combination with pre-treatment of cells with hypoxia increased growth of tumor foci in the lungs after i.v. injection and increased the development of spontaneous metastases from orthotopically implanted breast tumor cells in female NOD-SCID mice. Primary tumors lacking Rb demonstrated enriched protein levels of genes identified in our arrays when compared to negative control tumors. These results show that Rb is a negative modulator of hypoxia-regulated genetic programs by virtue of its direct effects on the HIF-complex. Understanding the HIF complex and the molecular mechanisms controlling the progression from benign tumors to metastasized and lethal forms will allow us to develop more specific breast cancer therapies. Citation Format: Mandeep K. Takhar, Mark P. Labrecque, Kevin J. Tam, Anne Haegert, Robert H. Bell, Manuel Altamirano-Dimas, Colin C. Collins, Gratien G. Prefontaine, Michael E. Cox, Kevin L. Bennewith, Timothy V. Beischlag. The retinoblastoma protein regulates hypoxia-inducible factor-1α-mediated transcriptional programs, tumor cell invasiveness, tumor growth and metastasis in human breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2921.
migratoryand invasiveproperties tobecomemesenchymal stemcells.EMT has been implicated in the init... more migratoryand invasiveproperties tobecomemesenchymal stemcells.EMT has been implicated in the initiation of metastasis for cancer progression. METHODS: Exosomes were isolated by ultracentrifugation from T24 or UMUC3 invasive bladder cancer cell conditioned media. T24 exosomes were added to urothelial cells for 4, 6, 24 or 48 hours. RNA or protein was collected from the urothelial cells and qRT-PCR or western blotting was performed to measure expression of EMT markers. T24or UMUC3-derived exosomes were added to urothelial cells and plated in transwell inserts for migration and invasion assays. RESULTS: Urothelial cells treated with T24 or UMUC3 bladder cancer exosomes showed an increased expression in several mesenchymal markers, including a-SMA, S100A4, and Snail, as compared to PBS treated cells. Moreover, treatment of urothelial cells with T24or UMUC3-derived exosomes resulted in decreased expression of epithelial markers, including e-cadherin and b-catenin, as compared to the control, PBS treated cells. T24and UMUC3-derived exosomes also increased the migration and invasion of urothelial cells, and this was blocked by heparin pre-treatment. We further showed that exosomes isolated from patient urine and barbotage samples were able to induce the expression of several mesenchymal markers in recipient urothelial cells. CONCLUSIONS: In thisstudy,weestablished thatexosomes from invasive bladder cancer cells are able to induce EMT in recipient urothelial cells. We further showed that exosomes from invasive bladder cancer cells can promote migration and invasion of recipient urothelial cells. We also demonstrated that the effect on migration and invasion is mediated by heparin. Finally, we established that exosomes isolated from bladder cancer patients hada similar effect on the expression ofmesenchymalmarkers in urothelial cells as the exosomes isolated from bladder cancer cell lines.
Low-grade serous ovarian carcinoma (LGSOC) is a rare tumor subtype with high case fatality rates ... more Low-grade serous ovarian carcinoma (LGSOC) is a rare tumor subtype with high case fatality rates in patients with metastatic disease. There is a pressing need to develop effective treatments using newly available preclinical models for therapeutic discovery and drug evaluation. Here, we use multiomics integration of wholeexome sequencing, RNA sequencing, and mass spectrometry-based proteomics on 14 LGSOC cell lines to elucidate novel biomarkers and therapeutic vulnerabilities. Comparison of LGSOC cell line data with LGSOC tumor data enabled predictive biomarker identification of MEK inhibitor (MEKi) efficacy, with KRAS mutations found exclusively in MEKi-sensitive cell lines and NRAS mutations found mostly in MEKi-resistant cell lines. Distinct patterns of Catalogue of Somatic Mutations in Cancer mutational signatures were identified in MEKi-sensitive and MEKi-resistant cell lines. Deletions of CDKN2A/B and MTAP genes were more frequent in cell lines than tumor samples and possibly represent key driver events in the absence of KRAS/NRAS/BRAF mutations. These LGSOC cell lines were representative models of the molecular aberrations found in LGSOC tumors. For prediction of in vitro MEKi efficacy, proteomic data provided better discrimination than gene expression data. Condensin, minichromosome maintenance, and replication factor C protein complexes were identified as potential treatment targets in MEKi-resistant cell lines. This study suggests that CDKN2A/B or MTAP deficiency may be exploited using synthetically lethal treatment strategies, highlighting the importance of using proteomic data as a tool for molecular drug prediction. Multiomics approaches are crucial to improving our understanding of the molecular underpinnings of LGSOC and applying this information to develop new therapies. Significance: These findings highlight the utility of global multiomics to characterize LGSOC cell lines as research models, to determine biomarkers of MEKi resistance, and to identify potential novel therapeutic targets.
To avoid over-or under-treatment of primary prostate tumours, there is a critical need for molecu... more To avoid over-or under-treatment of primary prostate tumours, there is a critical need for molecular signatures to discriminate indolent from aggressive, lethal disease. Reprogrammed energy metabolism is an important hallmark of cancer, and abnormal metabolic characteristics of cancers have been implicated as potential diagnostic/ prognostic signatures. While genomic and transcriptomic heterogeneity of prostate cancer is well documented and associated with tumour progression, less is known about metabolic heterogeneity of the disease. Using a panel of high fidelity patientderived xenograft (PDX) models derived from hormone-naïve prostate cancer, we demonstrated heterogeneity of expression of genes involved in cellular energetics and macromolecular biosynthesis. Such heterogeneity was also observed in clinical, treatment-naïve prostate cancers by analyzing the transcriptome sequencing data. Importantly, a metabolic gene signature of increased one-carbon metabolism or decreased proline degradation was identified to be associated with significantly decreased biochemical disease-free patient survival. These results suggest that metabolic heterogeneity of hormone-naïve prostate cancer is of biological and clinical importance and motivate further studies to determine the heterogeneity in metabolic flux in the disease that may lead to identification of new signatures for tumour/ patient stratification and the development of new strategies and targets for therapy of prostate cancer.
Urologic Oncology-seminars and Original Investigations, Apr 1, 2015
To assess whether Bcl-2, an inhibitor of the apoptotic cascade, can predict response to neoadjuva... more To assess whether Bcl-2, an inhibitor of the apoptotic cascade, can predict response to neoadjuvant chemotherapy in patients with urothelial cancer of the bladder (UCB). Methods: Bcl-2 expression was analyzed in 2 different tissue microarrays (TMAs). One TMA was constructed of primary tumors and their corresponding lymph node (LN) metastases from 152 patients with chemotherapy-naive UCB treated by cystectomy and pelvic lymphadenectomy (chemotherapy-naive TMA cohort). The other TMA was constructed of tumor samples obtained from 55 patients with UCB before neoadjuvant chemotherapy (transurethral resection of the bladder cancer) and after cystectomy with pelvic lymphadenectomy (residual primary tumor [ypTþ], n ¼ 38); residual LN metastases [ypNþ], n ¼ 24) (prechemotherapy/postchemotherapy TMA cohort). Bcl-2 overexpression was defined as 10% or more cancer cells showing cytoplasmic immunoreactivity. Results: In both TMA cohorts, Bcl-2 overexpression was significantly (P o 0.05) more frequent in LN metastases than in primary tumors (chemotherapy-naive TMA group: 18/148 [12%] in primary tumors vs. 39/143 [27%] in metastases; postchemotherapy TMA: ypTþ 7/35 [20%] vs. ypNþ 11/19 [58%]). In the neoadjuvant setting, patients with Bcl-2 overexpression in transurethral resection of the bladder cancer specimens showed significantly (P ¼ 0.04) higher ypT stages and less regression in their cystectomy specimens than did the control group, and only one-eighth (13%) had complete tumor regression (ypT0 ypN0). In survival analyses, only histopathological parameters added significant prognostic information. Conclusions: Bcl-2 overexpression in chemotherapy-naive primary bladder cancer is related to poor chemotherapy response and might help to select likely nonresponders.
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Papers by Robert Bell