Giving

Home

Cancer Biology

KRAS Engages AGO2 to Enhance Cellular Transformation

Nearly a third of all cancers have mutations in the RAS family of genes, including KRAS; however therapeutic targeting of RAS family has remained elusive. While screening for KRAS interacting proteins, we uncovered an interaction between KRAS and Argonaute 2 (AGO2) and found that AGO2 interacted with both mutated and normal KRAS. Endogenously, KRAS and AGO2 co-sedimented and co-localized in the endoplasmic reticulum. Functionally, AGO2 knockdown attenuated cell proliferation in mutant KRAS-dependent cells and AGO2 overexpression enhanced KRASG12V-mediated transformation. Using AGO2−/− cells, we demonstrated that the KRAS-AGO2 interaction is required for maximal mutant KRAS expression and cellular transformation. Mechanistically, oncogenic KRAS attenuated AGO2-mediated gene silencing. Overall, the functional interaction with AGO2 extends KRAS function beyond its canonical role in signaling. These findings suggest interrupting the KRAS-AGO2 interaction as a potential therapeutic strategy (Cell Rep. 2016 Feb 16;14(6):1448-61).

BET Bromodomain Inhibitors Enhance Efficacy and Disrupt Resistance to AR Antagonists in the Treatment of Prostate Cancer

Next-generation anti-androgen therapies, such as enzalutamide and abiraterone, have had a profound impact on the management of metastatic castration-resistant prostate cancer (mCRPC). However, mCRPC patients invariably develop resistance to these agents. We developed a series of clonal cell lines from enzalutamide-resistant prostate tumor xenografts to study the molecular mechanism of resistance and test their oncogenic potential under various treatment conditions. Androgen receptor (AR) signaling was maintained in these cell lines that acquired potential resistance mechanisms, including expression of AR-variant 7 (AR-v7) and glucocorticoid receptor. BET bromodomain inhibitors were shown previously to attenuate AR signaling in mCRPC; here, we demonstrated the efficacy of bromodomain and extraterminal (BET) inhibitors in enzalutamide-resistant prostate cancer models. AR antagonists, enzalutamide, and ARN509 exhibit enhanced prostate tumor growth inhibition when combined with BET inhibitors, JQ1 and OTX015, respectively. Taken together, these data provide a compelling preclinical rationale to combine BET inhibitors with AR antagonists to subvert resistance mechanisms (Mol Cancer Res. 2016 Apr;14(4):324-31).

Overexpression of the Long Non-coding RNA SChLAP1 Independently Predicts Lethal Prostate Cancer

The long noncoding RNA SChLAP1 is overexpressed in a subset of prostate cancers (PCa), and high SChLAP1 expression by in situ hybridization (ISH) independently predicts biochemical recurrence after radical prostatectomy. Importantly, although biochemical recurrence is a significant clinical outcome, it is not a validated surrogate for PCa-related mortality. Thus, we evaluated the association between SChLAP1 expression and development of lethal PCa in a large cohort of American men with PCa and long-term follow-up. SChLAP1 ISH was performed on tissue microarrays containing representative formalin-fixed, paraffin-embedded PCa tissue from all patients and scored using a semi-quantitative method (ISH score range 0-400). Hazard ratios (HRs) for the association between SChLAP1 expression and time to development of lethal PCa were estimated using multivariable Cox regression analysis. Of the 937 patients evaluated, 89 (9.5%) had high SChLAP1 expression (ISH score ≥100), which in patients treated with radical prostatectomy was strongly associated with development of lethal PCa independent of age, Gleason score, pathologic stage, and PTEN status (HR 2.2, 95% confidence interval 1.1-4.1). These results suggest that SChLAP1 may be a useful tissue-based biomarker for identifying PCa patients at higher risk of lethal progression (Eur Urol. 2015 Dec 23. pii: S0302-2838(15)01211-7).

The landscape of long noncoding RNAs in the human transcriptome

Long noncoding RNAs (lncRNAs) are emerging as important regulators of tissue physiology and disease processes including cancer. To delineate genome-wide lncRNA expression, we curated 7,256 RNA sequencing (RNA-seq) libraries from tumors, normal tissues and cell lines comprising over 43 Tb of sequence from 25 independent studies. We applied ab initio assembly methodology to this data set, yielding a consensus human transcriptome of 91,013 expressed genes. Over 68% (58,648) of genes were classified as lncRNAs, of which 79% were previously unannotated. About 1% (597) of the lncRNAs harbored ultraconserved elements, and 7% (3,900) overlapped disease-associated SNPs. To prioritize lineage-specific, disease-associated lncRNA expression, we employed non-parametric differential expression testing and nominated 7,942 lineage- or cancer-associated lncRNA genes. The lncRNA landscape characterized here may shed light on normal biology and cancer pathogenesis and may be valuable for future biomarker development. (Nat Genet. 2015 Jan 19. doi: 10.1038/ng.3192).

Transcriptome meta-analysis of lung cancer reveals recurrent aberrations in NRG1 and Hippo pathway genes

Lung cancer is emerging as a paradigm for disease molecular subtyping, facilitating targeted therapy based on driving somatic alterations. Here we perform transcriptome analysis of 153 samples representing lung adenocarcinomas, squamous cell carcinomas, large cell lung cancer, adenoid cystic carcinomas and cell lines. By integrating our data with The Cancer Genome Atlas and published sources, we analyse 753 lung cancer samples for gene fusions and other transcriptomic alterations. We show that higher numbers of gene fusions is an independent prognostic factor for poor survival in lung cancer. Our analysis confirms the recently reported CD74-NRG1 fusion and suggests that NRG1, NF1 and Hippo pathway fusions may play important roles in tumours without known driver mutations. In addition, we observe exon-skipping events in c-MET, which are attributable to splice site mutations. These classes of genetic aberrations may play a significant role in the genesis of lung cancers lacking known driver mutations. (Nat Commun. 2014 Dec 22;5:5893).

The long non-coding RNA PCAT-1 promotes prostate cancer cell proliferation through cMyc

Long non-coding RNAs (lncRNAs) represent an emerging layer of cancer biology, contributing to tumor proliferation, invasion, and metastasis. Here, we describe a role for the oncogenic lncRNA PCAT-1 in prostate cancer proliferation through cMyc. We find that PCAT-1-mediated proliferation is dependent on cMyc protein stabilization, and using expression profiling, we observed that cMyc is required for a subset of PCAT-1-induced expression changes. The PCAT-1-cMyc relationship is mediated through the post-transcriptional activity of the MYC 3' untranslated region, and we characterize a role for PCAT-1 in the disruption of MYC-targeting microRNAs. To further elucidate a role for post-transcriptional regulation, we demonstrate that targeting PCAT-1 with miR-3667-3p, which does not target MYC, is able to reverse the stabilization of cMyc by PCAT-1. This work establishes a basis for the oncogenic role of PCAT-1 in cancer cell proliferation and is the first study to implicate lncRNAs in the regulation of cMyc in prostate cancer. (Neoplasia. 2014 Nov 20;16(11):900-8).

New drug target for treating CRPC

Small-molecule inhibitors that target the amino-terminal bromodomains of BRD4, JQ1 and I-BET762, have been shown to exhibit anti-proliferative effects in a range of malignancies. In a recent study we showed that AR-signaling-competent human CRPC cell lines are preferentially sensitive to bromodomain and extraterminal (BET) inhibition. BRD4 physically interacted with the N-terminal domain of AR and this interaction can be disrupted by JQ1. Similar to the direct AR antagonist MDV3100, JQ1 disrupted AR recruitment to target gene loci and abrogated BRD4 localization to AR target loci and AR-mediated gene transcription. In vivo, BET bromodomain inhibition was more efficacious than direct AR antagonism in CRPC xenograft mouse models. Taken together, these studies provide a novel epigenetic approach for the concerted inhibition of oncogenic drivers in advanced prostate cancer. We generated several novel compounds to identify highly potent BET inhibitors that have optimal pharmaceutical properties for clinical development and tested their binding affinities to BRD proteins. Many of them showed low nanomolar (10-50nM) binding efficiency to target proteins.  We also tested these novel BET inhibitors in various in vitro assays and found that they displayed greater efficacy than other known inhibitors such as JQ1 (Nature. 2014 Jun 12;510(7504):278-82).

PCAT-1 regulates BRCA2 and Controls Homologous Recombination in Cancer

Recent studies have identified lncRNAs that are induced by genotoxic stress as well as being involved in the repair of DNA damage; however, the role of lncRNAs in the regulation of double strand break (DSB) repair remains unclear. Earlier we observed that PCAT-1 expression was a prostate cancer outlier associated with low levels of BRCA2. We therefore hypothesized that PCAT-1 mediated the repression of BRCA2, and thus PCAT-1 may be implicated in the dysregulation of HR upon genotoxic stress. We found an inverse relationship between PCAT-1 and BRCA2 in two independent cohorts of human prostate cancer samples. Using 58 prostate cancer tissues and 20 prostate cancer xenografts derived from human specimens, we found that increasing PCAT-1 expression correlated with decreased BRCA2 expression. Together, these data suggest that PCAT-1 expression antagonizes BRCA2 expression. This report is the first to demonstrate a role for lncRNAs in the regulation of DSBs in prostate cancer and suggests a new mechanistic basis for impaired HR in this disease (Cancer Res. 2014 Mar 15;74(6):1651-60).

The role of EED in the orchestration of polycomb group complexes

Polycomb Repressive Complexes 1 and 2 (PRC1 and 2) play a critical role in the epigenetic regulation of transcription during cellular differentiation, stem cell pluripotency, and neoplastic progression. We showed that the Polycomb Group protein EED, a core component of PRC2, physically interacts with and functions as part of PRC1. Components of PRC1 and PRC2 compete for EED binding. EED functions to recruit PRC1 to H3K27me3 loci and enhances PRC1-mediated H2A ubiquitin E3 ligase activity. We made the unexpected observation that EED, previously considered a critical component of PRC2, is instead a shared component of PRC2 and PRC1 that functions to interchange these epigenetic complexes at sites of histone modification. This study markedly enhances our understanding of how PRC2 and PRC1 coordinate epigenetic regulation and may have implications in therapeutically targeting these master regulators of transcription. (Nat Commun. 2014;5:3127).

The long noncoding RNA SChLAP1 promotes aggressive prostate cancer and antagonizes the SWI/SNF complex.

Prostate cancers remain indolent in the majority of individuals but behave aggressively in a minority. The molecular basis for this clinical heterogeneity remains incompletely understood. Here we characterized a long noncoding RNA termed SChLAP1 (second chromosome locus associated with prostate-1; also called LINC00913) that is overexpressed in a subset of prostate cancers. SChLAP1 levels independently predicted poor outcomes, including metastasis and prostate cancer-specific mortality. In vitro and in vivo gain-of-function and loss-of-function experiments indicated that SChLAP1 is critical for cancer cell invasiveness and metastasis. Mechanistically, SChLAP1 antagonizes the genome-wide localization and regulatory functions of the SWI/SNF chromatin-modifying complex. These results suggest that SChLAP1 contributes to the development of lethal cancer at least in part by antagonizing the tumor-suppressive functions of the SWI/SNF complex. (Nat Genet. 2013 Sep 29. doi: 10.1038/ng.2771).

Epigenetic regulation and dysfunction in cancer

Accumulating evidence supports a central role for epigenetic processes in the development of cancer. Altered chromatin states are hallmarks of tumor progression and chromatin modifiers including histone methyltransferases (HMTases) are key players in this process. Polycomb Repressive Complexes 1 and 2 (PRC1 and 2) play a critical role in the epigenetic regulation of transcription during cellular differentiation, stem cell pluripotency, and neoplastic progression.  EZH2 is the enzymatic component of PRC2 that catalyzes the tri-methylation of H3K27 resulting in the repression of target genes. We and others have shown that EZH2 is elevated in aggressive forms of prostate and breast cancer as well as many other solid tumors. MMSET is another HMTase that mediates H3K36 dimethylation that is associated with active transcription and like EZH2, MMSET is overexpressed in diverse solid tumors. Using next generation sequencing-based approach, we found that the EZH2-MMSET HMTase axis is coordinated by a microRNA network and that the oncogenic functions of EZH2 require MMSET activity. Together, these results suggest that the EZH2-MMSET HMTase axis coordinately functions as a master regulator of transcriptional repression, activation, and oncogenesis and may represent an attractive therapeutic target in cancer. (Mol Cell. 2013 Jan 10;49(1):80-93).

The role of sarcosine metabolism in prostate cancer progression

Earlier, metabolomic profiling of prostate cancer (PCa) progression identified markedly elevated levels of sarcosine (N-methyl glycine) in metastatic PCa and modest but significant elevation of the metabolite in PCa urine. Recently, we examined the role of key enzymes associated with sarcosine metabolism in PCa progression. Consistent with our earlier report, sarcosine levels were significantly elevated in PCa urine sediments compared to controls, with a modest area under the receiver operating characteristic curve of 0.71. In addition, the expression of sarcosine biosynthetic enzyme, glycine N-methyltransferase (GNMT), was elevated in PCa tissues, while sarcosine dehydrogenase (SARDH) and pipecolic acid oxidase (PIPOX), which metabolize sarcosine, were reduced in prostate tumors. Consistent with this, GNMT promoted the oncogenic potential of prostate cells by facilitating sarcosine production, while SARDH and PIPOX reduced the oncogenic potential of prostate cells by metabolizing sarcosine. Accordingly, addition of sarcosine, but not glycine or alanine, induced invasion and intravasation in an in vivo PCa model. In contrast, GNMT knockdown or SARDH overexpression in PCa xenografts inhibited tumor growth. Taken together, these studies substantiate the role of sarcosine in PCa progression. (Neoplasia. 2013 May;15(5):491-501).

RNA-Seq identifies targetable kinases in cancer

A study led by Dr. Chandan Kumar analyzed transcriptome sequencing data from a compendium of 482 cancer and benign samples from 25 different tissue types to assess the complete landscape of a cancer’s “kinome” expression and determine which kinases are activated in specific tumor types. Protein kinases represent the most effective class of therapeutic targets in cancer; therefore, determination of kinase aberrations is a major focus of cancer genomic studies. Here, researchers found frequent outlier kinase expression in breast cancer included therapeutic targets like ERBB2 and FGFR4 whereas MET, AKT2, and PLK2 were expressed in pancreatic cancer. Outlier expression of polo-like kinases was observed in a subset of KRAS-dependent pancreatic cancer cell lines, and conferred increased sensitivity to the pan-PLK inhibitor BI-6727. These results suggest that outlier kinases are effective therapeutic targets and can be readily identified through RNA sequencing of tumors. The results of this study was published in Cancer Discovery.

Read more from the UMHS Press Release

Epigenetic regulation and dysfunction in cancer

Accumulating evidence supports a central role for epigenetic processes in the development of cancer. Altered chromatin states are hallmarks of tumor progression and chromatin modifiers including histone methyltransferases (HMTases) are key players in this process. Polycomb Repressive Complexes 1 and 2 (PRC1 and 2) play a critical role in the epigenetic regulation of transcription during cellular differentiation, stem cell pluripotency, and neoplastic progression.  EZH2 is the enzymatic component of PRC2 that catalyzes the tri-methylation of H3K27 resulting in the repression of target genes. We and others have shown that EZH2 is elevated in aggressive forms of prostate and breast cancer as well as many other solid tumors. MMSET is another HMTase that mediates H3K36 dimethylation that is associated with active transcription and like EZH2, MMSET is overexpressed in diverse solid tumors. Using next generation sequencing-based approach, we found that the EZH2-MMSET HMTase axis is coordinated by a microRNA network and that the oncogenic functions of EZH2 require MMSET activity. Together, these results suggest that the EZH2-MMSET HMTase axis coordinately functions as a master regulator of transcriptional repression, activation, and oncogenesis and may represent an attractive therapeutic target in cancer. (Mol Cell. 2012 Nov 13. doi:pii: S1097-2765(12)00861-1).

Expressed pseudogenes in cancer

Pseudogenes are a class of non-coding RNA transcripts that are dysfunctional relatives of known functional genes that have lost their protein coding ability and often not expressed. Aberrant expression of several functional non-coding RNA in cancer has been previously described, however genome-wide expression of pseudogenes had not been reported for any cancer type. We developed a pseudogene expression pipeline to analyze a large compendium of paired-end next generation sequencing (RNASeq) data generated from 293 samples, comprising 13 different epithelial cancers. A number of pseudogene candidates displaying cancer-specific expression for various cancer types were identified. This study is the first large-scale analysis of pseudogene expression in human cancer using transcriptome sequencing data. (Cell. 2012 Jun 22;149(7):1622-34)

lncRNA PCAT-1 implicated in prostate cancer

Long noncoding RNAs (lncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease and to reveal uncharacterized aspects of tumor biology.  In a recent study by our group, we applied high-throughput transcriptome sequencing (RNA-Seq) on a large cohort of >100 prostate cancer samples in order to define the RNA landscape in this disease.  We found that nearly 20% of polyadenylated RNA species represent unannotated transcripts, including 121 intergenic, unannotated lncRNAs whose aberrant expression in prostate cancer rank highly as some of the most differentially-expressed RNAs in this disease.  We further validated and characterized several novel lncRNAs, including one prostate-specific lncRNA termed PCAT-1 (Prostate Cancer Associated Transcript-1).  PCAT-1 upregulation marked a set of aggressive prostate cancers and stratified against EZH2, a prostate cancer oncogene also upregulated in a set of aggressive cancers.  In vitro, PCAT-1 expression was necessary for cancer cell proliferation and overexpression of PCAT-1 was sufficient to increase benign prostate cell proliferation.  Mechanistic studies revealed that PCAT-1 is a transcriptional repressor of several known tumor suppressor genes, including BRCA2.  (Nat Biotechnol. 2011 Jul 31;29(8):742- 9). 

ERG Inhibitory Peptide

Transcription factors are proteins that modulate gene expression in a cell and play a key role in the development of a number of cancers, and therapeutically targeting them has remained a challenge. In prostate cancer, the recurrent gene fusion between the ETS transcription factor ERG and TMPRSS2 plays a critical role in prostate oncogenesis. We identified a consensus peptide from a phage display library that interacts specifically with the DNA binding domain of ERG. The interactive interface was mapped to 9-amino acid residues the ETS domain that is critical for ERG activity as a transcription factor. X-ray crystallography confirmed that the interaction residues are accessible for interaction. These peptides were found to efficiently disrupt ERG-mediated protein-protein interactions, transcription, DNA damage, and cell invasion. Furthermore, an inhibitory “retroinverso peptidomimetic” peptide suppressed tumor growth, intravasation, and metastasis in an animal model. Taken together, our results suggest that transcription factors have specific residues that are important for protein-protein interactions that may be amenable to therapeutic targeting.



Print

Affiliations: