Sunita Shankar

Genomic loss of microRNA-101 leads to overexpression of histone methyltransferase EZH2 in cancer.

Enhancer of zeste homolog 2 (EZH2) is a mammalian histone methyltransferase that contributes to the epigenetic silencing of target genes and regulates the survival and metastasis of cancer cells. EZH2 is overexpressed in aggressive solid tumors by mechanisms that remain unclear. Here we show that the expression and function of EZH2 in cancer cell lines are inhibited by microRNA-101 (miR-101). Analysis of human prostate tumors revealed that miR-101 expression decreases during cancer progression, paralleling an increase in EZH2 expression. One or both of the two genomic loci encoding miR-101 were somatically lost in 37.5% of clinically localized prostate cancer cells (6 of 16) and 66.7% of metastatic disease cells (22 of 33). We propose that the genomic loss of miR-101 in cancer leads to overexpression of EZH2 and concomitant dysregulation of epigenetic pathways, resulting in cancer progression.

An Integrated Network of Androgen Receptor, Polycomb, and TMPRSS2-ERG Gene Fusions in Prostate Cancer Progression

Chromosomal rearrangements fusing the androgen-regulated gene TMPRSS2 to the oncogenic ETS transcription factor ERG occur in approximately 50% of prostate cancers, but how the fusion products regulate prostate cancer remains unclear. Using chromatin immunoprecipitation coupled with massively parallel sequencing, we found that ERG disrupts androgen receptor (AR) signaling by inhibiting AR expression, binding to and inhibiting AR activity at gene-specific loci, and inducing repressive epigenetic programs via direct activation of the H3K27 methyltransferase EZH2, a Polycomb group protein. These findings provide a working model in which TMPRSS2-ERG plays a critical role in cancer progression by disrupting lineage-specific differentiation of the prostate and potentiating the EZH2-mediated dedifferentiation program.

Functionally recurrent rearrangements of the MAST kinase and Notch gene families in breast cancer.

Breast cancer is a heterogeneous disease that has a wide range of molecular aberrations and clinical outcomes. Here we used paired-end transcriptome sequencing to explore the landscape of gene fusions in a panel of breast cancer cell lines and tissues. We observed that individual breast cancers have a variety of expressed gene fusions. We identified two classes of recurrent gene rearrangements involving genes encoding microtubule-associated serine-threonine kinase (MAST) and members of the Notch family. Both MAST and Notch-family gene fusions have substantial phenotypic effects in breast epithelial cells. Breast cancer cell lines harboring Notch gene rearrangements are uniquely sensitive to inhibition of Notch signaling, and overexpression of MAST1 or MAST2 gene fusions has a proliferative effect both in vitro and in vivo. These findings show that recurrent gene rearrangements have key roles in subsets of carcinomas and suggest that transcriptome sequencing could identify individuals with rare, targetable gene fusions.

Deep sequencing reveals distinct patterns of DNA methylation in prostate cancer

Beginning with precursor lesions, aberrant DNA methylation marks the entire spectrum of prostate cancer progression. We mapped the global DNA methylation patterns in select prostate tissues and cell lines using MethylPlex-next-generation sequencing (M-NGS). Hidden Markov model-based next-generation sequence analysis identified ∼68,000 methylated regions per sample. While global CpG island (CGI) methylation was not differential between benign adjacent and cancer samples, overall promoter CGI methylation significantly increased from ~12.6% in benign samples to 19.3% and 21.8% in localized and metastatic cancer tissues, respectively (P-value < 2 × 10(-16)). We found distinct patterns of promoter methylation around transcription start sites, where methylation occurred not only on the CGIs, but also on flanking regions and CGI sparse promoters. Among the 6691 methylated promoters in prostate tissues, 2481 differentially methylated regions (DMRs) are cancer-specific, including numerous novel DMRs. A novel cancer-specific DMR in the WFDC2 promoter showed frequent methylation in cancer (17/22 tissues, 6/6 cell lines), but not in the benign tissues (0/10) and normal PrEC cells. Integration of LNCaP DNA methylation and H3K4me3 data suggested an epigenetic mechanism for alternate transcription start site utilization, and these modifications segregated into distinct regions when present on the same promoter. Finally, we observed differences in repeat element methylation, particularly LINE-1, between ERG gene fusion-positive and -negative cancers, and we confirmed this observation using pyrosequencing on a tissue panel. This comprehensive methylome map will further our understanding of epigenetic regulation in prostate cancer progression.

Characterization of KRAS Rearrangements in Metastatic Prostate Cancer

UNLABELLED Using an integrative genomics approach called amplification breakpoint ranking and assembly analysis, we nominated KRAS as a gene fusion with the ubiquitin-conjugating enzyme UBE2L3 in the DU145 cell line, originally derived from prostate cancer metastasis to the brain. Interestingly, analysis of tissues revealed that 2 of 62 metastatic prostate cancers harbored aberrations at the KRAS locus. In DU145 cells, UBE2L3-KRAS produces a fusion protein, a specific knockdown of which attenuates cell invasion and xenograft growth. Ectopic expression of the UBE2L3-KRAS fusion protein exhibits transforming activity in NIH 3T3 fibroblasts and RWPE prostate epithelial cells in vitro and in vivo. In NIH 3T3 cells, UBE2L3-KRAS attenuates MEK/ERK signaling, commonly engaged by oncogenic mutant KRAS, and instead signals via AKT and p38 mitogen-activated protein kinase (MAPK) pathways. This is the first report of a gene fusion involving the Ras family, suggesting that this aberration may drive metastatic progression in a rare subset of prostate cancers. SIGNIFICANCE This is the first description of an oncogenic gene fusion of KRAS, one of the most studied proto-oncogenes. KRAS rearrangement may represent the driving mutation in a rare subset of metastatic prostate cancers, emphasizing the importance of RAS-RAF-MAPK signaling in this disease.

Characterization of bone metastases from rapid autopsies of prostate cancer patients

Purpose: Bone is the most common metastatic site for prostate cancer, and osseous metastases are the leading cause of morbidity from this disease. Recent autopsy studies prove that 100% of men who die of prostate cancer have bone involvement. Understanding the biology of prostate cancer and its evolution to an incurable androgen-independent phenotype requires an understanding of the genetic and cellular alterations that lead to the seeding and proliferation of tumor foci in bone, as well as the microenvironment in which these metastases arise. No intensive studies, however, have been conducted on osseous metastatic tissues from patients with metastatic prostate cancer due to lack of access to such tissues for profiling and other research. Experimental Design: We show, for the first time, a reproducible methodology to obtain high quality clinical tumor tissues metastatic to the bone. This technique allowed the procurement of viable metastatic tumor tissue from involved bones in 13 recent autopsies conducted at the University of Michigan and analyzed the gene expression of these tissues using real-time PCR and microarrays. Results: We present here the discovery of nonossified bone metastases from multiple patients with advanced prostate cancer and their subsequent characterization and comparison to nonosseous metastases from the same patients. Conclusion: This represents a versatile and practical approach that may be employed to characterize the steps in metastasis and the phenotypic characteristics of osseous metastasis of prostate cancer and to profile RNA, DNA, and cDNA from tumor samples metastatic to the bone. Clin Cancer Res; 17(12); 3924–32. ©2011 AACR.

KRAS protein stability is regulated through SMURF2: UBCH5 complex-mediated β-TrCP1 degradation.

Attempts to target mutant KRAS have been unsuccessful. Here, we report the identification of Smad ubiquitination regulatory factor 2 (SMURF2) and UBCH5 as a critical E3:E2 complex maintaining KRAS protein stability. Loss of SMURF2 either by small interfering RNA/short hairpin RNA (siRNA/shRNA) or by overexpression of a catalytically inactive mutant causes KRAS degradation, whereas overexpression of wild-type SMURF2 enhances KRAS stability. Importantly, mutant KRAS is more susceptible to SMURF2 loss where protein half-life decreases from >12 hours in control siRNA-treated cells to <3 hours on Smurf2 silencing, whereas only marginal differences were noted for wild-type protein. This loss of mutant KRAS could be rescued by overexpressing a siRNA-resistant wild-type SMURF2. Our data further show that SMURF2 monoubiquitinates UBCH5 at lysine 144 to form an active complex required for efficient degradation of a RAS-family E3, β-transducing repeat containing protein 1 (β-TrCP1). Conversely, β-TrCP1 is accumulated on SMURF2 loss, leading to increased KRAS degradation. Therefore, as expected, β-TrCP1 knockdown following Smurf2 siRNA treatment rescues mutant KRAS loss. Further, we identify two conserved proline (P) residues in UBCH5 critical for SMURF2 interaction; mutation of either of these P to alanine also destabilizes KRAS. As a proof of principle, we demonstrate that Smurf2 silencing reduces the clonogenic survival in vitro and prolongs tumor latency in vivo in cancer cells including mutant KRAS-driven tumors. Taken together, we show that SMURF2:UBCH5 complex is critical in maintaining KRAS protein stability and propose that targeting such complex may be a unique strategy to degrade mutant KRAS to kill cancer cells.

KRAS Engages AGO2 to Enhance Cellular Transformation.

Oncogenic mutations in RAS provide a compelling yet intractable therapeutic target. Using co-immunoprecipitation mass spectrometry, we uncovered an interaction between RAS and Argonaute 2 (AGO2). Endogenously, RAS and AGO2 co-sediment and co-localize in the endoplasmic reticulum. The AGO2 N-terminal domain directly binds the Switch II region of KRAS, agnostic of nucleotide (GDP/GTP) binding. Functionally, AGO2 knockdown attenuates cell proliferation in mutant KRAS-dependent cells and AGO2 overexpression enhances KRAS(G12V)-mediated transformation. Using AGO2-/- cells, we demonstrate that the RAS-AGO2 interaction is required for maximal mutant KRAS expression and cellular transformation. Mechanistically, oncogenic KRAS attenuates AGO2-mediated gene silencing. Overall, the functional interaction with AGO2 extends KRAS function beyond its canonical role in signaling.

An Essential Role for Argonaute 2 in EGFR-KRAS Signaling in Pancreatic Cancer Development

KRAS and EGFR are known essential mediators of pancreatic cancer development. In addition, KRAS and EGFR have both been shown to interact with and perturb the function of Argonaute 2 (AGO2), a key regulator of RNA-mediated gene silencing. Here, we employed a genetically engineered mouse model of pancreatic cancer to define the effects of conditional loss of AGO2 in KRASG12D-driven pancreatic cancer. Genetic ablation of AGO2 does not interfere with development of the normal pancreas or KRASG12D-driven early precursor pancreatic intraepithelial neoplasia (PanIN) lesions. Remarkably, however, AGO2 is required for progression from early to late PanIN lesions, development of pancreatic ductal adenocarcinoma (PDAC), and metastasis. AGO2 ablation permits PanIN initiation driven by the EGFR-RAS axis, but rather than progressing to PDAC, these lesions undergo profound oncogene-induced senescence (OIS). Loss of Trp53 (p53) in this model obviates the requirement of AGO2 for PDAC development. In mouse and human pancreatic tissues, increased expression of AGO2 and elevated co-localization with RAS at the plasma membrane is associated with PDAC progression. Furthermore, phosphorylation of AGO2Y393 by EGFR disrupts the interaction of wild-type RAS with AGO2 at the membrane, but does not affect the interaction of mutant KRAS with AGO2. ARS-1620, a G12C-specific inhibitor, disrupts the KRASG12C-AGO2 interaction specifically in pancreatic cancer cells harboring this mutant, demonstrating that the oncogenic KRAS-AGO2 interaction can be pharmacologically targeted. Taken together, our study supports a biphasic model of pancreatic cancer development: an AGO2-independent early phase of PanIN formation reliant on EGFR-RAS signaling, and an AGO2-dependent phase wherein the mutant KRAS-AGO2 interaction is critical to prevent OIS in PanINs and allow progression to PDAC.

Abstract LB-058: Novel interactions of the RAS oncoprotein

Approximately 30% of all cancers harbor activating mutations in the RAS family of small GTPase proteins, making it one of the most common oncogenic aberrations in humans. Normal RAS proteins (H, K or N-RAS) localize to the inner cell membrane and transduce extracellular growth signals by cycling between an “active” GTP-bound state and “inactive” GDP-bound state, through interactions with various “GTPase activating proteins” (GAPs) that promote RAS mediated GTP hydrolysis. Oncogenic mutants of RAS lose their catalytic activity or association with the GAP proteins, resulting in constitutively active GTP-bound state that signals through direct interactions with effector kinases like RAF and PI3K and activate the MEK/ERK and/or Akt, leading to activation of hallmark cancer pathways including growth factor independence, uncontrolled cell proliferation, evasion of apoptosis and immune responses, increased metabolism as well as metastases. Although RAS is the most frequently mutated gene driving multifarious pathways of oncogenesis, our knowledge of protein interactions involving RAS proteins have been largely limited to RAS binding domains in RAF/PI3K/RalGDS. Targeting mutant RAS proteins or its direct effectors, or pathways activated by RAS effectors remains a challenging endeavor for treating RAS driven cancers. Towards the goal of a thorough understanding of RAS biology through a comprehensive identification of its interactors, we performed IP-Mass Spectrometric analysis of pan-RAS immunoprecipitates from multiple cell lines. Interestingly in our experiments, apart from the well-known interactor RAF, we found evidence of several novel RAS interacting proteins, including many with DNA and RNA binding motifs. Our study validates these findings through cell-free protein interaction analyses and explores the possible biological effects of these novel RAS interactions in mutant KRAS driven cellular transformation. Note: This abstract was not presented at the meeting. Citation Format: Sunita Shankar, Rohit Malik, Vishal Kothari, Yasuyuki Hosono, Sethuramasundaram Pitchiaya, Shanker Kalyana-Sundaram, Anastasia Yocum, June Escara-Wilke, Harika Gundlapalli, Krishnapriya Chinnaswamy, Matthew Shuler, Anton Poliakov, Xiaoju Wang, Vishalakshi Krishnan, Yasmine White, Ari Firestone, Xuhong Cao, Saravana M. Dhanasekaran, Jeanne Stuckey, Gideon Bollag, Kevin Shannon, Nils G. Walter, Chandan Kumar-Sinha, Arul Chinnaiyan. Novel interactions of the RAS oncoprotein. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-058. doi:10.1158/1538-7445.AM2015-LB-058