Jason A. Somarelli

The role of epithelial plasticity in prostate cancer dissemination and treatment resistance

Nearly 30,000 men die annually in the USA of prostate cancer, nearly uniformly from metastatic dissemination. Despite recent advances in hormonal, immunologic, bone-targeted, and cytotoxic chemotherapies, treatment resistance and further dissemination are inevitable in men with metastatic disease. Emerging data suggests that the phenomenon of epithelial plasticity, encompassing both reversible mesenchymal transitions and acquisition of stemness traits, may underlie this lethal biology of dissemination and treatment resistance. Understanding the molecular underpinnings of this cellular plasticity from preclinical models of prostate cancer and from biomarker studies of human metastatic prostate cancer has provided clues to novel therapeutic approaches that may delay or prevent metastatic disease and lethality over time. This review will discuss the preclinical and clinical evidence for epithelial plasticity in this rapidly changing field and relate this to clinical phenotype and resistance in prostate cancer while suggesting novel therapeutic approaches.

Alternative splicing in multiple sclerosis and other autoimmune diseases.

Alternative splicing is a general mechanism for regulating gene expression that affects the RNA products of more than 90% of human genes. Not surprisingly, alternative splicing is observed among gene products of metazoan immune systems, which have evolved to efficiently recognize pathogens and discriminate between “self” and “non-self”, and thus need to be both diverse and flexible. In this review we focus on the specific interface between alternative splicing and autoimmune diseases, which result from a malfunctioning of the immune system and are characterized by the inappropriate reaction to self-antigens. Despite the widespread recognition of alternative splicing as one of the major regulators of gene expression, the connections between alternative splicing and autoimmunity have not been apparent. We summarize recent findings connecting splicing and autoimmune disease, and attempt to find common patterns of splicing regulation that may advance our understanding of autoimmune diseases and open new avenues for therapy.

Cellular migration and invasion uncoupled: Increased migration is not an inexorable consequence of epithelial-to-mesenchymal transition

ABSTRACT Metastatic dissemination requires carcinoma cells to detach from the primary tumor and invade through the basement membrane. To acquire these characteristics, epithelial tumor cells undergo epithelial-to-mesenchymal transitions (EMT), whereby cells lose polarity and E-cadherin-mediated cell-cell adhesion. Post-EMT cells have also been shown, or assumed, to be more migratory; however, there have been contradictory reports on an immortalized human mammary epithelial cell line (HMLE) that underwent EMT. In the context of carcinoma-associated EMT, it is not yet clear whether the change in migration and invasion must be positively correlated during EMT or whether enhanced migration is a necessary consequence of having undergone EMT. Here, we report that pre-EMT rat prostate cancer (PC) and HMLE cells are more migratory than their post-EMT counterparts. To determine a mechanism for increased epithelial cell migration, gene expression analysis was performed and revealed an increase in epidermal growth factor receptor (EGFR) expression in pre-EMT cells. Indeed, inhibition of EGFR in PC epithelial cells slowed migration. Importantly, while post-EMT PC and HMLE cell lines are less migratory, both remain invasive in vitro and, for PC cells, in vivo. Our study demonstrates that enhanced migration is not a phenotypic requirement of EMT, and migration and invasion can be uncoupled during carcinoma-associated EMT.

EMT and MET: necessary or permissive for metastasis?

Epithelial‐to‐mesenchymal transition (EMT) and its reverse mesenchymal‐to‐epithelial transition (MET) have been suggested to play crucial roles in metastatic dissemination of carcinomas. These phenotypic transitions between states are not binary. Instead, carcinoma cells often exhibit a spectrum of epithelial/mesenchymal phenotype(s). While epithelial/mesenchymal plasticity has been observed preclinically and clinically, whether any of these phenotypic transitions are indispensable for metastatic outgrowth remains an unanswered question. Here, we focus on epithelial/mesenchymal plasticity in metastatic dissemination and propose alternative mechanisms for successful dissemination and metastases beyond the traditional EMT/MET view. We highlight multiple hypotheses that can help reconcile conflicting observations, and outline the next set of key questions that can offer valuable insights into mechanisms of metastasis in multiple tumor models.

Fluorescence-based alternative splicing reporters for the study of epithelial plasticity in vivo

Alternative splicing generates a vast diversity of protein isoforms from a limited number of protein-coding genes, with many of the isoforms possessing unique, and even contrasting, functions. Fluorescence-based splicing reporters have the potential to facilitate studies of alternative splicing at the single-cell level and can provide valuable information on phenotypic transitions in almost real time. Fibroblast growth factor receptor 2 (FGFR2) pre-mRNA is alternatively spliced to form the epithelial-specific and mesenchymal-specific IIIb and IIIc isoforms, respectively, which are useful markers of epithelial-mesenchymal transitions (EMT). We have used our knowledge of FGFR2 splicing regulation to develop a fluorescence-based reporter system to visualize exon IIIc regulation in vitro and in vivo. Here we show the application of this reporter system to the study of EMT in vitro in cell culture and in vivo in transgenic mice harboring these splicing constructs. In explant studies, the reporters revealed that FGFR2 isoform switching is not required for keratinocyte migration during cutaneous wound closure. Our results demonstrate the value of the splicing reporters as tools to study phenotypic transitions and cell fates at single cell resolution. Moreover, our data suggest that keratinocytes migrate efficiently in the absence of a complete EMT.

Mesenchymal-Epithelial Transition in Sarcomas Is Controlled by the Combinatorial Expression of MicroRNA 200s and GRHL2.

ABSTRACT Phenotypic plasticity involves a process in which cells transiently acquire phenotypic traits of another lineage. Two commonly studied types of phenotypic plasticity are epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET). In carcinomas, EMT drives invasion and metastatic dissemination, while MET is proposed to play a role in metastatic colonization. Phenotypic plasticity in sarcomas is not well studied; however, there is evidence that a subset of sarcomas undergo an MET-like phenomenon. While the exact mechanisms by which these transitions occur remain largely unknown, it is likely that some of the same master regulators that drive EMT and MET in carcinomas also act in sarcomas. In this study, we combined mathematical models with bench experiments to identify a core regulatory circuit that controls MET in sarcomas. This circuit comprises the microRNA 200 (miR-200) family, ZEB1, and GRHL2. Interestingly, combined expression of miR-200s and GRHL2 further upregulates epithelial genes to induce MET. This effect is phenocopied by downregulation of either ZEB1 or the ZEB1 cofactor, BRG1. In addition, an MET gene expression signature is prognostic for improved overall survival in sarcoma patients. Together, our results suggest that a miR-200, ZEB1, GRHL2 gene regulatory network may drive sarcoma cells to a more epithelial-like state and that this likely has prognostic relevance.

Snail promotes resistance to enzalutamide through regulation of androgen receptor activity in prostate cancer

Treatment with androgen-targeted therapies can induce upregulation of epithelial plasticity pathways. Epithelial plasticity is known to be important for metastatic dissemination and therapeutic resistance. The goal of this study is to elucidate the functional consequence of induced epithelial plasticity on AR regulation during disease progression to identify factors important for treatment-resistant and metastatic prostate cancer. We pinpoint the epithelial plasticity transcription factor, Snail, at the nexus of enzalutamide resistance and prostate cancer metastasis both in preclinical models of prostate cancer and in patients. In patients, Snail expression is associated with Gleason 9-10 high-risk disease and is strongly overexpressed in metastases as compared to localized prostate cancer. Snail expression is also elevated in enzalutamide-resistant prostate cancer cells compared to enzalutamide-sensitive cells, and downregulation of Snail re-sensitizes enzalutamide-resistant cells to enzalutamide. While activation of Snail increases migration and invasion, it is also capable of promoting enzalutamide resistance in enzalutamide-sensitive cells. This Snail-mediated enzalutamide resistance is a consequence of increased full-length AR and AR-V7 expression and nuclear localization. Downregulation of either full-length AR or AR-V7 re-sensitizes cells to enzalutamide in the presence of Snail, thus connecting Snail-induced enzalutamide resistance directly to AR biology. Finally, we demonstrate that Snail is capable of mediating-resistance through AR even in the absence of AR-V7. These findings imply that increased Snail expression during progression to metastatic disease may prime cells for resistance to AR-targeted therapies by promoting AR activity in prostate cancer.

Survival outcomes in cancer patients predicted by a partial EMT gene expression scoring metric

Metastasis is a significant contributor to morbidity and mortality for many cancer patients and remains a major obstacle for effective treatment. In many tissue types, metastasis is fueled by the epithelial-to-mesenchymal transition (EMT)-a dynamic process characterized by phenotypic and morphologic changes concomitant with increased migratory and invasive potential. Recent experimental and theoretical evidence suggests that cells can be stably halted en route to EMT in a hybrid E/M phenotype. Cells in this phenotype tend to move collectively, forming clusters of circulating tumor cells that are key tumor-initiating agents. Here, we developed an inferential model built on the gene expression of multiple cancer subtypes to devise an EMT metric that characterizes the degree to which a given cell line exhibits hybrid E/M features. Our model identified drivers and fine-tuners of epithelial-mesenchymal plasticity and recapitulated the behavior observed in multiple in vitro experiments across cancer types. We also predicted and experimentally validated the hybrid E/M status of certain cancer cell lines, including DU145 and A549. Finally, we demonstrated the relevance of predicted EMT scores to patient survival and observed that the role of the hybrid E/M phenotype in characterizing tumor aggressiveness is tissue and subtype specific. Our algorithm is a promising tool to quantify the EMT spectrum, to investigate the correlation of EMT score with cancer treatment response and survival, and to provide an important metric for systematic clinical risk stratification and treatment. Cancer Res; 77(22); 6415-28. ©2017 AACR.

Distinct routes to metastasis: plasticity-dependent and plasticity-independent pathways.

The cascade that culminates in macrometastases is thought to be mediated by phenotypic plasticity, including epithelial–mesenchymal and mesenchymal–epithelial transitions (EMT and MET). Although there is substantial support for the role of EMT in driving cancer cell invasion and dissemination, much less is known about the importance of MET in the later steps of metastatic colonization. We created novel reporters, which integrate transcriptional and post-transcriptional regulation, to test whether MET is required for metastasis in multiple in vivo cancer models. In a model of carcinosarcoma, metastasis occurred via an MET-dependent pathway; however, in two prostate carcinoma models, metastatic colonization was MET independent. Our results provide evidence for both MET-dependent and MET-independent metastatic pathways.

Whole Genomic Copy Number Alterations in Circulating Tumor Cells from Men with Abiraterone or Enzalutamide-Resistant Metastatic Castration-Resistant Prostate Cancer

Purpose: Beyond enumeration, circulating tumor cells (CTCs) can provide genetic information from metastatic cancer that may facilitate a greater understanding of tumor biology and enable a precision medicine approach. Experimental Design: CTCs and paired leukocytes from men with metastatic castration-resistant prostate cancer (mCRPC) were isolated from blood through red cell lysis, CD45 depletion, and flow sorting based on EpCAM/CD45 expression. We next performed whole genomic copy number analysis of CTCs and matched patient leukocytes (germline) using array-based comparative genomic hybridization (aCGH) from 16 men with mCRPC, including longitudinal and sequential aCGH analyses of CTCs in the context of enzalutamide therapy. Results: All patients had mCRPC and primary or acquired resistance to abiraterone acetate or enzalutamide. We compiled copy gains and losses, with a particular focus on those genes highly implicated in mCRPC progression and previously validated as being aberrant in metastatic tissue samples and genomic studies of reference mCRPC datasets. Genomic gains in >25% of CTCs were observed in AR, FOXA1, ABL1, MET, ERG, CDK12, BRD4, and ZFHX3, while common genomic losses involved PTEN, ZFHX3, PDE4DIP, RAF1, and GATA2. Analysis of aCGH in a sample with sequential enzalutamide-resistant visceral progression showed acquired loss of AR amplification concurrent with gain of MYCN, consistent with evolution toward a neuroendocrine-like, AR-independent clone. Conclusions: Genomic analysis of pooled CTCs in men with mCRPC suggests a reproducible, but highly complex molecular profile that includes common aberrations in AR, ERG, c-MET, and PI3K signaling during mCRPC progression, which may be useful for predictive biomarker development. Clin Cancer Res; 23(5); 1346–57. ©2016 AACR.