Michael E. Feigin

Organoid Models of Human and Mouse Ductal Pancreatic Cancer

Pancreatic cancer is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable methods to identify and interrogate pathways involved in pancreatic tumorigenesis are urgently needed. We established organoid models from normal and neoplastic murine and human pancreas tissues. Pancreatic organoids can be rapidly generated from resected tumors and biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Orthotopically transplanted neoplastic organoids recapitulate the full spectrum of tumor development by forming early-grade neoplasms that progress to locally invasive and metastatic carcinomas. Due to their ability to be genetically manipulated, organoids are a platform to probe genetic cooperation. Comprehensive transcriptional and proteomic analyses of murine pancreatic organoids revealed genes and pathways altered during disease progression. The confirmation of many of these protein changes in human tissues demonstrates that organoids are a facile model system to discover characteristics of this deadly malignancy.

Polarity proteins regulate mammalian cell-cell junctions and cancer pathogenesis

The epithelial cells of multicellular organisms form highly organized tissues specialized for the tasks of protection, secretion, and absorption, all of which require tight regulation of the core processes of cell polarity and tissue architecture. Disruption of these core processes is a critical feature of epithelial tumors. Cell polarity and tissue architecture are intimately linked, as proteins controlling cell shape are also responsible for proper localization and assembly of cell-cell junctions and three-dimensional tissue organization. The extracellular matrix underlying epithelial tissues supports tissue architecture and suppresses malignant growth through regulation of cell adhesion and activation of protective signaling cascades. Emerging evidence is uncovering the mechanisms by which polarity pathways alter the way epithelial cells organize and interact with the tissue microenvironment to promote aberrant growth and invasion during tumorigenesis. We discuss how cell polarity pathways regulate cell-cell junctions and highlight the new insights gained by investigating the role played by polarity pathways during the transformation of epithelial cells.

Mislocalization of the Cell Polarity Protein Scribble Promotes Mammary Tumorigenesis and Is Associated with Basal Breast Cancer

Scribble (SCRIB) localizes to cell-cell junctions and regulates establishment of epithelial cell polarity. Loss of expression of SCRIB functions as a tumor suppressor in Drosophila and mammals; conversely, overexpression of SCRIB promotes epithelial differentiation in mammals. Here, we report that SCRIB is frequently amplified, mRNA overexpressed, and protein is mislocalized from cell-cell junctions in human breast cancers. High levels of SCRIB mRNA are associated with poor clinical prognosis, identifying an unexpected role for SCRIB in breast cancer. We find that transgenic mice expressing a SCRIB mutant [Pro 305 to Leu (P305L)] that fails to localize to cell-cell junctions, under the control of the mouse mammary tumor virus long terminal repeat promoter, develop multifocal hyperplasia that progresses to highly pleomorphic and poorly differentiated tumors with basal characteristics. SCRIB interacts with phosphatase and tensin homolog (PTEN) and the expression of P305L, but not wild-type SCRIB, promotes an increase in PTEN levels in the cytosol. Overexpression of P305L, but not wild-type SCRIB, activates the Akt/mTOR/S6K signaling pathway. Human breast tumors overexpressing SCRIB have high levels of S6K but do not harbor mutations in PTEN or PIK3CA, identifying SCRIB amplification as a mechanism of activating PI3K signaling in tumors without mutations in PIK3CA or PTEN. Thus, we demonstrate that high levels of mislocalized SCRIB functions as a neomorph to promote mammary tumorigenesis by affecting subcellular localization of PTEN and activating an Akt/mTOR/S6kinase signaling pathway.

ErbB receptors and cell polarity: New pathways and paradigms for understanding cell migration and invasion

The ErbB family of receptor tyrosine kinases is involved in initiation and progression of a number of human cancers, and receptor activation or overexpression correlates with poor patient survival. Research over the past two decades has elucidated the molecular mechanisms underlying ErbB-induced tumorigenesis, which has resulted in the development of effective targeted therapies. ErbB-induced signal transduction cascades regulate a wide variety of cell processes, including cell proliferation, apoptosis, cell polarity, migration and invasion. Within tumors, disruption of these core processes, through cooperative oncogenic lesions, results in aggressive, metastatic disease. This review will focus on the ErbB signaling networks that regulate migration and invasion and identify a potential role for cell polarity pathways during cancer progression.

RGS19 regulates Wnt–β-catenin signaling through inactivation of Gαo

The Wnt–β-catenin pathway controls numerous cellular processes, including differentiation, cell-fate decisions and dorsal-ventral polarity in the developing embryo. Heterotrimeric G-proteins are essential for Wnt signaling, and regulator of G-protein signaling (RGS) proteins are known to act at the level of G-proteins. The functional role of RGS proteins in the Wnt–β-catenin pathway was investigated in mouse F9 embryonic teratocarcinoma cells. RGS protein expression was investigated at the mRNA level, and each RGS protein identified was overexpressed and tested for the ability to regulate the canonical Wnt pathway. Expression of RGS19 specifically was found to attenuate Wnt-responsive gene transcription in a time- and dose-dependent manner, to block cytosolic β-catenin accumulation and Dishevelled3 (Dvl3) phosphorylation in response to Wnt3a and to inhibit Wnt-induced formation of primitive endoderm (PE). Overexpression of a constitutively active mutant of Gαo rescued the inhibition of Lef-Tcf-sensitive gene transcription caused by RGS19. By contrast, expression of RGS19 did not inhibit activation of Lef-Tcf gene transcription when induced in response to Dvl3 expression. However, knockdown of RGS19 by siRNA suppressed canonical Wnt signaling, suggesting a complex role for RGS19 in regulating the ability of Wnt3a to signal to the level of β-catenin and gene transcription.

G-protein–coupled receptor GPR161 is overexpressed in breast cancer and is a promoter of cell proliferation and invasion

Significance Recent advances in DNA sequencing and bioinformatics tools have allowed the large-scale characterization of genetic alterations in human tumors. We exploited genomics data to identify commonly occurring alterations in G-protein–coupled receptors (GPCRs) in triple-negative breast cancer (TNBC), a subset of breast cancer with poor prognosis and lacking effective targeted therapy. We have discovered that the orphan GPCR, G-protein–coupled receptor 161 (GPR161), is overexpressed specifically in TNBC and correlates with poor prognosis. We identify GPR161 as a regulator of mammary epithelial cell proliferation and invasion in a mammalian target of rapamycin signaling-pathway–dependent manner. Down-regulation of GPR161 in a TNBC-derived cell line impairs cell growth, suggesting that GPR161 is a drug target for breast cancer. Triple-negative breast cancer (TNBC) accounts for 20% of breast cancer in women and lacks an effective targeted therapy. Therefore, finding common vulnerabilities in these tumors represents an opportunity for more effective treatment. Despite the growing appreciation of G-protein–coupled receptor (GPCR)-mediated signaling in cancer pathogenesis, very little is known about the role GPCRs play in TNBC. Using genomic information of human breast cancer, we have discovered that the orphan GPCR, G-protein–coupled receptor 161 (GPR161) is overexpressed specifically in TNBC and correlates with poor prognosis. Knockdown of GPR161 impairs proliferation of human basal breast cancer cell lines. Overexpression of GPR161 in human mammary epithelial cells increases cell proliferation, migration, intracellular accumulation of E-cadherin, and formation of multiacinar structures in 3D culture. GPR161 forms a signaling complex with the scaffold proteins β-arrestin 2 and Ile Gln motif containing GTPase Activating Protein 1, a regulator of mammalian target of rapamycin complex 1 and E-cadherin. Consistently, GPR161 amplified breast tumors and cells overexpressing GPR161 activate mammalian target of rapamycin signaling and decrease Ile Gln motif containing GTPase Activating Protein 1 phosphorylation. Thus, we identify the orphan GPCR, GPR161, as an important regulator and a potential drug target for TNBC.

Dysregulation of cell polarity proteins synergize with oncogenes or the microenvironment to induce invasive behavior in epithelial cells.

Changes in expression and localization of proteins that regulate cell and tissue polarity are frequently observed in carcinoma. However, the mechanisms by which changes in cell polarity proteins regulate carcinoma progression are not well understood. Here, we report that loss of polarity protein expression in epithelial cells primes them for cooperation with oncogenes or changes in tissue microenvironment to promote invasive behavior. Activation of ErbB2 in cells lacking the polarity regulators Scribble, Dlg1 or AF-6, induced invasive properties. This cooperation required the ability of ErbB2 to regulate the Par6/aPKC polarity complex. Inhibition of the ErbB2-Par6 pathway was sufficient to block ErbB2-induced invasion suggesting that two polarity hits may be needed for ErbB2 to promote invasion. Interestingly, in the absence of ErbB2 activation, either a combined loss of two polarity proteins, or exposure of cells lacking one polarity protein to cytokines IL-6 or TNFα induced invasive behavior in epithelial cells. We observed the invasive behavior only when cells were plated on a stiff matrix (Matrigel/Collagen-1) and not when plated on a soft matrix (Matrigel alone). Cells lacking two polarity proteins upregulated expression of EGFR and activated Akt. Inhibition of Akt activity blocked the invasive behavior identifying a mechanism by which loss of polarity promotes invasion of epithelial cells. Thus, we demonstrate that loss of polarity proteins confers phenotypic plasticity to epithelial cells such that they display normal behavior under normal culture conditions but display aggressive behavior in response to activation of oncogenes or exposure to cytokines.

Harnessing the genome for characterization of G‐protein coupled receptors in cancer pathogenesis

G‐protein coupled receptors (GPCRs) mediate numerous physiological processes and represent the targets for a vast array of therapeutics for diseases ranging from depression to hypertension to reflux. Despite the recognition that GPCRs can act as oncogenes and tumour suppressors by regulating oncogenic signalling networks, few drugs targeting GPCRs are utilized in cancer therapy. Recent large‐scale genome‐wide analyses of multiple human tumours have uncovered novel GPCRs altered in cancer. However, work aiming to determine which GPCRs from these lists are the drivers of tumourigenesis, and hence valid therapeutic targets, comprises a formidable challenge. The present review highlights recent studies providing evidence that GPCRs are relevant targets for cancer therapy through their effects on known cancer signalling pathways, tumour progression, invasion and metastasis, and the microenvironment. Furthermore, the review also explores how genomic analysis is beginning to highlight GPCRs as therapeutic targets in the age of personalized medicine.

Recurrent noncoding regulatory mutations in pancreatic ductal adenocarcinoma.

The contributions of coding mutations to tumorigenesis are relatively well known; however, little is known about somatic alterations in noncoding DNA. Here we describe GECCO (Genomic Enrichment Computational Clustering Operation) to analyze somatic noncoding alterations in 308 pancreatic ductal adenocarcinomas (PDAs) and identify commonly mutated regulatory regions. We find recurrent noncoding mutations to be enriched in PDA pathways, including axon guidance and cell adhesion, and newly identified processes, including transcription and homeobox genes. We identified mutations in protein binding sites correlating with differential expression of proximal genes and experimentally validated effects of mutations on expression. We developed an expression modulation score that quantifies the strength of gene regulation imposed by each class of regulatory elements, and found the strongest elements were most frequently mutated, suggesting a selective advantage. Our detailed single-cancer analysis of noncoding alterations identifies regulatory mutations as candidates for diagnostic and prognostic markers, and suggests new mechanisms for tumor evolution.

Challenges and Opportunities in Modeling Pancreatic Cancer

The ability to faithfully model complex processes lies at the heart of experimental biology. Although a reductionist approach necessarily reduces this complexity, it is nevertheless required for untangling the contributions and interactions of the various system components. It has long been appreciated that cancer is a complex process that involves positive and negative interactions between tumor cells, normal host tissue, and the associated cells of the tumor microenvironment. However, accurate models for studying these complex interactions in vitro have remained elusive. We seek to generate models of mouse and human pancreatic cancer that are relevant to disease biology and useful for elucidating poorly understood facets of this deadly disease. The ability to model, manipulate, and predict the therapeutic response of an individuals disease outside their body represents the promise of precision medicine. Therefore, these models are patient-specific and allow the identification of new biomarkers and novel treatment modalities for rapid translation to the clinic. In this perspective we will discuss recent advances in modeling pancreatic cancer in vitro, the discoveries these models have enabled, and future challenges and opportunities awaiting further investigation.