Martin H. Hager

Oncosome Formation in Prostate Cancer: Association with a Region of Frequent Chromosomal Deletion in Metastatic Disease

Oncosomes have recently been described as membrane-derived microvesicles secreted by cancer cells, which transfer oncogenic signals and protein complexes across cell boundaries. Here, we show the rapid formation and secretion of oncosomes from DU145 and LNCaP human prostate cancer cells. Oncosome formation was stimulated by epidermal growth factor receptor activation and also by overexpression of membrane-targeted Akt1. Microvesicles shed from prostate cancer cells contained numerous signal transduction proteins and were capable of activating rapid phospho-tyrosine and Akt pathway signaling, and stimulating proliferation and migration, in recipient tumor cells. They also induced a stromal reaction in recipient normal cells. Knockdown of the actin nucleating protein Diaphanous Related Formin 3 (DRF3/Dia2) by RNA interference enhanced rates of oncosome formation, indicating that these structures resemble, and may be identical to, nonapoptotic membrane blebs, a feature of the amoeboid form of cell motility. Analysis of primary and metastatic human prostate tumors using 100K single nucleotide polymorphism arrays revealed a significantly higher frequency of deletion of the locus encoding DRF3 (DIAPH3) in metastatic tumors (P = 0.001) in comparison with organ-confined tumors. Fluorescence in situ hybridization confirmed increased chromosomal loss of DIAPH3 in metastatic tumors in a different cohort of patients (P = 0.006). These data suggest that microvesicles shed from prostate cancer cells can alter the tumor microenvironment in a manner that may promote disease progression. They also show that DRF3 is a physiologically relevant protein that seems to regulate this process.

Large Oncosomes in Human Prostate Cancer Tissues and in the Circulation of Mice with Metastatic Disease

Oncosomes are tumor-derived microvesicles that transmit signaling complexes between cell and tissue compartments. Herein, we show that amoeboid tumor cells export large (1- to 10-μm diameter) vesicles, derived from bulky cellular protrusions, that contain metalloproteinases, RNA, caveolin-1, and the GTPase ADP-ribosylation factor 6, and are biologically active toward tumor cells, endothelial cells, and fibroblasts. We describe methods by which large oncosomes can be selectively sorted by flow cytometry and analyzed independently of vesicles <1 μm. Structures resembling large oncosomes were identified in the circulation of different mouse models of prostate cancer, and their abundance correlated with tumor progression. Similar large vesicles were also identified in human tumor tissues, but they were not detected in the benign compartment. They were more abundant in metastases. Our results suggest that tumor microvesicles substantially larger than exosome-sized particles can be visualized and quantified in tissues and in the circulation, and isolated and characterized using clinically adaptable methods. These findings also suggest a mechanism by which migrating tumor cells condition the tumor microenvironment and distant sites, thereby potentiating advanced disease.

DIAPH3 governs the cellular transition to the amoeboid tumour phenotype

Therapies for most malignancies are generally ineffective once metastasis occurs. While tumour cells migrate through tissues using diverse strategies, the signalling networks controlling such behaviours in human tumours are poorly understood. Here we define a role for the Diaphanous‐related formin‐3 (DIAPH3) as a non‐canonical regulator of metastasis that restrains conversion to amoeboid cell behaviour in multiple cancer types. The DIAPH3 locus is close to RB1, within a narrow consensus region of deletion on chromosome 13q in prostate, breast and hepatocellular carcinomas. DIAPH3 silencing in human carcinoma cells destabilized microtubules and induced defective endocytic trafficking, endosomal accumulation of EGFR, and hyperactivation of EGFR/MEK/ERK signalling. Silencing also evoked amoeboid properties, increased invasion and promoted metastasis in mice. In human tumours, DIAPH3 down‐regulation was associated with aggressive or metastatic disease. DIAPH3‐silenced cells were sensitive to MEK inhibition, but showed reduced sensitivity to EGFR inhibition. These findings have implications for understanding mechanisms of metastasis, and suggest that identifying patients with chromosomal deletions at DIAPH3 may have prognostic value.

VentX trans-Activates p53 and p16ink4a to Regulate Cellular Senescence

Cell senescence is a process of irreversible arrest of cell proliferation and plays an important role in tumor suppression. Recent studies showed that Wnt inhibition is a trigger of cellular senescence. Using methods of reverse genetics, we recently identified VentX, a human homolog of the vertebrate Xenopus Vent family of homeobox genes, as a novel Wnt repressor and a putative tumor suppressor in lymphocytic leukemia. Here, we show that VentX is a direct transcriptional activator of p53-p21 and p16ink4a-Rb tumor suppression pathways. Ectopic expression of VentX in cancer cells caused an irreversible cell cycle arrest with a typical senescence-like phenotype. Conversely, inhibition of VentX expression by RNA interference ameliorated chemotherapeutic agent-induced senescence in lymphocytic leukemia cells. The results of our study further reveal the mechanisms underlying tumor suppression function of VentX and suggest a role of VentX as a potential target in cancer prevention and treatment.

Regulation of microtubule dynamics by DIAPH3 influences amoeboid tumor cell mechanics and sensitivity to taxanes

Taxanes are widely employed chemotherapies for patients with metastatic prostate and breast cancer. Here, we show that loss of Diaphanous-related formin-3 (DIAPH3), frequently associated with metastatic breast and prostate cancers, correlates with increased sensitivity to taxanes. DIAPH3 interacted with microtubules (MT), and its loss altered several parameters of MT dynamics as well as decreased polarized force generation, contractility, and response to substrate stiffness. Silencing of DIAPH3 increased the cytotoxic response to taxanes in prostate and breast cancer cell lines. Analysis of drug activity for tubulin-targeted agents in the NCI-60 cell line panel revealed a uniform positive correlation between reduced DIAPH3 expression and drug sensitivity. Low DIAPH3 expression correlated with improved relapse-free survival in breast cancer patients treated with chemotherapeutic regimens containing taxanes. Our results suggest that inhibition of MT stability arising from DIAPH3 downregulation enhances susceptibility to MT poisons, and that the DIAPH3 network potentially reports taxane sensitivity in human tumors.

Scaffold attachment factor B1 regulates the androgen receptor in concert with the growth inhibitory kinase MST1 and the methyltransferase EZH2

The androgen receptor (AR) is a transcription factor that employs many diverse interactions with coregulatory proteins in normal physiology and in prostate cancer (PCa). The AR mediates cellular responses in association with chromatin complexes and kinase cascades. Here we report that the nuclear matrix protein, scaffold attachment factor B1 (SAFB1), regulates AR activity and AR levels in a manner that suggests its involvement in PCa. SAFB1 mRNA expression was lower in PCa in comparison with normal prostate tissue in a majority of publicly available RNA expression data sets. SAFB1 protein levels were also reduced with disease progression in a cohort of human PCa that included metastatic tumors. SAFB1 bound to AR and was phosphorylated by the MST1 (Hippo homolog) serine-threonine kinase, previously shown to be an AR repressor, and MST1 localization to AR-dependent promoters was inhibited by SAFB1 depletion. Knockdown of SAFB1 in androgen-dependent LNCaP PCa cells increased AR and prostate-specific antigen (PSA) levels, stimulated growth of cultured cells and subcutaneous xenografts and promoted a more aggressive phenotype, consistent with a repressive AR regulatory function. SAFB1 formed a complex with the histone methyltransferase EZH2 at AR-interacting chromatin sites in association with other polycomb repressive complex 2 (PRC2) proteins. We conclude that SAFB1 acts as a novel AR co-regulator at gene loci where signals from the MST1/Hippo and EZH2 pathways converge.

Trading in your spindles for blebs: the amoeboid tumor cell phenotype in prostate cancer

Prostate cancer (PCa) remains a principal cause of mortality in developed countries. Because no clinical interventions overcome resistance to androgen ablation therapy, management of castration resistance and metastatic disease remains largely untreatable. Metastasis is a multistep process in which tumor cells lose cell-cell contacts, egress from the primary tumor, intravasate, survive shear stress within the vasculature and extravasate into tissues to colonize ectopic sites. Tumor cells reestablish migratory behaviors employed during nonneoplastic processes such as embryonic development, leukocyte trafficking and wound healing. While mesenchymal motility is an established paradigm of dissemination, an alternate, ‘amoeboid’ phenotype is increasingly appreciated as relevant to human cancer. Here we discuss characteristics and pathways underlying the phenotype, and highlight our findings that the cytoskeletal regulator DIAPH3 governs the mesenchymal-amoeboid transition. We also describe our identification of a new class of tumor-derived microvesicles, large oncosomes, produced by amoeboid cells and with potential clinical utility in prostate and other cancers.

Abstract 486: The novel tumor suppressor DIAPH3 governs transition to an amoeboid phenotype

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: We previously reported that loss of DIAPH3, proximal to RB1 on chromosome 13q and encoding the cytoskeletal regulator Diaphanous-related Formin-3 (DRF3), is associated with metastatic prostate cancer. Here we examine further the impact of genomic DIAPH3 loss on motility and metastasis, in multiple cancers. Methods: We employed publically available genomic data sets (including the Genomic Identification of Significant Targets in Cancer platform), Oncomine expression profiles, Affymetrix Human Genome-wide SNP arrays, and tissue microarrays to determine DIAPH3 copy number and DRF3 expression in carcinomas and with disease progression. In cultured cancer cells, biochemical and cell biological approaches in two- and three-dimensional growth conditions were used to assess mechanisms underlying phenotypic changes associated with DRF3 deficiency. The in vivo impact of DIAPH3 loss was investigated with a murine experimental metastasis model. Results: Analysis of somatic copy number alterations along chromosome 13q revealed a region of significant focal deletion, in which the DIAPH3 locus was central, common to hepatocellular, breast, and prostate carcinomas. Employing prostate cancer as a disease-relevant model, we detected reduced DIAPH3 copy number in multiple, independent patient cohorts. DIAPH3 deletions accumulated during disease progression, were strongly associated with metastatic disease, and were prevalent in disseminated tumor cells from patient bone marrow aspirates. Tissue microarrays confirmed reduced expression of DRF3 in metastases relative to normal tissue and primary tumor. In vitro, stable silencing of DRF3 in prostate cancer and HRAS-transformed human mammary epithelial cells dramatically altered cell morphology, evoking an ‘amoeboid’ morphological phenotype. Accordingly, biochemical hallmarks of amoeboid behavior, as well as enhanced migration, invasion, and chemotaxis, were associated with DRF3 loss. Consistent with its role as a regulator of cytoskeletal dynamics, DRF3 depletion destabilized microtubules, thereby perturbing endocytic receptor trafficking. EGFR was observed to accumulate in endosomes and was hyperactive, coinciding with persistent signaling through the MEK-ERK pathway. Inhibition of MEK reversed the amoeboid phenotype stimulated by DRF3 loss. Enforced expression of DRF3 accelerated down-regulation of EGFR and suppressed amoeboid morphology. Phosphorylation was observed to modulate DRF3 function. In a murine metastasis model, the formation of pulmonary lesions was markedly potentiated by DRF3 silencing, supporting a role for the protein in mitigation of tumor cell dissemination. Conclusions: Genomic deletions at the DIAPH3 locus are prevalent in multiple carcinomas and are strongly associated with metastasis. DRF3 is a metastasis suppressor that prevents transition to an amoeboid tumor cell phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 486. doi:1538-7445.AM2012-486

HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN K IS A NOVEL REGULATOR OF ANDROGEN RECEPTOR TRANSLATION IN PROSTATE CANCER

Since the association of miR-125b with CaP is totally unknown, the purpose of this study was to understand the role of this miRNA in CaP. METHODS: LNCaP and cds1 cells cultured in androgendepleted medium were transfected with synthetic miR-125b and antimiR-125b, respectively, and cell proliferation was assessed using the WST-1 assay. Chromatin immunoprecipitation (ChIP) assay was used to determine the loading of androgen receptor (AR) onto the 5’-DNA region of miR-125b. To test whether BAK1 mRNA is a direct target of miR-125b, a luciferase assay was performed in AR-negative DU145 cells to evaluate the effects of miR-125b on the 3’-untranslated region (3’-UTR) of the BAK1 gene. RESULTS: Synthetic miR-125b stimulated the growth of LNCaP cells in the absence of androgens. These cells retained typical morphological features. In contrast, in cds cells, inhibition of miR125b activity using synthetic anti-miR-125b inhibited proliferation of