Cancer Letters

Cancer Letters

Volume 408, 1 November 2017, Pages 73-81
Cancer Letters

Original Article
Manumycin A suppresses exosome biogenesis and secretion via targeted inhibition of Ras/Raf/ERK1/2 signaling and hnRNP H1 in castration-resistant prostate cancer cells

https://doi.org/10.1016/j.canlet.2017.08.020Get rights and content

Highlights

  • There are no known approved drugs targeting exosome biogenesis and secretion by castration-resistant prostate cancer (CRPC) cells.

  • MA suppresses exosome biogenesis and secretion via inhibition of Ras/Raf/MEK/ERK1/2 signaling in CRPC cells.

  • The Ras-dependent MA suppression of exosome biogenesis and secretion is partly mediated via an ERK-dependent inhibition of hnRNP H1 in CRPC cells.

  • MA is a potential adjuvant therapeutic drug in patients presenting with CRPC.

Abstract

Emerging evidence links exosomes to cancer progression by the trafficking of oncogenic factors and neoplastic reprogramming of stem cells. This necessitates identification and integration of functionally validated exosome-targeting therapeutics into current cancer management regimens. We employed quantitative high throughput screen on two libraries to identify exosome-targeting drugs; a commercially available collection of 1280 pharmacologically active compounds and a collection of 3300 clinically approved compounds. Manumycin-A (MA), a natural microbial metabolite, was identified as an inhibitor of exosome biogenesis and secretion by castration-resistant prostate cancer (CRPC) C4-2B, but not the normal RWPE-1, cells. While no effect was observed on cell growth, MA attenuated ESCRT-0 proteins Hrs, ALIX and Rab27a and exosome biogenesis and secretion by CRPC cells. The MA inhibitory effect is primarily mediated via targeted inhibition of the Ras/Raf/ERK1/2 signaling. The Ras-dependent MA suppression of exosome biogenesis and secretion is partly mediated by ERK-dependent inhibition of the oncogenic splicing factor hnRNP H1. Our findings suggest that MA is a potential drug candidate to suppress exosome biogenesis and secretion by CRPC cells.

Introduction

Prostate cancer (PC) is the most common and the second leading cause of cancer-related deaths among American males. Despite the initial response to various treatment regimens [1], some PC patients inevitably progress to castration-resistant PC (CRPC). Thus, there is a need to develop new, highly effective therapeutic agents to circumvent advanced disease.

Recently, the breadth of knowledge about biogenesis, ‘cargo’ contents and intercellular communication of diverse types of cancer-derived extracellular vesicles (EVs) in a variety of physiologic contexts [2] has expanded considerably. EVs encompass a broad range of secreted vesicles, including exosomes, microvesicles (MVs), and apoptotic blebs [3]. In addition to their own selective markers, the membranous nano-sized exosomes harbor surface markers indicative of their cellular origin. The exosome ‘cargo’ contains a wide variety of RNAs (including mRNAs, non-coding RNAs), proteins, DNA and lipids [4]. The endosomal sorting complex required for transport (ESCRT) machinery and their associated proteins, such as tumor susceptibility gene 101 (TSG101), hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and ALG-2 interacting protein X (Alix), are pivotal to biogenesis, cargo sorting and secretion of exosomes [5].

Exosomes are implicated in cell-cell communication and modulation of the biology of recipient cells [6]. The PC-derived exosomes are detected in the prostatic secretions, seminal fluid, tissue, urine, and blood [7], implicating their clinical utility as ‘liquid biopsies’ in the diagnosis and prognosis of PC. The tumor-derived exosomes promote growth, angiogenesis, and metastasis of recipient cells [8]. We recently demonstrated that trafficking of oncogenic factors by PC cell-derived exosomes subvert tumor microenvironment and prime oncogenic reprogramming of tumor-tropic PC patients' derived adipose stem cells, leading to tumor clonal expansion in vivo [9]. However, currently, there are no drugs that selectively target pathways involved in exosome biogenesis and secretion by cancer cells and their uptake by recipient cells. Such drugs may prove to be clinically effective for the prevention and/or treatment of advanced PC.

The family of heterogeneous nuclear ribonucleoproteins (hnRNPs) regulates pre-mRNA biogenesis, metabolism, and transport [10]. As a bona fide component of the nuclear matrix, the hnRNP H/F subfamily (hnRNP H1, hnRNP H2, hnRNP F, and hnRNP 2H9) are characterized by the possession of the quasi-RNA binding recognition motif (qRRM). Notably, we recently demonstrated the selective expression and growth promotion ability of the oncogenic slicing factor hnRNP H1 in CRPC cells via transcriptional upregulation of androgen receptor (AR) and its splice variant AR-V7 [11]. However, the functional role of hnRNPs, including hnRNP H1, in the biogenesis and/or section of exosomes remains elusive.

Ras proteins are small GTPases that function as molecular switches by alternating between inactive GDP-bound to active GTP-bound states. Active Ras (GTP bound Ras) binds to and activates downstream effectors, such as PI3K, RAF/MEK/ERK pathway, and initiates a cascade of cellular events related to tumor and non-tumor pathologies [12], [13]. Although Ras mutations in prostate cancer are infrequent, they play a pivotal role in multiple pathways that have been implicated in prostate cancer growth, transformation, differentiation, and stress responses to androgen independence [14].

The present study provides evidence for the first time that Manumycin-A (MA) suppresses exosome biogenesis and secretion in CRPC cells via inhibition of Ras signaling pathway and hnRNP H1 expression. The shRNA silencing of hnRNP H1 attenuated the endogenous levels of Alix, Rab27a, and Ras, suggesting that hnRNP H1 is pivotal to MA-mediated inhibition exosome biogenesis and secretion in CRPC cells.

Section snippets

Materials

RPMI 1640, K-SFM, penicillin/streptomycin solution, fetal bovine serum (FBS) were from Invitrogen (Camarillo, CA). Manumycin A (MA) and GW4869 were purchased from Cayman Chemical Company (Denver, CO). The NCGC Pharmaceutical Collection (NPC) was custom assembled at National Center for Advancing Translational Sciences (NCATS, NIH, Bethesda, MD). The Ras activation assay kit (17-218) was purchased from Millipore (Darmstadt, Germany). U0126 and SB203580 were from Promega (Madison, WI) and SP600125

High throughput screen (HTS) for CD63-GFP biogenesis

Two libraries were screened to examine a variety of compounds on exosome biogenesis; the LOPAC, a commercially available collection of 1280 pharmacologically active compounds, and the NPC (NCATS Pharmaceutical Collection, NIH), a collection of 3300 clinically approved compounds were screened for this assay. CD63-GFP-expressing C4-2B cells were generated and the screen was implemented in a dose response mode. The compounds were selected on the basis of GFP signal. One hundred and twenty-eight

Discussion

Exosomes and other microvesicles are released into the extracellular space under both physiological and disease conditions by virtually all cells [2]. Exosomes are essentially involved in cellular communications through the trafficking of cytosolic components and membrane proteins, from donor cells into recipient cells [5]. The exosome-mediated transfer of such factors is implicated in cancer development, progression, and clonal expansion by provoking immune suppression, angiogenesis,

Authors contributions

Conception and design: A. B. Abdel-Mageed, D. Mondal and M. Ferrer.

Development of Methodology: A. Datta, H. Kim, M. Lal, L. McGee, A. Johnson, A. Moustafa, J. C. Jones.

Grant support

This work was supported by grants from the National Center for Advancing Translational Sciences (NCATS, National Institutes of Health (1UH2TR000928-01; 1UH3TR000928-04), to A.B.A.

Acknowledgements

We thank Jessica A Daigle for editing the manuscript.

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    Authors contributed equally to this work.

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