Competitive enhancement of HGF-induced epithelial scattering by accessory growth factors

Abstract

HGF signaling induces epithelial cells to disassemble cadherin-based adhesion and increase cell motility and invasion, a process termed epithelial–mesenchymal transition (EMT). EMT plays a major role in cancer metastasis, allowing individual cells to detach from the primary tumor, invade local tissue, and colonize distant tissues with new tumors. While invasion of vascular and lymphatic networks is the predominant route of metastasis, nerves also can act as networks for dissemination of cancer cell to distant sites in a process termed perineual invasion (PNI). Signaling between nerves and invasive cancer cells remains poorly understood, as does cellular decision making that selects the specific route of invasion. Here we examine how HGF signaling contributes to PNI using reductionist culture model systems. We find that TGFβ, produced by PC12 cells, enhances scattering in response to HGF stimulation, increasing both cell–cell junction disassembly and cell migration. Further, gradients of TGFβ induce migratory mesenchymal cells to undergo chemotaxis towards the source of TGFβ. Interestingly, VEGF suppresses TGFβ-induced enhancement of scattering. These results have broad implications for how combinatorial growth factor signaling contributes to cancer metastasis, suggesting that VEGF and TGFβ might modulate HGF signaling to influence route selection during cancer progression.

Introduction

A central feature of malignant cells is their ability to detach from the primary tumor, invade through local tissues, migrate to distant sites, and colonize distant tissues with secondary tumors. The traditional view is that cancer cells first infiltrate vascular and lymphatic networks en route to metastatic locations. Additional routes of cancer invasion exist, although these remain much more poorly understood than vascular and lymphatic invasion, or VLI. One important example of an alternative route for metastatic cells occurs when detached tumor cells invade and colonize nerve bundles, a process termed perineural invasion, or PNI [1]. In certain cancer types, such as prostate [2] and pancreatic cancers [3], PNI is thought to be the primary route for establishment of metastatic disease.

Cellular signaling between cancer cells and nerves is required for PNI. Although it is proposed that nerves produce factors that allow cancer cells to home their migration towards nerves, resulting in nerve colonization [4], signaling systems required for PNI remain undefined. Further, although specific tumor types are associated with either VLI or PNI, how tumor cells select VLI or PNI as a primary route of invasion is also unknown. Signal transduction pathways that are activated in invasive cells and that drive invasive behavior are likely candidates. Hepatocyte growth factor (HGF) signaling plays an important role in cancer progression [5]. HGF binds and activates the c-met receptor tyrosine kinase [6], whose overexpression or mutation is tightly associated with cancer progression, particularly cancer metastasis [5]. Consistent with a role in cancer invasion, HGF signaling induces epithelial–mesenchymal transition (EMT) [7], [8]. However, it is unclear whether HGF signaling can specify a particular route of invasion.

Cancer cell lines can be grown with primary cultures of dorsal root ganglion, resulting in enhanced cell migration and colonization of nerve tissues [9]. Such models are likely to prove powerful in the dissection of signal transduction pathways that drive or permit PNI. However, perturbations in cellular signaling pathways that are inherent to cancer cells confound dissecting signaling that drives PNI. In this report, we employ a reductionist co-culture model system to examine how HGF signaling impacts cellular behaviors associated with PNI. Instead of a cancer cell line as a model for invasive cells, we use MDCK cells, a cell line derived from normal canine kidney tissue and that expresses c-met [7]. MDCK cells are co-cultured with PC12 cells, a rat adrenal pheochromocytoma cell line that differentiates into nerves when stimulated with growth factors. We find that PC12 cells exacerbate HGF-induced scattering, an effect that can be reproduced with conditioned medium or with purified transforming growth factor β (TGFβ). In addition to increasing MDCK cell scattering in response to HGF, TGFβ acts as a chemoattractant. The effects of TGFβ occur when cells adhere to collagen; on plastic it is vascular endothelial growth factor (VEGF) that enhances scattering. Interestingly, VEGF and TGFβ oppose each other in driving increased scattering. On collagen, VEGF blocks TGFβ increases in HGF-induced scattering, while on plastic TGFβ blocks VEGF increases in HGF-induced scattering. We propose a simple working model whereby signaling by additional growth factors (TGFβ or VEGF) predisposes metastatic cells to select a specific route of invasion.