Senescent Peritoneal Mesothelial Cells Promote Ovarian Cancer Cell Adhesion

Abstract

Adhesion of ovarian cancer cells to the peritoneal mesothelium is a key step in the malignant progression of the disease. In an in vitro study, we showed that the adherence of ovarian cancer cells (of the OVCAR-3, SKOV-3, and A2780 cell lines) to senescent human omentum-derived peritoneal mesothelial cells (HOMCs) was greater than to early passage cells. The process was mediated primarily by the increased interaction of the ␣5␤1 integrin on cancer cells with HOMC-associated fibronectin (FN). In comparison with early passage HOMCs, senescent cells exhibited increased FN mRNA expression levels and produced significantly more FN. To assess the effect of senescence-associated oxidative stress on FN release, HOMCs were rendered senescent by exposure to an oxidant, tert-butyl hydroperoxide. Treatment with tert-butyl hydroperoxide resulted in a significant increase in HOMC FN mRNA and protein expression levels. The effect of oxidative stress on FN synthesis was found to be mediated by transforming growth factor-␤1, whose signaling pathway was controlled at upstream and downstream levels by p38 MAPK. The activity of p38 MAPK increased markedly in senescent HOMCs. Treatment of HOMCs with antioxidants significantly attenuated senescence-associated increases in p38 MAPK activity, production of both transforming growth factor-␤1 and FN, and ovarian cancer cell adhesion. These data indicate that oxidative stress that accompanies senescence may increase Ovarian cancer is still associated with high mortality. The poor outcome is related to a large extent to metastatic spreading of cancer cells within the peritoneal cavity. Attachment of malignant cells to the peritoneal mesothelium is thought to be a critical step in peritoneal dissemination of the disease. Available data indicate that the process is mediated by interactions between extracellular matrix components produced by mesothelial cells and the corresponding adhesion molecules on ovarian cancer cells. A key role is thought to be played by CD44 and ␤1 integrin receptors and their ligands-hyaluronan, fibronectin (FN), and vitronectin. [2][3][4][5][6] FN is a ubiquitous constituent of extracellular matrix. Secreted by cells as a soluble dimer, it is then processed and assembled into insoluble fibrils at the cell surface. 7 FN is involved in many cellular functions including the maintenance of cell shape, tissue repair, cell differentiation, adhesion, and migration. These processes are primarily mediated by binding of the Arg-Gly-Asp (RGD) sequence of the FN molecule to integrin receptors (especially to ␣5␤1 and ␣ V ␤3).8 It has been demonstrated that FN released by peritoneal mesothelial cells stimulates ovarian cancer cell motility in vitro.9 Moreover, competitive inhibition of FN by RGD-containing peptides has been reported to decrease peritoneal spreading of ovarian cancer cells in mice. 10 More recently it has been demonstrated that the attachment of ovarian cancer cells to the peritoneum results in up-regulation of their matrix metalloproteinase-2, which cleaves mesot...

<em>In vitro</em> Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

“…Previously, several groups [8][9][10][11][12][13][14] had used endpoint assays to show that ovarian cancer cells attach to and invade into mesothelial cell monolayers. This assay is unique in that it uses fluorescently labeled cells to distinguish tumor cells from mesothelial cells, so that the dynamics of these two cell populations can be monitored throughout the assay.…”

“…Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells 8 , and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells) 9,10 . This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis.…”

“…Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers [8][9][10][11][12][13][14][15][16] , and was first described in a report showing that ovarian tumor cells utilize an integrin -dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid 17 . This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction.…”

Mesothelial cells promote early ovarian cancer metastasis through fibronectin secretion

“…Rieppi et al revealed that conditioned media (CM) of primary human mesothelial cells induced migration of OvCa cell lines through a gelatin-coated Boyden chamber (8), and a later paper demonstrated that mesothelial cells promote OvCa adhesion (9). Collectively, these findings were the first evidence that mesothelial cells actively participate in the establishment of the OvCa metastatic niche.…”

Detection of novel biomarkers for ovarian cancer with an optical nanotechnology detection system enabling label-free diagnostics

“…Fibronectin is a matrix adhesion protein of known involvement in ovarian cancer, and fibronectin synthesis has been hypothesized to be up-regulated in response to oxidative stress occurring during early malignant progression of the disease. 49 One pathological study measuring immunoreactivity of fibronectin in a cohort of 211 German ovarian cancer patients found a significant association with tumor stage and growth fraction, 50 while another recent study found increased fibronectin plasma levels in a number of gynecological cancers, but not ovarian cancer. 51 These conflicting data further highlight the need for novel protein biomarker assay panels that can quantify multiple relevant biomarkers both accurately and cost-effectively.…”