| Cancer is still one of the principle diseases that seriously threaten human health. Most of the previous studies on cancer initiation and progression are performed with an oncogene- and tumor suppressor gene-centric view. In recent years, more and more evidence has been provided to support the view that cancer-stroma crosstalk plays a critical role in the multiple steps of carcinogenesis and cancer progression. Study on cancer-stroma crosstalk has been a hot spot in the field of cancer biology and will not only broaden our basic knowledge of cancer development, but also provide some promising novel cancer biomarkers and therapeutical targets.HAb18G/CD147 is a novel cancer target identified in our lab. It is a member of CD147 family, a cellular adhension molecule family, and belongs to the immunoglobulin super family. It has been found to be highly expressed in most of the cancer tissues, while expressed negatively or at a very low level in normal tissues. It has been proved to be an independent predictor of the poor prognosis of post-resection patients with hepatocellular carcinoma (HCC). Using it as a drug target, a radioimmunotherapeutic agent LICARTIN, a national class 1 new drug developed in our lab, has showed a significant suppressing efficacy on tumor progression in HCC patients, and can significantly decrease the tumor recurrence/metastasis rate and prolong the patients'survival in post-liver transplantation patients with advanced HCC. Preliminary study showed that HAb18G/CD147 in HCC cells could induce the secretion of matrix metalloproteinases (MMPs) and promote the invasion of HCC cells. But the blocking effects of HAb18 monoclonal antibody (mAb), a specific mAb against HAb18G/CD147, on the function of HAb18G/CD147 remains unclear. The function of HAb18G/CD147 in the cancer-stroma crosstalk has also not been demonstrated. The present study aims to determine the blocking effects of HAb18 mAb on cancer invasion and to explore the function of HAb18G/CD147 in transforming fibroblasts to cancer-associated fibroblasts, a major cell type in the cancer-stoma crosstalk. This study also tries to examine the function of HAb18G/CD147 in HCC cell migration via involved into the cancer-stroma crosstalk. The study is composed of three parts.Part 1. HAb18 mAb blocking the function of HAb18G/CD147 in HCC cellsThe results of competitive binding test showed that HAb18 mAb could effectively bind to HAb18G/CD147 expressed in HCC cells. The inhibition effects of HAb18 mAb treatment on HCC cells are comparable with those of gene silence of HAb18G/CD147 by small interfering RNA (siRNA) transfection. Gelatinase zymography and In-vitro invasion assay showed that HAb18 mAb and siRNA could both significantly inhibit the secretion of MMPs by HCC cells (2 HCC cell lines, FHCC-98 and MHCC97-H cells) and the invasion of the cells (P < 0.01). In the coculture of HCC cells and fibroblasts, the cellular invasive potential was obviously enhanced. Downregulation of HAb18G/CD147 in HCC cells could significantly inhibit the secretion of MMPs and the invasion of the cells (P < 0.01). Co-silence of two gelatinases, MMP-2 and MMP-9, either in HCC cells or in fibroblasts could both suppress the invasion of the cells in the coculture of HCC cells and fibroblasts (P < 0.01), but the latter showed a stronger effects (P < 0.01). Silencing the expression of HAb18G/CD147 in HCC cells showed a comparable effect on the cellular invasive potential with co-silencing MMP-2 and MMP-9 in HCC cells and fibroblasts did. These results suggest that HCC-associated HAb18G/CD147 can regulate the invasive potential of HCC cells via modulating the secretion of MMPs by HCC cells and by surrounding fibroblasts, while the regulation of fibroblasts plays a more important role in HCC invasion. HAb18 mAb can effectively bind to HAb18G/CD147 in HCC cells and block its function in HCC invasion and metastasis.Part 2. Experimental study on the function of HAb18G/CD147 in"activating"stromal fibroblastsα-SMA is a well-known marker expressed in the cancer-associated fibroblasts. Gelatinase zymography and Western-blot analysis showed that the secretion of MMPs and the expression ofα-SMA were obviously enhanced in the coculture of HCC cells and fibroblasts. Silencing HAb18G/CD147 in HCC cells obviously decreased the expression ofα-SMA in the coculture, suggesting that HAb18G/CD147 may be involved into the induction ofα-SMA expression and the activation of fibroblasts via the interaction of HCC cells and fibroblasts. Gelatinase zymography and Western-blot analysis showed that stimulating fibroblasts with recombination HAb18G/CD147 extracellular protion (rEP) could upregulate the secretion of MMPs and the expression ofα-SMA and HAb18G/CD147 itself. The results of immunofluorenscence staining showed that the fluorenscence indensity representing HAb18G/CD147 in fibroblasts obviously increased when the fibroblasts cocultured with HCC cells. rEP could also stimulate the expression of HAb18G/CD147 andα-SMA in fibroblasts and induce the cells to present a contracted appearance. These results indicate that HAb18G/CD147 may be involved into the interaction of HCC cells and fibroblasts via inducingα-SMA expression and promote the fibroblasts to be transformed to the cancer-associated fibroblasts with the feature of myofibroblasts. The positive feedback regulation of HAb18G/CD147 expression in fibroblast might have some role in this process.Part 3. Preliminary exploration of the involvement of HAb18G/CD147 in HCC cell migrationThe results of cell migration test showed that silencing HAb18G/CD147 with RNA interference or blocking it with HAb18 mAb in HCC cells could both significantly inhibit the migration of the cells on a Matrigel-coated surface (P < 0.05) and the adhension of the cells to Matrigel (P < 0.05), suggesting that HAb18G/CD147 is involved into the cell migration and the adhension of HCC cells to the extracellular matrix (ECM) or basemembrane. Immunofluorenscent analysis showed that HAb18G/CD147 could co-locate with F-actin in the lamellipodia of HCC cells under the treatment of TPA, a stimulator of lamellipodia. When HCC cells were treated with cytochalasin D (CyD), HAb18G/CD147 also co-located with F-actin. Together with that actin could be co-immunoprecipitated with HAb18G/CD147, these results suggest that HAb18G/CD147 may participate in the formation of lamellipodia via the interaction with the cytoskeletal protein. Immunofluorescent analysis showed that HAb18G/CD147 could also co-localize wth membrane type 1 matrix metalloproteinase (MT1-MMP), a key regulator of cell migration, in HCC cells. The co-immunoprecipitation test showed a possible interaction between HAb18G/CD147 and MT1-MMP. These results suggest HAb18G/CD147 may be an adhension molecule connecting the ECM and the cytoskeletal protein in the process of cell migration. It is involved into the adhension of HCC cells to ECM and the formation of lamellipodia. In addition, the potential interaction between HAb18G/CD147 and MT1-MMP might play some role in the cell migration.All the above results suggest an important role of HAb18G/CD147 in cancer-stroma crosstalk. Its regulation of MMPs secretion by fibroblasts plays a more critical role in the HCC cell invasion than that by HCC cells. The HAb18 mAb could effectively block its function in HCC cells. This is the first time to determine the function of cancer-associated HAb18G/CD147 in"activating"fibroblasts via the interaction between HCC cells and fibroblasts,and to examine the possible function of HAb18G/CD147 in the adhesion of HCC cells to ECM, the formation of lamellipodia via interacting with actin, and the cell migration. The present study founds a basis for the further demonstration of the function of HAb18G/CD147, a novel cancer target, in cancer-stroma crosstalk and provides some evidence for its further application. |
PDF Full Text Request