Menashe Bar-Eli, Ph.D.
Professor of Cancer Biology
The focus of my laboratory is to study the molecular biology of melanoma metastasis. The molecular changes associated with the transition of melanoma cells from radial growth phase (RGP) to vertical growth phase (VGP, metastatic phenotype) are not very well defined.
One tumor cell property essential for metastasis is the expression of cell surface adhesion molecules that mediate cell-to-cell or cell-to-matrix interactions. MCAM/MUC18 is a 113-kDa cell surface glycoprotein originally discovered on malignant melanoma cells. Our laboratory has shown that the expression of MCAM/MUC18 by human melanoma cells directly correlates with their metastatic potential in nude mice, and that so far no MCAM/MUC18-negative cell lines have been found to be metastatic. Furthermore, enforced expression of MCAM in MCAM-negative cells rendered them highly tumorigenic and increased their metastatic potential in vivo.
While upregulation of the MCAM gene consists of gain of function, we have recently demonstrated that the progression of melanoma is associated with loss of c-KIT. We found that re-expression of the c-KIT receptor in highly metastatic cells inhibited their tumorigenic and metastatic potential in nude mice. Moreover, the ligand for c-KIT, SCF, inhibited the growth and induced apoptosis in melanoma cell lines expressing c-KIT but not in melanocytes, under both in vitro and in vivo conditions. These data imply that melanoma cells expressing c-KIT may allow malignant melanoma cells to escape SCF/c-KIT-mediated apoptosis, hence contributing to tumor growth and eventually metastasis.
In other studies we have shown that transition of melanoma cells to the metastatic phenotype is associated with increased activity of the metalloproteinase (MMP-2) that can be regulated by IL-8.
We found that the highly metastatic cells do not express the transcriptional factor AP-2. Because all the above three genes (MCAM, c-KIT and MMP-2) contribute to the metastatic phenotype, and since all the three are regulated by AP-2, and since other important genes involved in the progression of human melanoma such as E-cadherin, HER-2, VEGF, FAS/APO-1, bcl-2 and Kai-1, are also regulated by AP-2, we hypothesized that loss of AP-2 could be a "major switch" in the development of malignant melanoma. We were able to demonstrate that loss of AP-2 expression resulted in loss of c-KIT and upregulation of MUC18. Furthermore, re-introduction of AP-2 into the highly metastatic cells caused inhibition of tumor growth and significant reduction in their metastatic potential in nude mice. Using cDNA microchip, we recently identified the thrombin receptor PAR-1 to be a target for regulation by AP-2. We found that loss of AP-2 resulted in overexpression of PAR-1 in metastatic melanoma cells, which in turn contributes, to invasion and metastasis. Based on our data, we propose the notion that AP-2 serves a key regulator of melanoma metastasis.
In other studies we have recently demonstrated that dominant-negative CREB can inhibit growth and metastasis of melanoma via regulation of MMP-2 and MUC18 gene expression. In addition, we also demonstrated that CREB and its associated proteins act as survival factors for human melanoma cells, thus, providing a mechanism, for the first time, on how overexpression of CREB in melanoma cells may contribute to the acquisition of the metastatic phenotype.
In recent studies, we found that PAF, which is secreted by cells in the tumor microenvironment, stimulates the phosphorylation and activation of CREB in metastatic melanoma cells. Based on our data on the involvement of MUC18 and IL-8 in the progression of human melanoma, we recently developed two fully humanized antibodies to target these molecules. Treatment of melanoma bearing nude mice with fully human IL-8 (ABX-IL-8) or fully human anti-MUC18 (ABX-MA1) reduced melanoma growth and inhibited their metastatic potential.
Douglas D. Boyd, Ph.D.
Professor of Cancer Biology
My laboratory investigates the transcriptional control of genes that contribute to the spread of cancer. One of these genes, the urokinase receptor (u-PAR), is overexpressed in colon cancer. We are using transgenic mice and DNaseI hypersensitivity assays to identify novel regions driving tissue-specific u-PAR expression both in healthy mice and animals genetically induced for colon cancer, the latter allowing an analysis of transcriptional requirements in colon cancer in vivo.
We also study the regulation of the expression of the MMP-9 metalloproteinase since this gene product also contributes to cancer spread. We reported that MTA1, a multiprotein complex with chromatin remodeling activity, binds directly to the MMP-9 promoter and represses its expression. To further investigate the role of the chromatin environment, we have employed recombinant technology to genomically integrate MMP-9 promoter-reporter constructs in a site-specific manner. This technology creates a refined system to investigate the contribution of the chromatin environment to MMP-9 gene expression. Moreover, we are currently using this system to screen compound libraries for candidate agents that repress MMP-9 expression.
For both MMP-9 and u-PAR transcription, we are employing expression cloning and gene trap strategies to identify novel regulators of both u-PAR and MMP-9 expression, and, to date, we have identified two hitherto unknown regulators of their expression, i.e., KLF4 and SM22, respectively.
Finally, we have identified a novel gene (ZNF306) encoding a transcription factor that contributes to colon cancer progression based on our data with human cancer cells forced to over-express, or conversely, knocked down for this DNA-binding protein. Presently, we are using animal models to determine if the ZNF306 transgene causes malignancy in APC +/Min mice which normally only form adenomas and whether mice null for this zinc finger protein become resistant to azoxymethane-induced carcinogenesis. Using cyclic amplification and selection of targets (CAST-ing), we have identified a ZNF306 DNA-binding site and we are currently determining if ZNF306 is a “master” regulator of an expressed gene program that contributes to the progression of this malignancy.