Studies are conducted to define the mechanisms involved in tumor growth and metastasis and to develop new animal models of human cancers. We have found that a basement membrane extract (Matrigel) when premixed with human breast and prostate tumor cells (which do not grow well in mice) promotes tumor incidence and growth. We have been able to culture new highly differentiated human tumor cell lines from the tumors grown in mice, including certain colon and prostate cell lines. Laminin, a major basement membrane component, has been found to promote the malignant phenotype of tumor cells. Laminin-adherent melanoma and colon cancer cells are more malignant than either the nonadherent cells or the parental cells. Various biologically active laminin-derived synthetic peptides have been identified. One peptide A5G27 from the alpha-5 chain blocks breast cancer growth and angiogenesis in vivo by binding in a dominant-negative manner to its receptor. We have now begun to further develop this peptide as a therapeutic (working with a peptide chemist) and to define its signaling pathways. We have also identified the potential mechanism of A5G27 activity using WiDr human colorectal carcinoma cells. WiDr cells bound to the laminin A5G27 peptide via the heparin-like and chondroitin sulfate B glycosaminoglycan side chains of CD44. Cell binding to FGF2 was blocked by laminin peptide A5G27 but not by either a scrambled version of this peptide or by another laminin peptide known to bind cell surface proteoglycans. FGF2 signaling involving tyrosine phosphorylation was also blocked by laminin peptide A5G27 but was not affected by peptide controls. Finally, we have shown that peptide A5G27 directly blocks FGF2 binding to heparin. Peptide A5G27 has sequence homology to a region on FGF2 that binds heparin and the FGF receptor and is important in FGF2 structure. We conclude that peptide A5G27 inhibits metastasis and angiogenesis by blocking FGF2 binding to the heparan sulfate side chains of CD44 variant 3, thus decreasing FGF2 bioactivity. We are now investigating the sequence in FGF2 that is homologous to see if it has similar biological activity. Another peptide, LQVQLSIR, increases metastases in various tumor cell types (breast cancer, melanoma, and ovarian cancer) and binds to the receptor CD44. This is the only site on laminin found to date that promotes tumor metastasis. The receptor for this peptide has already been shown by others to be important in metastasis in a broad range of tumor cell types. We also find that CD44 is higher in melanoma cells that are adhesion-selected to this peptide, and when these peptide-adhesion-selected cells are injected into mice, more metastases are observed in the absence of added peptide. Using antibody array technology, we have identified genes regulated by this peptide. One of these genes, fibronectin, is upregulated by this peptide in all tumor cell types tested to date. We now have a better understanding of how this peptide promotes tumor metastasis. Our goal is to define the molecular mechanisms involved in tumor growth and metastases. Our approach is to (1) select for and isolate highly malignant cells, (2) define their cellular laminin receptors, (3) identify additional sequences on laminin that promote or reduce the malignant phenotype, (4) identify genes involved in malignancy, and (5) define molecules in bone responsible for prostate and breast cancer organ-specific metastases. We work with several models, including B16F10 melanoma cells, breast, prostate, and salivary gland tumor cells. ? ? Both breast and prostate cancers metastasize preferentially to bone. The reason for this organ-specific metastasis is not clear. Some researchers believe that the bone cell environment has growth factors that support the growth of these tumors. We found that breast and prostate tumor cells preferentially migrate and invade in vitro to extracts of bone over extracts of other tissues, such as liver, lung, or brain. We purified the 35-kDa active factor and identified it as osteonectin. Purified osteonectin specifically promoted the migration and invasion in vitro of several breast and prostate cancer cell lines but was inactive with melanoma and fibrosarcoma cells. Furthermore, we found that osteonectin increases protease activity in bone-metastasizing cancer cells but not in melanoma, fibrosarcoma, or 3T3 cells. We have set up a new model to measure metastasis to bone using green fluorescent-labeled breast cancer cells. We find that cells transfected with osteonectin form fewer bone metastases, possibly due to a decrease in platelet aggregation. Understanding the factors that cause the cells to metastasize to bone may lead to new therapeutic approaches aimed at blocking metastatic disease. ?
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