Promotion Of Metastatic Processes By Laminin And Laminin

研究旨在确定肿瘤生长和转移的机制，并开发新的人类癌症动物模型。我们发现基底膜提取物（Matrigel）与人乳腺和前列腺肿瘤细胞（在小鼠中生长不好）预混后，可促进肿瘤的发生和生长。我们已经能够从小鼠肿瘤中培养出新的高度分化的人类肿瘤细胞系，包括某些结肠和前列腺细胞系。层粘连蛋白是一种主要的基底膜成分，已被发现可促进肿瘤细胞的恶性表型。粘连蛋白粘附的黑色素瘤和结肠癌细胞比非粘连细胞或亲代细胞更恶性。各种生物活性层粘连蛋白衍生的合成肽已被确定。α -5链上的一种肽A5G27通过以显性负性方式与其受体结合，在体内阻断乳腺癌的生长和血管生成。我们现在已经开始进一步开发这种肽作为治疗（与肽化学家合作）并确定其信号通路。我们还利用WiDr人类结直肠癌细胞确定了A5G27活性的潜在机制。WiDr细胞通过CD44的类肝素和硫酸软骨素B糖胺聚糖侧链与层粘连蛋白A5G27肽结合。层粘连蛋白肽A5G27可以阻断细胞与FGF2的结合，但这种肽的混乱版本或另一种已知与细胞表面蛋白聚糖结合的层粘连蛋白肽都不能阻断细胞与FGF2的结合。涉及酪氨酸磷酸化的FGF2信号也被层粘连蛋白肽A5G27阻断，但不受肽控制的影响。最后，我们已经证明肽A5G27直接阻断FGF2与肝素的结合。肽A5G27与FGF2上一个结合肝素和FGF受体的区域序列同源，在FGF2结构中起重要作用。我们得出结论，肽A5G27通过阻断FGF2与CD44变体3硫酸肝素侧链的结合，从而降低FGF2的生物活性，从而抑制转移和血管生成。我们现在正在研究FGF2中的同源序列，看看它是否具有类似的生物活性。另一种肽，LQVQLSIR，增加各种肿瘤细胞类型（乳腺癌、黑色素瘤和卵巢癌）的转移，并与受体CD44结合。这是迄今为止发现的层粘连蛋白上唯一一个促进肿瘤转移的位点。这种肽的受体已经被其他人证明在多种肿瘤细胞类型的转移中起重要作用。我们还发现CD44在这种肽粘附选择的黑色素瘤细胞中更高，当这些肽粘附选择的细胞注射到小鼠体内时，在没有添加肽的情况下观察到更多的转移。利用抗体阵列技术，我们已经鉴定出受该肽调控的基因。其中一种基因，纤维连接蛋白，在迄今为止测试的所有肿瘤细胞类型中都被这种肽上调。我们现在对这种肽如何促进肿瘤转移有了更好的了解。我们的目标是确定参与肿瘤生长和转移的分子机制。我们的方法是：(1)选择并分离高度恶性细胞，(2)确定它们的细胞层粘连蛋白受体，(3)确定层粘连蛋白上促进或减少恶性表型的其他序列，(4)确定与恶性肿瘤有关的基因，(5)确定骨中负责前列腺癌和乳腺癌器官特异性转移的分子。我们使用了几种模型，包括B16F10黑色素瘤细胞、乳腺、前列腺和唾液腺肿瘤细胞。

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.