Metastatic Processes--Laminin /Laminin Peptide Promotion

研究的目的是明确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，当基底膜提取物(Matrigel)与人乳腺和前列腺肿瘤细胞(它们在小鼠体内生长不好)预混合时，会促进肿瘤的发生和生长。我们已经能够从小鼠体内生长的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。层粘连蛋白是基底膜的主要成分，已被发现促进肿瘤细胞的恶性表型。层粘连蛋白粘附性黑色素瘤和结肠癌细胞比非粘附性黑色素瘤细胞或亲代细胞更恶性。目前已鉴定出多种具有生物活性的层粘连蛋白合成肽。来自α5链的一种多肽A5G27通过与其受体以显性-负性方式结合，在体内阻止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多肽作为一种治疗手段(与多肽化学家合作)，并确定其信号通路。我们还利用WiDR人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。细胞与FGF2的结合可被层粘连蛋白多肽A5G27阻断，但不能被该多肽的杂乱版本或另一种已知与细胞表面蛋白多糖结合的层粘连蛋白多肽所阻断。涉及酪氨酸磷酸化的FGF2信号也被层粘连蛋白多肽A5G27阻断，但不受多肽对照的影响。最后，我们发现A5G27可以直接阻断FGF2与肝素的结合。A5G27多肽与FGF2上结合肝素和成纤维细胞生长因子受体的区域具有序列同源性，在FGF2结构中起重要作用。结论：A5G27通过阻断FGF2与CD44变异体3的硫酸乙酰肝素侧链的结合，从而降低FGF2的生物活性，从而抑制肿瘤的转移和血管生成。我们现在正在研究FGF2中的同源序列，以确定它是否具有类似的生物学活性。另一种多肽，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。这是迄今为止在层粘连蛋白上发现的唯一促进肿瘤转移的部位。这种多肽的受体已经被其他人证明在广泛的肿瘤细胞类型的转移中起重要作用。我们还发现，CD44在黏附选择这种肽的黑色素瘤细胞中更高，当这些黏附选择的细胞被注射到小鼠体内时，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，我们已经鉴定了受该肽调控的基因。到目前为止，这些基因之一，纤维连接蛋白，在所有测试的肿瘤细胞类型中都被这种多肽上调。我们现在对这种多肽如何促进肿瘤转移有了更好的了解。我们的目标是确定与肿瘤生长和转移有关的分子机制。我们的方法是(1)选择和分离高度恶性的细胞，(2)定义它们的细胞层粘连蛋白受体，(3)确定促进或减少恶性表型的层粘连蛋白的额外序列，(4)确定与恶性相关的基因，以及(5)确定骨中与前列腺癌和乳腺癌器官特异性转移有关的分子。我们研究了几种模型，包括B16F10黑色素瘤细胞、乳腺、前列腺和唾液腺肿瘤细胞。 乳腺癌和前列腺癌都优先向骨骼转移。这种器官特异性转移的原因尚不清楚。一些研究人员认为，骨细胞环境中有支持这些肿瘤生长的生长因子。我们发现，乳腺和前列腺肿瘤细胞在体外优先迁移和侵袭到骨的提取物，而不是其他组织的提取物，如肝、肺或脑。我们纯化了35 kDa的活性因子，并将其鉴定为骨结素。纯化的骨连蛋白在体外特异性地促进几种乳腺和前列腺癌细胞系的迁移和侵袭，但对黑色素瘤和纤维肉瘤细胞不起作用。此外，我们还发现，骨连蛋白增加了骨转移癌细胞中的蛋白水解酶活性，但在黑色素瘤、纤维肉瘤或3T3细胞中却没有。我们已经建立了一种新的模型，用绿色荧光标记的乳腺癌细胞来测量骨转移。我们发现，转染骨结素的细胞形成的骨转移较少，这可能是由于减少了血小板聚集。了解导致细胞转移到骨骼的因素可能会导致旨在阻止转移疾病的新的治疗方法。 我们希望定义与转移相关的调节事件，并开发治疗和诊断试剂。

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 non-adherent 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 which 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. We hope to define regulatory events involved in metastases and develop therapeutic and diagnostic reagents.