Metastatic Processes--Laminin /Laminin Peptide Promotion

转移过程--层粘连蛋白/层粘连蛋白肽促进

• 批准号: 7146107
• 负责人: HYNDA K KLEINMAN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7146107
• 关键词: angiogenesis; basement membrane; breast neoplasms; colon neoplasms; disease /disorder model; estrogens; green fluorescent proteins; human tissue; laminin; melanoma; metastasis; model design /development; neoplasm /cancer; neoplastic process; phenotype; prostate neoplasms; synthetic peptide; tissue /cell culture

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.

进行研究以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。我们已经发现，基底膜提取物（Matrigel）当与人乳腺和前列腺肿瘤细胞（其在小鼠中不能很好地生长）预混合时促进肿瘤发生和生长。我们已经能够培养新的高度分化的人类肿瘤细胞系从肿瘤生长在小鼠，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，已被发现促进肿瘤细胞的恶性表型。层粘连蛋白粘附的黑素瘤和结肠癌细胞比非粘附细胞或亲本细胞更恶性。已经鉴定了多种具有生物活性的层粘连蛋白衍生的合成肽。来自α 5链的一种肽A5 G27通过以显性负性方式结合其受体来阻断乳腺癌生长和体内血管生成。我们现在已经开始进一步开发这种肽作为治疗药物（与肽化学家合作），并确定其信号通路。我们还使用WiDr人结直肠癌细胞鉴定了A5 G27活性的潜在机制。WiDr细胞通过CD 44的肝素样和硫酸软骨素B糖胺聚糖侧链与层粘连蛋白A5 G27肽结合。细胞与FGF 2的结合被层粘连蛋白肽A5 G27阻断，但不被该肽的混杂形式或另一种已知结合细胞表面蛋白聚糖的层粘连蛋白肽阻断。涉及酪氨酸磷酸化的FGF 2信号传导也被层粘连蛋白肽A5 G27阻断，但不受肽对照的影响。最后，我们已经表明肽A5 G27直接阻断FGF 2与肝素的结合。肽A5 G27与FGF 2上结合肝素和FGF受体的区域具有序列同源性，并且在FGF 2结构中是重要的。我们的结论是，肽A5 G27通过阻断FGF 2与CD 44变体3的硫酸乙酰肝素侧链的结合，从而降低FGF 2的生物活性来抑制转移和血管生成。我们现在正在研究FGF 2中同源的序列，看看它是否具有相似的生物活性。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CD 44结合。这是迄今为止发现的促进肿瘤转移的层粘连蛋白上的唯一位点。该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是重要的。我们还发现，CD 44在粘附选择该肽的黑色素瘤细胞中更高，并且当这些肽粘附选择的细胞注射到小鼠中时，在没有添加肽的情况下观察到更多的转移。使用抗体阵列技术，我们已经确定了由这种肽调控的基因。其中一种基因纤连蛋白在迄今为止测试的所有肿瘤细胞类型中均被该肽上调。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）选择和分离高度恶性细胞，（2）确定其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）确定恶性肿瘤相关基因，（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。我们研究了几种模型，包括B16 F10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转移到骨。这种器官特异性转移的原因尚不清楚。一些研究人员认为，骨细胞环境具有支持这些肿瘤生长的生长因子。我们发现，乳腺癌和前列腺癌细胞在体外优先迁移和侵入骨提取物，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，并将其鉴定为骨粘连蛋白。纯化的骨粘连蛋白特异性地促进了几种乳腺癌和前列腺癌细胞系的体外迁移和侵袭，但对黑色素瘤和纤维肉瘤细胞无活性。此外，我们发现骨连接素增加骨转移癌细胞的蛋白酶活性，但在黑色素瘤，纤维肉瘤或3 T3细胞中不增加。我们已经建立了一个新的模型来衡量转移到骨使用绿色荧光标记的乳腺癌细胞。我们发现，转染骨连接素的细胞形成较少的骨转移，可能是由于血小板聚集减少。了解导致细胞转移到骨的因素可能会导致旨在阻断转移性疾病的新治疗方法。 我们希望能够确定转移中的调控事件，并开发治疗和诊断试剂。