Regulatory function of fyn in oral SCC invasion

口腔鳞状细胞癌(SCC)以侵袭和转移为特征，整合素受体参与这些过程。这项申请是我们之前资助的工作的继续，该工作重点是α-v-β6整合素和TN-C(TN-C)作为口腔鳞癌侵袭的潜在调节器的作用。α-v-β6纤维连接蛋白/TN-C整合素在正常口腔角质形成细胞中不存在，但在口腔鳞癌中高表达。我们在低侵袭性的SCC9细胞中表达了Beta6，并建立了高侵袭性的SCC9beta6细胞系。当将SCC9beta6细胞与纤维连接蛋白(FN)复合后，与SCC9细胞相比，在Tyr 397和Tyr 925处的Src家族激酶Fyn和粘着斑激酶的自磷酸化水平增加。Tyr 925而不是Tyr 397的自身磷酸化依赖于Fyn的激活。在SCC9beta6细胞中，MAPK通路(ERK1/2)的激活也增加。用SCC细胞和瘤周成纤维细胞(PTF)模拟体内条件。SCC9/PTF共培养形成丰富的FN基质，SCC9beta6/PTF共培养中FN基质减少。明胶酶谱显示SCC9和SCC9beta6细胞表达等量的基质金属蛋白酶MMP2和MMP9。相反，酪蛋白酶谱显示SCC9beta6细胞中MMP3活性增加。加入抗α-v-beta6抗体(10D5)或一般的基质金属蛋白酶抑制剂GM6001可恢复FN的组装，并抑制通过重建的基底膜的侵袭。这些结果表明，Alpha-v-beta6的表达以及Fyn和MMP3的激活调节了口腔鳞癌的侵袭和FN的基质组织。为了评估Fyn和MMP3在体内的激活，本应用解决了以下问题：1)Fyn活性的抑制是否影响口腔鳞癌的生长、侵袭和转移？2)成分活性Fyn的表达是否调节口腔鳞癌的生长、侵袭和转移？3)成分活性MMP3的表达是否调节口腔鳞癌的侵袭和转移？了解Fyn和MMP3如何调节口腔鳞状细胞癌行为可能会导致未来治疗干预的靶点。

DESCRIPTION: Oral squamous cell carcinoma (SCC) is characterized by invasion and metastasis, and integrin receptors participate in these processes. This application is a continuation of our previously funded work, which focused on the role of the alpha-v-beta6 integrin and tenascin-C (TN-C) as potential modulators of oral SCC invasion. The alpha-v-beta6 fibronectin/TN-C integrin is not found in normal oral keratinocytes but is highly expressed in oral SCC. We expressed beta6 in poorly invasive SCC9 cells and established the highly invasive SCC9beta6 cell line. When SCC9beta6 cells were plated on fibronectin (FN), autophosphorylation of the Src family kinase Fyn and focal adhesion kinase at Tyr 397 and Tyr 925 was increased as compared with SCC9 cells. Autophosphorylation at Tyr 925 but not Tyr 397 depended upon Fyn activation. Activation of the MAP kinase pathway (ERK1/2) was also increased in SCC9beta6 cells. SCC cells and peri-tumor fibroblasts (PTF) were used to simulate in vivo conditions. SCC9/PTF co-cultures organized a rich FN matrix, which was decreased in SCC9beta6/PTF co-cultures. Gelatin zymographs indicated SCC9 and SCC9beta6 cells expressed equivalent levels of matrix metalloproteinases MMP2 and MMP9. In contrast, casein zymography indicated increased MMP3 activation in the SCC9beta6 cells. Adding anti-alpha-v-beta6 antibodies (10D5) or the general MMP inhibitor GM6001 restored FN assembly and suppressed invasion through a reconstituted basement membrane. These results suggest that expression ofalpha-v-beta6 and activation of Fyn and MMP3 modulate oral SCC invasion and FN matrix organization. To evaluate Fyn and MMP3 activation in vivo, this application addresses these questions: 1) Does suppression of Fyn activity affect oral SCC growth, invasion, and metastasis? 2) Does expression of a constitutively active Fyn modulate oral SCC growth, invasion and metastasis? 3) Does expression of a constitutively active MMP3 modulate oral SCC invasion and metastasis? Understanding how Fyn and MMP3 may modulate oral SCC behavior may potentially lead to targets for future therapeutic intervention.