Regulatory function of fyn in oral SCC invasion

描述：口腔鳞状细胞癌（SCC）的特点是侵袭和转移，整合素受体参与这些过程。这项申请是我们先前资助的工作的延续，该工作集中在α-v-β 6整合素和生腱蛋白-C（TN-C）作为口腔SCC侵袭的潜在调节剂的作用。α-v-β 6纤连蛋白/TN-C整合素在正常口腔角质形成细胞中未发现，但在口腔SCC中高度表达。我们在低侵袭性SCC 9细胞中表达β 6，并建立了高侵袭性SCC 9 β 6细胞系。当SCC 9 β 6细胞接种在纤连蛋白（FN）上时，与SCC 9细胞相比，Src家族激酶Fyn和粘着斑激酶在Tyr 397和Tyr 925处的自磷酸化增加。Tyr 925而不是Tyr 397的自磷酸化依赖于Fyn活化。在SCC 9 β 6细胞中，MAP激酶通路（ERK 1/2）的激活也增加。SCC细胞和肿瘤周围成纤维细胞（PTF）用于模拟体内条件。SCC 9/PTF共培养物组织了丰富的FN基质，其在SCC 9 β 6/PTF共培养物中减少。明胶酶谱表明SCC 9和SCC 9 β 6细胞表达同等水平的基质金属蛋白酶MMP 2和MMP 9。相比之下，酪蛋白酶谱表明SCC 9 β 6细胞中MMP 3活化增加。加入抗α-v-β 6抗体（10 D5）或一般MMP抑制剂GM 6001恢复FN组装并抑制通过重建的基底膜的侵袭。这些结果表明，α-v-β 6的表达和Fyn和MMP 3的活化调节口腔SCC的侵袭和FN基质组织。为了评估体内Fyn和MMP 3的活化，本申请解决了这些问题：1）Fyn活性的抑制是否影响口腔SCC的生长、侵袭和转移？2)组成性活性Fyn的表达调节口腔鳞癌的生长、侵袭和转移吗？3)组成型活性MMP 3的表达调节口腔鳞癌的侵袭和转移吗？了解Fyn和MMP 3如何调节口腔SCC行为可能会导致未来治疗干预的目标。

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.