Regulatory function of fyn in oral SCC invasion

口腔鳞状细胞癌（SCC）具有侵袭和转移的特点，整合素受体参与了这一过程。该应用是我们之前资助的工作的延续，其重点是α -v- β - 6整合素和tenascin-C （TN-C）作为口腔SCC侵袭的潜在调节剂的作用。α -v- β 6纤维连接蛋白/TN-C整合素在正常口腔角质形成细胞中未发现，但在口腔鳞状细胞癌中高表达。我们在低侵袭性SCC9细胞中表达了beta6，并建立了高侵袭性SCC9beta6细胞系。SCC9beta6细胞经纤维连接蛋白（FN）处理后，Src家族激酶Fyn和黏附激酶Tyr 397和Tyr 925位点的自磷酸化水平较SCC9细胞升高。Tyr 925而不是Tyr 397的自磷酸化依赖于Fyn的激活。在SCC9beta6细胞中，MAP激酶通路（ERK1/2）的激活也增加。用SCC细胞和肿瘤周围成纤维细胞（PTF）模拟体内条件。SCC9/PTF共培养组织了丰富的FN基质，SCC9beta6/PTF共培养减少了FN基质。明胶酶谱显示SCC9和SCC9beta6细胞表达相同水平的基质金属蛋白酶MMP2和MMP9。相比之下，酪蛋白酶谱显示SCC9beta6细胞中MMP3激活增加。加入抗α -v- β - 6抗体（10D5）或通用MMP抑制剂GM6001，通过重组基膜恢复FN组装并抑制入侵。这些结果表明α -v- β 6的表达以及Fyn和MMP3的激活调节了口腔SCC的侵袭和FN基质的组织。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活性的抑制是否会影响口腔SCC的生长、侵袭和转移？2)本构活性Fyn的表达是否调节口腔SCC的生长、侵袭和转移？3)组成活性MMP3的表达是否调节口腔SCC的侵袭和转移？了解Fyn和MMP3如何调节口腔鳞状细胞癌的行为可能会为未来的治疗干预提供潜在的靶点。

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.