| Summary: | 碩士 === 國立成功大學 === 生物化學暨分子生物學研究所 === 101 === The leading cause of cancer death is cancer metastasis, which could be promoted by cancer pericellular fibronectin (FN)/endothelial CD26-mediated cell-cell adhesion within lung vasculatures. Understanding the molecular mechanisms of FN assembly on suspended cancer cells may facilitate anti-metastatic strategies. It has been reported that the activity of STAT3 is intimately associated with FN expression and cancer metastasis. However, whether STAT3 regulates suspended cancer pericellular fibronectin assembly to increase metastasis remains unclear. We have previously found that STAT3 activity (represented by pY705-STAT3) promotes FN assembly on suspended cancer cell surfaces. Interestingly, there is a more obvious 120 kDa non-STAT3 protein expressed in highly FN assembled metastatic cancer cells. After mass spectrometry analysis, it is hnRNP U that is cross-reacted with the anti-pY705-STAT3 polyclonal antibody. Here we showed that suspended cancer pericellular fibronectin assembly could also be promoted by hnRNP U. Consistently, we found that pY705-STAT3 was able to promote tyrosine phosphorylation of hnRNP U. Subsequently, we knocked down hnRNP U in AS2 cancer cells ectopically expressing the constitutively activated STAT3 (STAT3-S3C) and showed that the pY705-STAT3-promoted FN assembly was decreased, suggesting that hnRNP U is indeed involved in pY705-STAT3-promoted FN assembly. Next, to further study how STAT3 regulates hnRNP U phosphorylation, we found that both STAT3-S3C and DNA-binding mutant STAT3 (STAT3-S3D) promoted hnRNP U phosphorylation but not Y705F-mutated inactive STAT3 (STAT3-S3F). On the other hand, only STAT3-S3C enhances hnRNP U protein expression. These results suggested that pY705-STAT3 may promote hnRNP U phosphorylation through a unexpected non-canonical pathway. STAT3 are known to be able to activate metastasis-promoting c-Met. Therefore, we assumed that c-Met is involved in pY705-STAT3-triggered hnRNP U phosphorylation. We showed that pY705-STAT3 was indeed capable of promoting c-Met phosphorylation through non-canonical pathway, and inhibition of c-Met activity reduced hnRNP U phosphorylation. PP2A is reported to participate in both metastasis and the regulation of c-Met activity. We also found that STAT3 promoted PP2A expression. This thesis partially unveils the molecular mechanisms of pericellular FN on metastatic cancer cells in suspension is promoted by STAT3-regulated hnRNP U, and PP2A and c-Met participate in this mechanisms.
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