Please use this identifier to cite or link to this item: http://prr.hec.gov.pk/jspui/handle/123456789/12127
Title: Expression and Functional Analysis of Caveolar and SNARE Proteins in Cancer Progression
Authors: Raja, Sadaf Azad
Keywords: BioSciences
Biology
Issue Date: 2020
Publisher: COMSATS University, Islamabad.
Abstract: Membrane proteins are highly important players that help to maintain cellular integrity and control cell to cell interactions. One of the most intricate mechanisms of cell to cell communication is via vesicles formation and trafficking, carrying signaling proteins and molecules. Caveolar complex and SNARE (Soluble N-ethylmaleimidesensitive-factor attachment protein receptors) complex are among the membrane protein complexes that are functionally involved in vesicle trafficking and so are critical in cell to cell communication and development. Recent studies have observed an association of dysregulation of gene expression of CAV1and, DNM2 with cancer progression. Strategic components of SNARE machinery, Synaptobrevin2/Vesicle associated membrane protein-2 (v-SNARE) and Syntaxin 1A (t-SNARE) play a critical role in colon, lung and breast cancer progression and metastasis. In this study, we analyzed the relative expression of the STX1A, VAMP2, CAV1, and DNM2 for their possible association in the progression and metastasis of bladder cancer. The quantitative expression of the target genes was observed in human bladder cancer samples. The expression of STX1A, VAMP2, CAV1, and DNM2 were increased 5-fold, 2.9-fold, 8.88-folds and, 8.62-fold respectively. The overall expression of STX1A and VAMP2 found to be elevated significantly (P<0.0001) in high-grade tumors cells compared to normal and low-grade tumors. Similarly high-grade tumors had significantly higher expression of CAV1 (P<0.0001) and DNM2 (P<0.0001) compared to low-grade tumors. The correlation between the STX1A and VAMP2 was positive while between CAV1 and DNM2 the correlation was found strong positive. These data suggests that the increased expression of the key components of caveolar and SNARE complex might be responsible for the tumor grade and stage progression in bladder cancer. To further dissect the downstream signaling involved in vesicular trafficking we selected Cav-1 protein to provide an insight into its functional aspects. For this purpose we exploited a functional disruption imposed by a specific frame shift mutation at F160X resulting into truncated protein. Functional analyses of this Cav-1-mutant protein in MIA-PaCa-2, HT1376, HCT116, HT-29, and HCC827 showed that the Cav-1-mut cells have reduced growth, proliferation with improved mitochondrial respiration. The Cav-1-mut cells showed increased OCRs and reduced ECARs showing their reduced glycolytic behaviour compared to control cancer cells. The functional impact of this mutant Cav-1 might be the missing C-terminus including the important conserved phosphorylation site S168E, which is required for the translocation of the Cav-1 protein. As a structural component of caveolae, Cav-1 interacts with signaling molecules via a caveolin scaffolding domain (CSD) regulating cell signaling. Recent reports have shown that Cav-1 is a negative regulator in tumor metastasis. Therefore, we hypothesized that Cav-1 inhibits cell migration through its CSD. HeLa cells were engineered to overexpress Cav-1 (Cav-1 OE), Cav-1 without a functional CSD (∆CSD), or enhanced green fluorescent protein (EGFP) as a control. HeLa cell migration was suppressed in Cav-1 OE cells while ∆CSD showed increased migration. This altered migration corresponded to a decreased expression of zona occludens (ZO-1) with increased expression of vimentin. This shows that the CSD impacts the migratory phenotype in HeLa cells. Impact of Cav-1∆CSD on cellular migration was further confirmed in epithelial based colon cancer cell lines that have high (HCT116) and low (HT29) expression of this gene. To further elucidate the function of CSD we explored the JAK/STAT3 signaling account for the hypermigratory phenotype in the Cav-1∆CSD cells. Phosphorylated STAT3 was decreased in Cav-1 OE cells compared to control and ∆CSD cells; reducing STAT3 expression alone decreased cell migration. ∆CSD blunted HeLa proliferation by restricting the cells in G2/M phase of the cell cycle. Overexpressing the CSD peptide alone suppressed HeLa cell migration and inhibited phosphorylation of STAT3. These data suggests the importance of CSD as a negative regulator of STAT3 phosphorylation. Our functional investigations of Cav-1 specifically revealed that Cav-1 CSD may be critical in controlling the dynamic phenotype of cancer cells by facilitating the interactions of specific signal transduction pathways, regulating STAT3 and participating in a G2/M checkpoint. Modulating the CSD and targeting specific proteins may offer potential targets for novel therapies to control cancer metastasis.
Gov't Doc #: 19812
URI: http://prr.hec.gov.pk/jspui/handle/123456789/12127
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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