## 5 Discussion
Besides frequently being overexpressed in neoplastic cell surfaces and involved in invasion of cancer cells and metastatic dissemination, ITG\(\alpha\)v via its ligand association interacts with various cell proteases such as MMP2 which helps in enhancing the activity of tumor cells to degrade ECM and eventually migrate within the environment [49]. Several pathways are responsible for the migration of tumor cells. As we could see, transfection of this integrin siRNA caused reduction in protein expressions of phosphorylated AKT and MAPK as well as total MAPK (Figure 8c). This indicates that these pathways have a critical role in modulation of ITG\(\alpha\)v expression. However, apart from AKT and MAPK, the TGF-\(\beta\) pathway also plays an essential role in integrinsignaling and these pathways have been found to be co-related to each other. In 4T1 cells, research shows that TGF-\(\beta\) can induce EMT, cell migration, and phosphorylation of mothers against decapentaplegic homolog (Smad)-2 protein and transcriptional responses are dependent on the PI3K-AKT pathway [50]. Further, elevated endogenous levels of various MAPK proteins by this sub-unit have also been found responsible in increasing the tumor invasiveness [51]. Therefore, inhibiting these pathways with the help of antagonist such as ITG\(\alpha\)v siRNA could modulate the morphogenetic, transcriptional, and migratory activities of activated signaling pathways such as TGF-\(\beta\), AKT, and MAPK in 4T1 models (in vitro and in vivo). While \(\alpha\)v integrin can form heterodimers with other \(\beta\) integrin units as well as connect with additional cell cytoskeletal proteins [52], disrupting its function could possibly decrease the association with these molecules, which could further reduce the invasion of the tumor cells. Therefore, its knockdown could be vital in reducing the metastatic dissemination of the cancer in breasts and eventually reducing the cell invasion. Integrin \(\alpha\)6 (ITG\(\alpha\)6), widely upregulated in tumors, participates in migration and invasion in cancer cells and as compared to normal cells, has a high level of protein and mRNA expressions [26; 53], causing poor prognosis and survival. Transfection of CA-ITG\(\alpha\)6 siRNA slightly decreased cell viability (~72%) in MCF-7 cells (Figure 2a) with cytotoxicity of ~11% (Table 2). ITG\(\alpha\)6 siRNA transfection also showed the decrease expression of phosphorylated and total AKT and MAPK proteins in MCF-7 cells (Figure 8a). A study performed by [PERSON] et al. (2008) showed ITG\(\alpha\)6 was necessary for MCF-7 tumorigenicity and overexpression of \(\alpha\)6 has been found to provide growth and survival of tumor cells. Further, within this cell line, the sub population of MSS-derived cells attribute the stemness, which is stem cell characteristics and ITG\(\alpha\)6 is one of its markers for identification. Therefore, by reducing the expression of this integrin via these proteins the invasive and migration properties of these cells could be reduced [54]. On the other hand, in MDA-MB-231 and 4T1 cells (Figure 2b,c respectively) no visible decrease in the viability was observed. As the level of \(\alpha\)6 integrin is very high in TNBC and in metastatic breast cancers, the transfection at smaller dose may not be sufficient to carry out the reduction in cell viability in the respective MDA-MB-231 and 4T1 cell lines. Additionally, ITG\(\alpha\)6 is found in two transcript variants [26; 53; 54]. Hence, with one of the variants more expressed than the other, it may be possible that this pre-designed siRNA is not sufficient to reduce the activity of both the variants in the cells, leading to unaffected cell cytotoxicity. Reduction in levels of phosphorylated and total AKT and MAPK in MDA-MB-231 and in 4T1 cells (Figure 8b,c respectively) suggest MAPK and AKT pathways are critical in overregulated \(\alpha\)6.