## 5 Discussion
They play an essential role in causing chemoresistance in breast cancer via \(\alpha\)6 [26], and by controlling their expression by down regulating molecules such as ITG\(\alpha\)6 they could prove beneficial for chemotherapeutics. However, at the same time targeting these pathways may not be enough to reduce the cell viability in certain high proliferative and metastatic in vitro cells. Cross talks might affect more downstream molecules of AKT and MAPK pathway, leading to increased viability, and involvement of other pathways and molecules such as growth factors, TGF-\(\beta\), hedgehog, cell adhesion, and p63 might play critical role [55; 56]. Studies show upon knockdown of ITG\(\alpha\)6, reduction in the cell migration, invasion, and pulmonary metastasis can be observed. This can be seen in our in vivo model as well, where clear reduction in the tumor volume was observed post second dose of siRNA-CA treatment (Day 11 onwards) with maximum fold reduction of 2.65 on Day 17 (Table 5) and small tumors were observed at the end of the treatment (Figure 12b). As we can see from Figure 10, the trend for outgrowth of tumor over the time for ITG\(\alpha\)6 siRNA complexed with CA nanoparticle was smaller compared to CA nanoparticle and untreated group. Statistical analysis performed between nanoparticle treated and siRNA-CA nanoparticle treated groups showed significant difference (*) with \(p<0.05\) on selected time points. Research shows apart from targeting main cell proliferation pathways such as PI3K/AKT or ERK/MAPK one of the ways the reduction of ITG\(\alpha\)6 expression could take place is also by inhibiting cells to enter S phase. This is done by up-regulating p27, a cyclin protein, which results in down-regulation of cyclin E/CDK2 complexes [57]. Integrin \(\beta\)1 (ITG\(\beta\)1), the largest subgroup of integrin family, plays a vital role in breast cancer. Responsible for tumor proliferation, causing the switch of tumor cells from the dormant stage to metastatic stage, and providing resistance to tumor cells against adjuvant therapy and ionization radiation [58], targeting this component of integrin could be a promising strategy in reducing the tumor metastasis and increasing the efficiency in therapy. Based on our MTT results significant reduction in the cell viability after 48 h of transfection with this siRNA in all three cell lines was seen. While MCF-7 showed cell viability ~63% (Figure 3a) and cytotoxicity of ~32% (Table 2), MDA-MB-231 showed cell viability of ~86% (Figure 3b) and cytotoxicity ~10% (Table 3) and 4T1 showed cell viability of ~81% and cytotoxicity of ~12.5% (Table 4). The reduced expression of phosphorylated and total MAPK and AKT levels was observed in MCF-7, MDA-MB-231, and 4T1 cells (Figure 8a-c respectively). In animal model, tumor regression analysis also showed decrease in the tumor volume upon delivery of this integrin siRNA sub-unit along with CA nanoparticles (Figure 11). The significant difference was observed post first dose of treatment till the mice were sacrificed with highest fold decrease of 6.3 in tumor volume (Table 6) and smaller tumor compared to CA group (Figure 12c). From these results the essential role of this integrin subunit in various types and stages of breast cancer and its significant correlation with AKT and MAPK pathways can be inferred. Previous studies have shown knockdown of \(\beta\)1 in MDA-MB-231 cell line decreased the expression levels of \(\beta\)1-associated major \(\alpha\) subunits and decreased the phosphorylation levels of FAK, thereby disrupting the focal adhesion and inhibiting mobility [59]. ITG\(\beta\)1 is involved in bidirectional signaling where the domain interacts with cytoskeleton as well as the intracellular proteins in the cell. This helps ITG\(\beta\)1 to contribute towards several tumor generating steps such as initiation, its survival, progression, reversion, and eventually metastasis.