Semaphorin 3A upregulates FOXO 3a-dependent MelCAM expression leading to attenuation of breast tumor growth and angiogenesis.

Semaphorin 3A (Sema 3A), a member of semaphorin family, serves as a guidance clue during embryonic development and is known as a candidate tumor suppressor that attenuates breast tumor progression by binding with its co-receptor, neuropilin-1 (NRP-1). However, the underlying mechanism by which Sema 3A suppresses breast tumor growth is still unexplored. In this study, we report that Sema 3A regulates phosphorylation and nuclear translocation of phosphatase and tensin homolog (PTEN) and FOXO 3a. Moreover, Sema 3A controls NRP-1-mediated PTEN-dependent FOXO 3a activation. Overexpression of PTEN and FOXO 3a enhances Sema 3A-induced attenuation of breast cancer cell migration. Chromatin immunoprecipitation and electrophoretic mobility shift assay data revealed that FOXO 3a regulates MelCAM at the transcriptional level. Furthermore, Sema 3A induces NRP-1-mediated MelCAM expression through PTEN and FOXO 3a. The data also showed that vascular endothelial growth factor-induced angiogenesis is inhibited by Sema 3A. Loss of or gain in function study revealed that Sema 3A modulates phosphorylation of PTEN and FOXO 3a and expression of MelCAM, leading to suppression of tumor growth and angiogenesis using in vivo mice model. Clinical specimen analysis revealed that reduced expression of Sema 3A and p-PTEN are correlated with enhanced breast cancer progression, further strengthening our in vitro and in vivo findings. Correlation of relapse-free survival of breast cancer patients (n=2878) with expression levels of Sema 3A, NRP-1, FOXO 3a and MelCAM were studied by Kaplan-Meier analysis. Statistical analysis revealed a close association between reduced expression of Sema 3A and MelCAM with that of poor patient's survival. Our study demonstrated a novel mechanism of regulation of tumor suppression by Sema 3A in coordination with a chain of tumor-suppressor genes, which in turn inhibits breast cancer cell migration, tumor growth and angiogenesis.

Hypoxia-driven osteopontin contributes to breast tumor growth through modulation of HIF1α-mediated VEGF-dependent angiogenesis.

Hypoxia is a salient feature of most solid tumors, and hypoxic adaptation of cancer cells has crucial implications in propagation of malignant clonal cell population. Osteopontin (OPN) has been identified as a hypoxia-responsive gene, but the mechanistic and regulatory role of OPN under hypoxia is less characterized. The present study identifies the existence of a positive inter-regulatory loop between hypoxia and OPN. We have shown that hypoxia induces OPN expression in breast cancer cells; however, the expression was found to be HIF1α independent. OPN enabled transcriptional upregulation of HIF1α expression both under normoxia and hypoxia, whereas stability of HIF1α protein in breast cancer cells remained unaffected. Moreover, we have shown that OPN induces integrin-linked kinase (ILK)/Akt-mediated nuclear factor (NF)-κB p65 activation leading to HIF1α-dependent vascular endothelial growth factor (VEGF) expression and angiogenesis in response to hypoxia. These in vitro data are biologically important as OPN expressing cells induce greater tumor growth and angiogenesis through enhanced expressions of proangiogenic molecules as compared with control. Immunohistochemical analysis of human breast cancer specimens revealed significant correlation between OPN and HIF1α but not HIF2α. Elevated expression of HIF1α and OPN was observed in pre-neoplastic and early stage infiltrating ductal carcinoma implicating the role of these proteins in neoplastic progression of breast cancer. Together, our results substantiate the prime role of OPN in cellular adaptation through ILK and NF-κB-mediated HIF1α-dependent VEGF expression in response to hypoxia that ultimately controls breast cancer progression and angiogenesis. Our study reinforces the fact that targeting OPN and its regulated signaling network hold important therapeutic implications.