br Transparency document br Introduction The high mortality
Introduction The high mortality rate of breast cancer (BCa) is mainly due to tumor cell dissemination from the primary lesion, leading to metastatic disease . Metastasis is largely driven by the activity of chemokines, which are chemo-attracting small cytokine molecules (8–10 kDa) [2,3]. CXCL13 is a widely-studied chemokine that functions through its receptor CXCR5 [4,5], and is known to promote downstream function through the activation of PI3K, Akt, and Src [6,7]. Crucially, CXCL13 participates in the processes of invasion and migration in various cancers [4,6,8]. Several cell types have been implicated as potential sources of CXCL13, namely follicular dendritic SPDP (fDCs) , macrophages , germinal center-T cells  as well as T follicular helper cells (TFH) . Interestingly, it has been reported that primary breast tumor cells also secrete CXCL13 . When CXCL13 and CXCR5 are co-expressed within breast tumors, this ligand-receptor pair forms an autocrine loop that favors epithelial-to-mesenchymal transition (EMT), elevated expression and activation of matrix metalloproteinase 9 (MMP9), which drives increased cellular migration and lymph node metastasis (LNM) . Therefore, it is of extreme importance to unveil the molecular mechanisms that govern CXCL13-CXCR5 co-expression to broaden our understanding of the process of BCa metastasis. CXCR5 expression is positively regulated by p65/RelA through its physical interaction with specific binding-sequences in the cxcr5 promoter . Involvement of RelA subunit of NFĸB in BCa progression has been well documented [, , , ]. Direct inhibition of the NFĸB-RelA activity in BCa cells induces apoptosis [17,19]. A recent report has shown that transcriptional repression of RelA by reducing nuclear phosphorylation of p65 and inhibition of NFκB signaling impedes BCa progression . Moreover, RelA regulates transcription of different EMT-related genes during BCa progression , however the molecular events driving co-expression of cxcr5 and cxcl13 remain unknown. On the other hand, Nrf2 induces cellular stress signals and also regulates transcriptional activities of many genes by binding on to the antioxidant response element (ARE) within their promoters [22,23]. It has sufficiently been addressed in literature that Nrf2 is involved in the regulation of cellular growth, apoptosis and tumorigenesis [24,25]. Interestingly, whether Nrf2 could be considered as a tumor suppressor or an oncogene is debated , suggesting both pro-oncogenic and anti-oncogenic activities of Nrf2 in BCa.
Materials and methods
Discussion CXCL13 and its associated signaling have gained immense importance in connection to BCa progression [5,7,12]. Besides fDCs, macrophages and TFH cells [9,10,12], researchers have also shown that primary breast tumor epithelial cells secrete CXCL13 . CXCL13 has been found to drive a poorer prognosis during BCa progression when it is co-expressed with its receptor CXCR5 within primary tumors . Co-expression of this chemokine ligand-receptor pair forms possible autocrine loop which favors EMT, migration and metastasis of BCa cells . Therefore, it is utmost important to understand the molecular mechanisms that drive co-expression of CXCL13 and CXCR5. Previously, it has been demonstrated in MCF7 BCa cells that RelA facilitates cxcr5 transcription by binding to specific sequences; this in turn is negatively regulated by p53 . RelA, being a multifunctional TF, regulates transcription of many genes during BCa progression [17,18,21]. In this study, we have demonstrated RelA binding to cxcl13 promoter drives its transcription. We sought to verify whether elevated RelA within primary tumor is significantly associated with CXCL13-CXCR5 co-expression. Analyses of tumor samples indicated that RelA overexpression is obligatory, though it does not exclusively correlate with CXCL13-CXCR5 co-expression (Fig. 1). This observation had raised the possibilities for negative regulation of cxcl13 and cxcr5 transcription beyond RelA-mediated positive induction. Careful analyses of “RelA overexpressed CXCL13-CXCR5 non-co-expressed” samples indicated differential negative regulation for cxcl13 and cxcr5.