The calcium-sensing receptor (CaSR), a member of the class-C G protein-coupled receptor family, is known to be expressed in ductal epithelial mammary cells [1]. This receptor is upregulated during lactation, and downregulated during pregnancy [2]. In preclinical models of breast cancer, CaSR contributes to tumor progression, suggesting an oncogenic potential of overexpressed CaSR in some cancer patients [3]. Similarly, other cancer types include CaSR as an altered element of their aberrant signaling circuits [4,5]. In the context of metastatic breast cancer [6], upregulation of CaSR expression is associated with the chemotactic behavior of cancer cells and their propensity to induce osteolysis [7]. CaSR also induce migration in MDA-MB-231, MCF7 and T47D breast tumor cells. In the case of highly metastatic MDA-MB-231 breast cancer cells, CaSR activates cell migration through the Gβγ-PI3K-mTORC2 pathway [[8], [9], [10]], raising interest on the characterization of the effector mechanisms activated by CaSR via mTORC2.

Given that PKC kinases are direct effectors of mTORC2 [11], and have been recognized as targets of diverse tumor promoters linked to cell motility and invasion driven by actin cytoskeleton reorganization [12], cellular processes critical to metastatic dissemination [13], we focused our attention on the protein kinase C (PKC) family as a source of potential effectors of CaSR signaling driving cell migration. This subgroup of AGC kinases comprises conventional (cPKC: α, β1, β2, and γ) and non-conventional PKCs. The second group is further divided into novel (nPKC) and atypical (aPKC) subgroups: (nPKC: δ, ε, η, and θ; aPKC: ζ and λ/ι). cPKCs are activated by intracellular calcium and the second messenger 1,2-diacylglycerol (DAG) [14,15], whereas non-conventional PKC isoforms do not require intracellular calcium but are activated by DAG in the case of nPKCs and by PIP3, in the case of aPKC isoforms, being PKCζ a prototypical effector of the PI3K pathway [16]. In addition, aPKCs are controlled by protein-protein interactions, with the intervention of their PTB1 domains, and their PDZ binding motifs [[17], [18], [19]]. Some of these serine/threonine kinases are bona-fide targets of mTORC2 activated as effectors of a wide range of G protein-coupled and tyrosine kinase receptors in a variety of cell types [17,20,21].

In the case of aPKCs as participants of oncogenic processes, accumulating evidence indicates that PKCζ plays a role on the activation of small GTPases of the Rho family, contributing to the metastatic behavior of several cancers [[22], [23], [24], [25]]. However, despite intense research on the involvement of aPKCs in metastatic cancer cell migration, the role of this subgroup of kinases and the RacGEFs putatively activated as their downstream effectors in the pathway triggered by CaSR to regulate cancer cell migration, have yet to be established. Knowing that CaSR promotes breast cancer cell migration through the Gβγ-PI3K-mTORC2 signaling cascade [8], here we reasoned that this receptor activates PKC isoforms as effectors of mTORC2 involved in cancer cell migration [11]. Therefore, we assessed the potential role of PKC isoforms, tested with inhibitor-based and knockdown strategies, in the migratory and invasive pathways resulting from the activation of Rac1 by CaSR. In addition, in breast cancer patient datasets, we analyzed which RacGEFs clustered as phosphoproteins with active PKCζ, identified by its phosphorylation at the turn motif. Our data expand our current knowledge on a relevant signaling axis sustaining CaSR-driven breast cancer cell migration and invasion.