Calmodulin modulates Akt activity in human breast cancer cell lines

Abstract

Growth factor-induced activation of Akt occurs in the majority of human breast cancer cell lines resulting in a variety of cellular outcomes, including suppression of apoptosis and enhanced survival. We demonstrate that epidermal growth factor (EGF)-initiated activation of Akt is mediated by the ubiquitous calcium sensing molecule, calmodulin, in the majority of human breast cancer cell lines. Specifically, in estrogen receptor (ER)-negative, but not ER-positive, breast cancer cells, Akt activation is abolished by treatment with the calmodulin antagonist, W-7. Suppression of calmodulin expression by siRNAs against all three calmodulin genes in c-Myc-overexpressing mouse mammary carcinoma cells results in significant inhibition of EGF-induced Akt activation. Additionally, transient expression of constitutively active Akt (Myr-Akt) can overcome W-7-mediated suppression of Akt activation. These results confirm the involvement of calmodulin in the Akt pathway. The calmodulin independence of EGF-initiated Akt signaling in some cells was not explained by calmodulin expression level. Additionally, it was not explained by ER status or activation, since removal of estrogen and ablation of the ER did not convert the ER-positive, W-7 insensitive, MCF-7 cell line to calmodulin dependent signaling. However, forced overexpression of either epidermal growth factor receptor (EGFR) or ErbB2 did partially restore calmodulin dependent EGF-stimulated Akt activation. This is consistent with observation that W-7 sensitive cells tend to be estrogen independent and express high levels of EGFR family members. In an attempt to address how calmodulin is regulating Akt activity, we looked at localization of fluorescently tagged Akt and calmodulin in MCF-7 and SK-BR-3 cells. We found that both Akt and calmodulin translocate to the membrane after EGF-stimulation, and this translocation to the same sub-cellular compartment is inhibited by the calmodulin inhibitor W-7. Thus, calmodulin may be regulating Akt activity by modulating its sub-cellular location and is a novel target in the poor prognosis, ER-negative subset of breast cancers.

Structural and Biophysical Characterization of the Interactions between Calmodulin and the Pleckstrin Homology Domain of Akt

“…Induction of cell survival is often initiated at the cell surface and is triggered by a wide range of receptor-ligand interactions (1)(2)(3)(4)(5). One of the most frequent cell survival pathways involves activation of Akt, a serine/threonine kinase that is critical in regulating apoptosis and oncogenesis.…”

“…It is believed that PI(3,4,5)P 3 binding to a conserved basic patch on the PHD induces a conformational change, allowing for phosphorylation of residues Thr-308 and Ser-473. In addition to PI(3,4,5)P 3 , it has been recently shown that Akt translocation to the PM and subsequent activation are also modulated by phosphatidylser-ine (PS) (17). PS interacts with specific residues in the PHD and regulatory domain of Akt, promoting binding of Akt to PI(3,4,5)P 3 (17).…”

“…However, functional isoform-specific differences concerning proliferation, apoptosis, and migration in human cancer cells have been identified (18 -21). A role for Akt1 (which will be referred to as Akt throughout this work) in breast cancer cells has been proposed (3,22). It has been found that epidermal growth factor (EGF)-induced membrane translocation and activation of Akt is modulated by calmodulin (CaM) (3,22), a ubiquitous calciumbinding protein expressed in all eukaryotic cells (23)(24)(25)(26)(27)(28).…”

“…A role for Akt1 (which will be referred to as Akt throughout this work) in breast cancer cells has been proposed (3,22). It has been found that epidermal growth factor (EGF)-induced membrane translocation and activation of Akt is modulated by calmodulin (CaM) (3,22), a ubiquitous calciumbinding protein expressed in all eukaryotic cells (23)(24)(25)(26)(27)(28). Perturbation of this targeting mechanism by CaM antagonists inhibited Akt-CaM translocation to the membrane and led to apoptotic cell death in tumorigenic mammary carcinoma cells (3).…”

“…It has been found that epidermal growth factor (EGF)-induced membrane translocation and activation of Akt is modulated by calmodulin (CaM) (3,22), a ubiquitous calciumbinding protein expressed in all eukaryotic cells (23)(24)(25)(26)(27)(28). Perturbation of this targeting mechanism by CaM antagonists inhibited Akt-CaM translocation to the membrane and led to apoptotic cell death in tumorigenic mammary carcinoma cells (3). These studies are consistent with the findings that CaM is overexpressed in breast tumors and breast cancer cell lines (29,30).…”