An integrin centered complex coordinates ion transport and pH to regulate f-actin organization and cell migration in breast cancer.

Abstract

Reciprocal signaling between the Tumor Microenvironment (TME) and cancer cells regulates abnormal proliferation, migration and pro-metastatic behavior. Major player in such interaction is integrin-mediated cell adhesion to the Extracellular Matrix (ECM). Integrin receptors organize signaling hubs constituted by multiprotein membrane complexes often comprising ion channels and transporters. We studied whether and how integrin-centered multiprotein complexes control cell behavior in Breast Cancer (BCa) cell populations with different molecular characteristics. BCa cells were cultured onto the ECM protein fibronectin (FN), to trigger {beta}1 integrin activation. Through biochemical, immunofluorescence and electrophysiological experiments we provide evidence of a novel signaling pathway that involves a {beta}1 integrin-centered plasma membrane complex formed by different transport proteins: the hERG1 K+ channel, the neonatal form of the Na+ channel NaV1.5 (nNaV1.5) and the Na+/H+ antiporter NHE1. The NHE1/hERG1/{beta}1/nNaV1.5 complex was found on the plasma membrane of BCa cells, and particularly of Triple Negative Breast Cancer (TNBCa). When engaged by cell adhesion to FN, such membrane complex recruited the cytoskeletal actin-binding protein a-actinin1 and stimulated NHE1-mediated cytoplasmic alkalinization. Thus, the multiprotein complex activation affected TNBCa migration and invasiveness by stimulating f-actin organization directly (through -actinin1) and indirectly (by intracellular alkalinization). The contribution of both hERG1 and nNaV1.5 was essential, as the adhesion-dependent signaling pathway and its functional consequences were inhibited by blocking either channel with, respectively, E4031 and TTX, or by applying RNA silencing procedures. The contribution of hERG1 to the structural integrity of the membrane complex appeared to be critical, as the adhesion-dependent signals were hampered by harnessing the hERG1/{beta}1 integrin complex with a single chain bispecific antibody (scDb-hERG1-{beta}1) which disrupts the macromolecular complex without blocking the K+ current, as well as by E4031, which impairs the complex formation by blocking the channel in the open state. In conclusion, we revealed that integrin-centered macromolecular complexes in BCa cells recruit a battery of ion transport proteins that cooperate in modulating different aspects of the downstream signals that lead to malignant behavior. This complex could be targeted to develop novel therapeutic strategies for one of the most difficult-to-treat cancers, i.e. TNBCa.