An integrin centered complex coordinates ion transport and pH to regulate f-actin organization and cell migration in breast cancer
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eLife Assessment
This study presents a valuable finding on the identification of a complex consisting of NHE1, hERG1, β1/integrin and NaV1.5 on the membrane of breast cancer cells. The evidence supporting the claims of the authors is somewhat incomplete. The inclusion of clarification of some experimental design and the amendment of cropping Western blot data would have strengthened the study. The work will be of interest to scientists working on breast cancer.
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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 β1 integrin activation. Through biochemical, immunofluorescence and electrophysiological experiments we provide evidence of a novel signaling pathway that involves a β1 integrin-centered plasma membrane complex formed by different transport proteins: the hERG1 K + channel, the neonatal form of the Na + channel Na V 1.5 (nNa V 1.5) and the Na + /H + antiporter NHE1. The NHE1/hERG1/β1/nNa V 1.5 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 α-actinin1) and indirectly (by intracellular alkalinization). The contribution of both hERG1 and nNa V 1.5 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/β1 integrin complex with a single chain bispecific antibody (scDb-hERG1-β1) 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.
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eLife Assessment
This study presents a valuable finding on the identification of a complex consisting of NHE1, hERG1, β1/integrin and NaV1.5 on the membrane of breast cancer cells. The evidence supporting the claims of the authors is somewhat incomplete. The inclusion of clarification of some experimental design and the amendment of cropping Western blot data would have strengthened the study. The work will be of interest to scientists working on breast cancer.
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Reviewer #1 (Public Review):
This manuscript by Capitani et al. extends previous studies of ion channel expression in triple-negative breast cancer cell lines. Probing four phenotypically different breast cancer cell lines, they used co-IP and confocal immunofluorescence (IF) colocalization to reveal that beta1 integrin forms a complex with the neonatal form of the Na+ channel NaV1.5 (nNaV1.5) and the Na+/H+ antiporter NHE1 in addition to previously reported hERG1. They used siRNA to show that silencing beta1 results in a co-depletion of hERG and Nav1.5, further supporting the conclusion that they form a complex; a complementary enhancement of Na current with increased hERG expression was also demonstrated. These data compellingly describe a complex of membrane proteins unregulated in breast cancer and thus present novel potential …
Reviewer #1 (Public Review):
This manuscript by Capitani et al. extends previous studies of ion channel expression in triple-negative breast cancer cell lines. Probing four phenotypically different breast cancer cell lines, they used co-IP and confocal immunofluorescence (IF) colocalization to reveal that beta1 integrin forms a complex with the neonatal form of the Na+ channel NaV1.5 (nNaV1.5) and the Na+/H+ antiporter NHE1 in addition to previously reported hERG1. They used siRNA to show that silencing beta1 results in a co-depletion of hERG and Nav1.5, further supporting the conclusion that they form a complex; a complementary enhancement of Na current with increased hERG expression was also demonstrated. These data compellingly describe a complex of membrane proteins unregulated in breast cancer and thus present novel potential targets for treatment.
There are several concerns with experimental approaches. How fluorescence measurements were compared and controlled among experiments was not described, and masks drawn to define membrane expression seemed arbitrary, and included in some cases large sections of cytoplasm. There are issues associated with the use of channel blocking agents and a bifunctional small-chain antibody that are not well rationalized. Why are they being used, to test what hypotheses or disrupt what processes? The extremely high concentrations of E-4031 (4000x IC50 for block), e.g., are not expected to have selective actions. The effects of E-4031 at high concentrations altering cytoskeleton properties associated with invasiveness (and thus cancer progression) are questionable. There are numerous problems with co-IPs together carried out together with knock-down, which in one case depleted the protein targeted by the primary IP antibody. Western blots (WB) were quantified by comparing treatment to control, which does not control for loading errors. The control and treated signals should be divided by the respective tubulin signals to control for loading errors. Then the treated value can be compared with the control.
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Reviewer #2 (Public Review):
The manuscript by Chiara Capitani and Annarosa Arcangeli reports the identification of a complex comprising NHE1,hERG1, β1 integrin, and NaV1.5 on the plasma membrane of breast cancer cells. The authors further investigated the mutual regulatory interactions among these proteins using Western blotting and co-immunoprecipitation assays. They also examined the downstream signaling pathways associated with this complex and assessed its impact on the malignant behavior of breast cancer cells.
Strengths
The manuscript used different breast cancer cell lines and combined Western blot, immunostaining, and electrophysiology to provide evidence for the proposed complex. The inhibitors are also used to test the requirement of channel activity to function in the development of breast cancer cells with in-vitro studies.
Reviewer #2 (Public Review):
The manuscript by Chiara Capitani and Annarosa Arcangeli reports the identification of a complex comprising NHE1,hERG1, β1 integrin, and NaV1.5 on the plasma membrane of breast cancer cells. The authors further investigated the mutual regulatory interactions among these proteins using Western blotting and co-immunoprecipitation assays. They also examined the downstream signaling pathways associated with this complex and assessed its impact on the malignant behavior of breast cancer cells.
Strengths
The manuscript used different breast cancer cell lines and combined Western blot, immunostaining, and electrophysiology to provide evidence for the proposed complex. The inhibitors are also used to test the requirement of channel activity to function in the development of breast cancer cells with in-vitro studies.
Weaknesses
The data shown in this manuscript include the western blots that are cropped and imaged separately to draw conclusions about protein levels and changes in immunoprecipitation. These cannot be done on separate, cropped blots but must be imaged together to make these comparisons.
Antibodies used for hERG, NaV1.5 and β1 integrin must be validated to work for IP using KO or KD cell lines for the respective proteins to demonstrate specificity. The same goes for all the immunofluorescence imaging shown in the manuscript as these are all key pieces of data to support the conclusions.
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