EHD2 overexpression promotes tumorigenesis and metastasis in triple-negative breast cancer by regulating store-operated calcium entry
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Curated by eLife
eLife assessment
This study, supported by reasonably solid evidence, will be of interest to breast cancer researchers. The finding that EHD2 promotes tumor growth and impacts store-operated calcium entry (SOCE) adds to our understanding of breast cancer cell physiology. If supported by further research, the study provides a rationale for using SOCE inhibitors in a subset of breast cancers, with high expression of EHD2 serving as a potential predictive biomarker for using SOCE inhibitors.
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- Cancer Biology (eLife)
Abstract
With nearly all cancer deaths a result of metastasis, elucidating novel pro-metastatic cellular adaptations could provide new therapeutic targets. Here, we show that overexpression of the EPS15-Homology Domain-containing 2 (EHD2) protein in a large subset of breast cancers (BCs), especially the triple-negative (TNBC) and HER2+ subtypes, correlates with shorter patient survival. The mRNAs for EHD2 and Caveolin-1/2, structural components of caveolae, show co-overexpression across breast tumors, predicting shorter survival in basal-like BC. EHD2 shRNA knockdown and CRISPR-Cas9 knockout with mouse Ehd2 rescue, in TNBC cell line models demonstrate a major positive role of EHD2 in promoting tumorigenesis and metastasis. Mechanistically, we link these roles of EHD2 to store-operated calcium entry (SOCE), with EHD2-dependent stabilization of plasma membrane caveolae ensuring high cell surface expression of the SOCE-linked calcium channel Orai1. The novel EHD2-SOCE oncogenic axis represents a potential therapeutic target in EHD2- and CAV1/2-overexpressing BC.
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Author Response
Reviewer #1 (Public Review):
This study elucidates a role of EHD2 as a tumor/metastasis promoting protein. Prior work has found varying results indicating that high expression of EHD2 is either associated with good or poor outcomes. In this work the authors find that EHD2 is expressed in both the nucleus and cytoplasm, and that high cytoplasmic to nuclear expression is associated with a poor prognosis. Using WT and either shRNA knockdown or CRISPR KO cells, they show that EHD2 promotes 3D growth, migration and invasion in vitro, and tumor growth and metastasis in vivo. Importantly, re-expression of EHD2 in KO cells rescues the loss of function phenotype. Mechanistically, the investigators show that the loss of EHD2 decreases the calveoli and that this decreases the Orai1/Stim induced calcium influx. Finally, they show …
Author Response
Reviewer #1 (Public Review):
This study elucidates a role of EHD2 as a tumor/metastasis promoting protein. Prior work has found varying results indicating that high expression of EHD2 is either associated with good or poor outcomes. In this work the authors find that EHD2 is expressed in both the nucleus and cytoplasm, and that high cytoplasmic to nuclear expression is associated with a poor prognosis. Using WT and either shRNA knockdown or CRISPR KO cells, they show that EHD2 promotes 3D growth, migration and invasion in vitro, and tumor growth and metastasis in vivo. Importantly, re-expression of EHD2 in KO cells rescues the loss of function phenotype. Mechanistically, the investigators show that the loss of EHD2 decreases the calveoli and that this decreases the Orai1/Stim induced calcium influx. Finally, they show that inhibitors of store operated calcium entry (SOCE) phenocopies the loss of EHD2. Together the data support a protumorigenic role for EHD2 via store-operated calcium entry and reinforce the utility of targeting calveoli and SOCE in tumors with high cytosolic EHD2. This study provides a rationale for using SOCE inhibitors in a subset of breast cancers, and a potential predictive biomarker for using SOCE inhibitors based on high expression of EHD2.
We are grateful for the positive comments. Since this paragraph is to be published in the event of our manuscript being accepted, we request the correction of one typo in the paragraph: “calveoli” should be “caveolae”.
Reviewer #2 (Public Review):
The manuscript by Luan et. al. describes the role of EHD2 in promoting breast tumor growth. They showed that EHD2 cytoplasmic staining predicts poor patient outcome. Both EHD2 KO or knockdown cells showed decreased cell migration/invasion abilities and significant reduction of tumor growth and metastasis in mice. The authors further showed that the levels of EHD2 and Cav1/2 correlate with each other. EHD2 KO cells showed defects on Ca2+ trafficking. Overexpressing the SOCE factor STIM1 partially rescued SOCE defects in EHD2 KO cells. Treatment of the SOCE inhibitor SKF96365 inhibited tumor cell migration in vitro and tumor growth in vivo.
Major strengths: The authors showed that EHD2 cytoplasmic levels predict patient survival and provided strong evidence that EHD2 knockout or knockdown inhibits tumor cell migration in vitro and tumor growth in vivo. The authors also showed that SKF96365, which inhibits SOCE, suppresses tumor growth in vivo.
Major weaknesses: The connection between EHD2 and SOCE is weak.
We are thankful to the reviewer for her/his assessment of the strengths in our manuscript and appreciate her/his pointing to its weaknesses. We agree that more studies will be needed to fully establish the connection of EHD2 to SOCE and have appropriately moderated our statements in the results and discussion sections of the manuscript. We have also added statements about the need for such future studies.
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eLife assessment
This study, supported by reasonably solid evidence, will be of interest to breast cancer researchers. The finding that EHD2 promotes tumor growth and impacts store-operated calcium entry (SOCE) adds to our understanding of breast cancer cell physiology. If supported by further research, the study provides a rationale for using SOCE inhibitors in a subset of breast cancers, with high expression of EHD2 serving as a potential predictive biomarker for using SOCE inhibitors.
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Reviewer #1 (Public Review):
This study elucidates a role of EHD2 as a tumor/metastasis promoting protein. Prior work has found varying results indicating that high expression of EHD2 is either associated with good or poor outcomes. In this work the authors find that EHD2 is expressed in both the nucleus and cytoplasm, and that high cytoplasmic to nuclear expression is associated with a poor prognosis. Using WT and either shRNA knockdown or CRISPR KO cells, they show that EHD2 promotes 3D growth, migration and invasion in vitro, and tumor growth and metastasis in vivo. Importantly, re-expression of EHD2 in KO cells rescues the loss of function phenotype. Mechanistically, the investigators show that the loss of EHD2 decreases the calveoli and that this decreases the Orai1/Stim induced calcium influx. Finally, they show that inhibitors of …
Reviewer #1 (Public Review):
This study elucidates a role of EHD2 as a tumor/metastasis promoting protein. Prior work has found varying results indicating that high expression of EHD2 is either associated with good or poor outcomes. In this work the authors find that EHD2 is expressed in both the nucleus and cytoplasm, and that high cytoplasmic to nuclear expression is associated with a poor prognosis. Using WT and either shRNA knockdown or CRISPR KO cells, they show that EHD2 promotes 3D growth, migration and invasion in vitro, and tumor growth and metastasis in vivo. Importantly, re-expression of EHD2 in KO cells rescues the loss of function phenotype. Mechanistically, the investigators show that the loss of EHD2 decreases the calveoli and that this decreases the Orai1/Stim induced calcium influx. Finally, they show that inhibitors of store operated calcium entry (SOCE) phenocopies the loss of EHD2. Together the data support a protumorigenic role for EHD2 via store-operated calcium entry and reinforce the utility of targeting calveoli and SOCE in tumors with high cytosolic EHD2. This study provides a rationale for using SOCE inhibitors in a subset of breast cancers, and a potential predictive biomarker for using SOCE inhibitors based on high expression of EHD2.
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Reviewer #2 (Public Review):
The manuscript by Luan et. al. describes the role of EHD2 in promoting breast tumor growth. They showed that EHD2 cytoplasmic staining predicts poor patient outcome. Both EHD2 KO or knockdown cells showed decreased cell migration/invasion abilities and significant reduction of tumor growth and metastasis in mice. The authors further showed that the levels of EHD2 and Cav1/2 correlate with each other. EHD2 KO cells showed defects on Ca2+ trafficking. Overexpressing the SOCE factor STIM1 partially rescued SOCE defects in EHD2 KO cells. Treatment of the SOCE inhibitor SKF96365 inhibited tumor cell migration in vitro and tumor growth in vivo.
Major strengths:
The authors showed that EHD2 cytoplasmic levels predict patient survival and provided strong evidence that EHD2 knockout or knockdown inhibits tumor cell …Reviewer #2 (Public Review):
The manuscript by Luan et. al. describes the role of EHD2 in promoting breast tumor growth. They showed that EHD2 cytoplasmic staining predicts poor patient outcome. Both EHD2 KO or knockdown cells showed decreased cell migration/invasion abilities and significant reduction of tumor growth and metastasis in mice. The authors further showed that the levels of EHD2 and Cav1/2 correlate with each other. EHD2 KO cells showed defects on Ca2+ trafficking. Overexpressing the SOCE factor STIM1 partially rescued SOCE defects in EHD2 KO cells. Treatment of the SOCE inhibitor SKF96365 inhibited tumor cell migration in vitro and tumor growth in vivo.
Major strengths:
The authors showed that EHD2 cytoplasmic levels predict patient survival and provided strong evidence that EHD2 knockout or knockdown inhibits tumor cell migration in vitro and tumor growth in vivo. The authors also showed that SKF96365, which inhibits SOCE, suppresses tumor growth in vivo.Major weaknesses:
The connection between EHD2 and SOCE is weak. -