EHD2 overexpression promotes tumorigenesis and metastasis in triple-negative breast cancer by regulating store-operated calcium entry

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.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting file.

Article and author information

Author details

  1. Haitao Luan

    Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, United States
    Competing interests
    No competing interests declared.
  2. Timothy A Bielecki

    Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, United States
    Competing interests
    No competing interests declared.
  3. Bhopal C Mohapatra

    Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, United States
    Competing interests
    No competing interests declared.
  4. Namista Islam

    Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, United States
    Competing interests
    No competing interests declared.
  5. Insha Mushtaq

    Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, United States
    Competing interests
    No competing interests declared.
  6. Aaqib M Bhat

    Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, United States
    Competing interests
    No competing interests declared.
  7. Sameer Mirza

    Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, United States
    Competing interests
    No competing interests declared.
  8. Sukanya Chakraborty

    Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, United States
    Competing interests
    No competing interests declared.
  9. Mohsin Raza

    Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, United States
    Competing interests
    No competing interests declared.
  10. Matthew D Storck

    Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, United States
    Competing interests
    No competing interests declared.
  11. Michael S Toss

    Department of Histopathology, Nottingham University Hospitals NHS Trust, Nottingham, United Kingdom
    Competing interests
    No competing interests declared.
  12. Jane L Meza

    Departments of Biostatistics, University of Nebraska Medical Center, Omaha, United States
    Competing interests
    No competing interests declared.
  13. Wallace B Thoreson

    Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7104-042X
  14. Donald W Coulter

    Department of Pediatrics, University of Nebraska Medical Center, Omaha, United States
    Competing interests
    No competing interests declared.
  15. Emad A Rakha

    Department of Histopathology, Nottingham University Hospitals NHS Trust, Nottingham, United Kingdom
    Competing interests
    No competing interests declared.
  16. Vimla Band

    Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, United States
    For correspondence
    vband@unmc.edu
    Competing interests
    Vimla Band, received funding from Nimbus Therapeutics for an unrelated project..
  17. Hamid Band

    Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, United States
    For correspondence
    hband@unmc.edu
    Competing interests
    Hamid Band, received funding from Nimbus Therapeutics for an unrelated project..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4996-9002

Funding

Department of Defence (W81XWH-17-1-0616 and W81XWH-20-1-0058)

  • Hamid Band

Department of Defence (W81XWH-20-1-0546)

  • Vimla Band

National Institutes of Health (R21CA241055 and R03CA253193)

  • Vimla Band

Fred and Pamela Buffett Cancer Center (Pilot grant)

  • Vimla Band

Fred and Pamela Buffett Cancer Center (Pilot grant)

  • Hamid Band

University of Nebraska Medical Center (Graduate Student Fellowships)

  • Timothy A Bielecki

University of Nebraska Medical Center (Graduate Student Fellowships)

  • Aaqib M Bhat

University of Nebraska Medical Center (Graduate Student Fellowships)

  • Sukanya Chakraborty

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: All mouse xenograft and treatment studies were pre-approved by the UNMC Institutional Animal Care and Use Committee (IACUC) under the IACUC protocol number 19-115-10-FC and conducted strictly according to the pre-approved procedures, in compliance with Federal and State guidelines.

Human subjects: Human tissues were collected and processed at the Nottingham University Hospital, United Kingdom. This study was approved by the Yorkshire & The Humber-Leeds East Research Ethics Committee (REC reference: 19/YH/0293) under the IRAS Project ID: 266925. Informed consent was obtained from all individuals prior to surgery for the use of their tissue materials in research. All samples were properly coded and anonymized in accordance with the approved protocols.

Reviewing Editor

  1. Jonathan A Cooper, Fred Hutchinson Cancer Center, United States

Publication history

  1. Received: June 22, 2022
  2. Accepted: January 10, 2023
  3. Accepted Manuscript published: January 10, 2023 (version 1)

Copyright

© 2023, Luan et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 350
    Page views
  • 79
    Downloads
  • 0
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Haitao Luan
  2. Timothy A Bielecki
  3. Bhopal C Mohapatra
  4. Namista Islam
  5. Insha Mushtaq
  6. Aaqib M Bhat
  7. Sameer Mirza
  8. Sukanya Chakraborty
  9. Mohsin Raza
  10. Matthew D Storck
  11. Michael S Toss
  12. Jane L Meza
  13. Wallace B Thoreson
  14. Donald W Coulter
  15. Emad A Rakha
  16. Vimla Band
  17. Hamid Band
(2023)
EHD2 overexpression promotes tumorigenesis and metastasis in triple-negative breast cancer by regulating store-operated calcium entry
eLife 12:e81288.
https://doi.org/10.7554/eLife.81288

Further reading

    1. Cancer Biology
    2. Microbiology and Infectious Disease
    Changkun Hu, Taylor Bugbee ... Nicholas Wallace
    Research Article

    Double strand breaks (DSBs) are one of the most lethal DNA lesions in cells. The E6 protein of beta-human papillomavirus (HPV8 E6) impairs two critical DSB repair pathways; homologous recombination (HR) and non-homologous end-joining (NHEJ). However, HPV8 E6 only delays DSB repair. How DSBs are repaired in cells with HPV8 E6 remains to be studied. We hypothesize that HPV8 E6 promotes a less commonly used DSB repair pathway, alternative end-joining (Alt-EJ). Using CAS9 based Alt-EJ reporters, we show that HPV8 E6 promotes Alt-EJ. Further, using small molecule inhibitors, CRISPR/CAS9 gene knockout, and HPV8 E6 mutant, we find that HPV8 E6 promotes Alt-EJ by binding p300, an acetyltransferase that facilitates DSB repair by HR and NHEJ. At least some of this repair occurs through a subset of Alt-EJ known as polymerase theta dependent end joining. Finally, whole genome sequencing analysis showed HPV8 E6 caused an increased frequency of deletions bearing the microhomology signatures of Alt-EJ. This study fills the knowledge gap of how DSB is repaired in cells with HPV8 E6 and the mutagenic consequences of HPV8 E6 mediated p300 destabilization. Broadly, this study supports the hypothesis that beta-HPV promotes cancer formation by increasing genomic instability.

    1. Cancer Biology
    Anthony Lozano, Francois-Régis Souche ... Damien Grégoire
    Research Article

    Quantitative differences in signal transduction are to date an understudied feature of tumour heterogeneity. The MAPK Erk pathway, which is activated in a large proportion of human tumours, is a prototypic example of distinct cell fates being driven by signal intensity. We have used primary hepatocyte precursors transformed with different dosages of an oncogenic form of Ras to model subclonal variations in MAPK signalling. Orthotopic allografts of Ras-transformed cells in immunocompromised mice gave rise to fast-growing aggressive tumours, both at the primary location and in the peritoneal cavity. Fluorescent labelling of cells expressing different oncogene levels, and consequently varying levels of MAPK Erk activation, highlighted the selection processes operating at the two sites of tumour growth. Indeed, significantly higher Ras expression was observed in primary as compared to secondary, metastatic sites, despite the apparent evolutionary trade-off of increased apoptotic death in the liver that correlated with high Ras dosage. Analysis of the immune tumour microenvironment at the two locations suggests that fast peritoneal tumour growth in the immunocompromised setting is abrogated in immunocompetent animals due to efficient antigen presentation by peritoneal dendritic cells. Furthermore, our data indicate that, in contrast to the metastatic-like outgrowth, strong MAPK signalling is required in the primary liver tumours to resist elimination by NK cells. Overall, this study describes a quantitative aspect of tumour heterogeneity and points to a potential vulnerability of a subtype of hepatocellular carcinoma as a function of MAPK Erk signalling intensity.