Breast cancer (BC) remains a major cause of cancer-related deaths, with less than 30% 5-year survival rate in patients with metastatic disease (https://www.acs.org/). Triple-negative BC (TNBC) presents a particularly difficult diagnosis with lack of targeted therapies. A better understanding of tumorigenesis- and metastasis-associated cellular adaptations could open novel approaches to improve the survival of metastatic BC patients.

EPS15-homology (EH) domain-containing (EHD) proteins (EHD1-4) are evolutionarily conserved lipid membrane-activated ATPases that regulate inward or outward vesicular traffic between the plasma membrane and intracellular organelles by controlling tubulation and scission of trafficking vesicles (Naslavsky and Caplan, 2011). Unlike other family members, which predominantly localize to endosomal and other intracellular compartments, EHD2 is known to primarily localizes to plasma membrane caveolae to maintain their stable membrane pool (Morén et al., 2012; Stoeber et al., 2012), suggesting a likely role in caveolae-associated cellular functions. Indeed, caveolae-dependent fatty acid uptake in adipocytes and eNOS-NO induced small blood vessel relaxation are impaired in Ehd2 knockout mice (Matthaeus et al., 2020; Matthaeus et al., 2019). EHD2-dependent stabilization of caveolae was also found to promote the cell surface expression of ATP-sensitive K⁺ channels and protect cardiomyocytes against ischemic injury (Yang et al., 2018). Caveolae are key to buffering the plasma membrane stress (Sinha et al., 2011) and EHD2 has been shown to positively regulate mechano-transduction through re-localization to the nucleus and regulation of gene transcription (Torrino et al., 2018).

Recent studies have painted a complex picture of the potential roles of EHD2 in cancer. Reduced EHD2 expression was reported in esophageal, colorectal, breast, and hepatocellular cancers (Li et al., 2013; Guan et al., 2021; Yang et al., 2015; Liu et al., 2016), with in vitro knockdown or overexpression studies supporting a tumor suppressive role for EHD2. On the contrary, EHD2 overexpression was found as a component of a mesenchymal signature in malignant gliomas with shorter survival, and knockdown analyses showed the EHD2 requirement for cell proliferation, migration, and invasion (Zhang et al., 2021). Higher EHD2 mRNA expression in papillary thyroid carcinomas was associated with extrathyroidal extension, lymph node metastasis, higher risk of recurrence, and presence of BRAF-V600E mutation (Kim et al., 2017). Studies of clear cell renal cell carcinoma also supported a positive role of EHD2 in tumorigenesis (Liu et al., 2019). A recent study provided a more mixed picture, with loss of EHD2 expression in TNBC cell lines enhancing their proliferation, migration, and invasion but low levels of EHD2 mRNA in TNBC patient tumors predicting better prognosis (Shen et al., 2020). Thus, a definitive role of EHD2 in oncogenesis and its mechanisms remain unclear.

Here, our comprehensive expression analyses in BC samples and in vitro and in vivo studies using EHD2 knockdown or knockout approaches in TNBC cell models provide definitive evidence for strong pro-tumorigenic and pro-metastatic role of EHD2. Our studies suggest a novel pro-oncogenic mechanism of EHD2, namely its requirement for efficient store-operated calcium entry (SOCE), a pathway known to promote tumorigenesis and metastasis in breast and other cancers (Mo and Yang, 2018; Yang et al., 2009).

Elucidating novel tumorigenesis- and metastasis-associated cellular adaptations could dictate new therapeutic options in BC. Here, we use TNBC cell models to elucidate a novel signaling axis linking EHD2 overexpression in BC to store-operated calcium entry (SOCE), a known pro-oncogenic and pro-metastatic pathway. Our studies support the potential for targeting the SOCE pathway in EHD2-overexpressing TNBC and other BC subtypes.

Our IHC analyses demonstrated high cytoplasmic EHD2 expression in a substantial proportion of breast tumors, associated with shorter BC-specific patient survival (Figure 3B) and higher tumor grade (Supplementary file 1-Table 1). A higher proportion of TNBC, luminal B and HER2 + patients exhibited high cytoplasmic EHD2 (Figure 3D, Supplementary file 1-Table 1). Our results differ from reports that reduction in EHD2 expression correlates with tumor progression in BC (Yang et al., 2015; Shi et al., 2015). Notably, a recent study, while it reported the depletion of EHD2 to increase the oncogenic traits of BC cell lines in vitro, found low EHD2 expression in breast tumors to specify good prognosis and better chemotherapy response (Shen et al., 2020). Several factors could account for the discordance, including the lack of validation of antibodies in prior studies, the high EHD2 expression in normal mammary adipocytes (Figure 1B), resulting in apparent reduction in EHD2 expression in tumor tissue using western blotting (Yang et al., 2015; Shen et al., 2020; Shi et al., 2015), and the possibility that EHD2 signals in prior studies represented nuclear EHD2, which we find is associated with positive prognostic factors (Figure 3B, Supplementary file 1-Table 1). Previous cell-line-based studies have shown that EHD2 shuttles between the nucleus and cytoplasm based on nuclear import and exit signals, the latter dependent on its SUMOylation, and that nuclear EHD2 repressed transcription from fused transactivation domains (Pekar et al., 2012). Another study found that application of mechanical stress to cells led to EHD2 release from caveolae, its SUMOylation and nuclear translocation where it regulated transcription of several genes including caveolar components, in a potential positive feedback mechanism (Torrino et al., 2018). However, no roles have thus far been assigned to the nuclear/cytoplasmic partitioning of EHD2 in oncogenesis. In view of the opposite prognostic significance of nuclear vs. non-nuclear EHD2 in our analyses of BC patient samples, it could be speculated that nuclear translocation may sequester EHD2 to inhibit its plasma membrane-associated pro-oncogenic role in SOCE, but rigorous studies will be needed to test this suggestion.

Analysis of mRNA expression in public BC databases (Figure 7A) and of protein levels in BC cell lines (Figure 7C) demonstrated high degree of EHD2 co-expression with caveolin-1/2, the structural elements of caveolae. This was noteworthy since EHD2 is known to regulate the stability of caveolae (Morén et al., 2012; Stoeber et al., 2012). Significantly, EHD2 and CAV1 or CAV2 mRNA overexpression predicted shorter patient survival specifically in the PAM50-defined basal BC (Figure 7B), consistent with the predominant basal (myoepithelial) cell expression of EHD2 in mouse mammary epithelium (Figure 1B and D). Additional analyses of publicly available cell line mRNA data in CCLE (Figure 2C) and of a subset of BC samples analyzed for EHD2 expression (Figure 2A), further reinforce the conclusion that high cytoplasmic EHD2 expression is a feature of BC with basal-like/triple-negative features.

Multi-pronged approaches using shRNA knockdown and CRISPR-Cas9 KO of EHD2 in TNBC cell models together with mouse Ehd2 rescue of EHD2-KO demonstrated that EHD2 is required for tumorigenesis and metastasis. We show that in vitro tumor cell growth under stringent conditions (tumorspheres; Figure 4C) and pro-metastatic traits of cell migration, invasion, and ECM degradation (Figures 4D–E–5B–D) are EHD2-dependent. In vivo, loss of EHD2 markedly impaired the orthotopic TNBC xenograft formation and metastasis (Figure 4F; Figure 5H), and tumor growth was rescued by exogenous mouse Ehd2 (Figure 5H). Notably, intravenous injections demonstrated the inability of EHD2-KO TNBC cells to form lung metastases (Figure 6). Although further studies are needed to fully dissect the steps at which EHD2 is critical in the metastatic process, collectively, our analyses conclusively demonstrate that extranuclear EHD2 overexpression in BC cells represents a key pro-tumorigenic and pro-metastatic adaptation.

Mechanistically, we link the EHD2 overexpression in BC cells to regulation of caveolae, whose stability is known to be controlled by EHD2 (Morén et al., 2012; Stoeber et al., 2012; Morén et al., 2019). This includes the strong EHD2 co-localization with CAV1/2 (Figure 7D), reduction in cell surface caveolae density using TIRF microscopy upon EHD2-KO (Figure 7E), and inhibition of TNBC cell migration upon CAV1-KO (Figure 7G), consistent with the previously documented pro-tumorigenic roles of CAV1 in TNBC (Badana et al., 2016; Zou et al., 2017). However, since our localization analyses show both EHD2 and CAV1 to also localize intracellularly besides at the plasma membrane (Figure 7D), and prior studies have identified pro-oncogenic roles for CAV1 localized in various intracellular locations (Simón et al., 2020), further studies will be needed to determine if the role of EHD2 we define here is exclusively related to regulation of the plasma membrane caveolae or might involve CAV1 in other compartments as well.

Caveolae serve as hubs for signaling (Lamaze et al., 2017). Among these, the SOCE pathway stood out as it is known to regulate multiple tumorigenic and pro-metastatic traits in TNBC (Pani and Singh, 2009; Bohórquez-Hernández et al., 2017), as with EHD2 depletion. Also, EHD2 interacts with Ca²⁺-binding proteins such as Ferlins (Posey et al., 2011) that are involved in Ca²⁺-dependent membrane repair and EHD2 was found to accumulate at sites of membrane repair in skeletal muscle models (Marg et al., 2012; Demonbreun et al., 2016). Indeed, our extensive analyses demonstrate that EHD2 is a major positive regulator of SOCE in TNBC cell models. This includes analyses of fluorescent dye-labeled cells and two distinct agents (thapsigargin or CPA) to release ER Ca²⁺ as stimuli (Figure 8A–B), and independent validation using cytoplasm- or plasma membrane-localized genetic reporters of Ca²⁺ (Figure 8E–F, Figure 8—figure supplement 1A–B). Reciprocally, CRISPR-activation of endogenous EHD2 expression in an EHD2-nonexpressing TNBC cell line upregulated SOCE (Figure 8G) and cell migration (Figure 5G). Complementing these, EHD2-KO reduces the STIM1-Orai1 interaction at the ER-plasma membrane contact sites as measured using fluorescent STIM1 (Figure 9A) and overexpression of STIM1 partially rescues the SOCE and cell migration defects in EHD2-KO TNBC (Figure 9E–F). Consistent with the established role of SOCE in the intracellular Ca²⁺ store refilling (Chung et al., 2017), which then serves as the source of the initial Ca²⁺ release upon appropriate stimuli (Tg or CPA in our studies), loss of EHD2 or CAV1 expression or treatment with SOCE inhibitors led to an impairment of the SOCE as well as the initial Ca²⁺ peak (Figure 8A–E; Figure 8H–I). Reciprocally, CRISPRa upregulation of EHD2 elevated both phases of Ca²⁺ flux (Figure 8G).

Orai1 is a major STIM1-interacting caveolae-resident SOCE channel (Bohórquez-Hernández et al., 2017; Sathish et al., 2012). Indeed, our cell surface biotinylation studies demonstrated that EHD2-KO specifically reduces the cell surface Orai1 levels (Figure 9C). Thus, our findings support a model whereby EHD2-dependent stabilization of cell surface caveolae ensures high cell surface levels of Orai1 to enable robust SOCE in TNBCs, which in turn promotes pro-tumorigenic and pro-metastatic behaviors of tumor cells (Mo and Yang, 2018; Yang et al., 2009). Consistent with this model, EHD2 deficiency reduced the cell surface levels of caveolae-associated ATP-sensitive K⁺ channels (Yang et al., 2018). However, further genetic studies to perturb Orai1 and its family members as well as other potential mediators of SOCE will be necessary to fully establish a causal role of the SOCE pathway in EHD2-dependent oncogenesis.

Finally, consistent with prior studies (Yang et al., 2009), chemical inhibition of SOCE markedly impaired the pro-metastatic traits of EHD2-overexpressing TNBCs, with a smaller impact on EHD2-KO cell lines (Figure 10A) and impaired the TNBC metastatic growth in vivo (Figure 10B). However, since SKF96365 targets additional Ca²⁺ channels besides Orai1 (Ramsey et al., 2006; Ding et al., 2012), further studies using more selective inhibitors together with genetic analyses will be needed.

Together, our studies support the idea that EHD2-overexpressing subsets of TNBC and other BC subtypes may be selectively amenable to SOCE targeting, with EHD2 and CAV1/2 overexpression as predictors of response.

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

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Author details

1. Haitao Luan

   Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   Contributed equally with

   Timothy A Bielecki and Bhopal C Mohapatra

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9831-167X

2. Timothy A Bielecki

   Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, United States

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing, Conceptualization

   Contributed equally with

   Haitao Luan and Bhopal C Mohapatra

   Competing interests

   No competing interests declared

3. Bhopal C Mohapatra

   1. Department of Genetics, Cell Biology and Anatomy, College of Medicine, University of Nebraska Medical Center, Omaha, United States
   2. Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, United States

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – review and editing, Conceptualization

   Contributed equally with

   Haitao Luan and Timothy A Bielecki

   Competing interests

   No competing interests declared

4. Namista Islam

   1. Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, United States
   2. Department of Genetics, Cell Biology and Anatomy, College of Medicine, University of Nebraska Medical Center, Omaha, United States

   Contribution

   Data curation, Formal analysis

   Competing interests

   No competing interests declared

5. Insha Mushtaq

   1. Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, United States
   2. Department of Pathology & Microbiology, College of Medicine, University of Nebraska Medical Center, Omaha, United States

   Contribution

   Data curation, Formal analysis

   Competing interests

   No competing interests declared

6. Aaqib M Bhat

   1. Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, United States
   2. Department of Genetics, Cell Biology and Anatomy, College of Medicine, University of Nebraska Medical Center, Omaha, United States

   Contribution

   Data curation, Formal analysis

   Competing interests

   No competing interests declared

7. Sameer Mirza

   Department of Genetics, Cell Biology and Anatomy, College of Medicine, University of Nebraska Medical Center, Omaha, United States

   Contribution

   Data curation, Formal analysis

   Competing interests

   No competing interests declared

8. Sukanya Chakraborty

   1. Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, United States
   2. Department of Genetics, Cell Biology and Anatomy, College of Medicine, University of Nebraska Medical Center, Omaha, United States

   Contribution

   Data curation, Formal analysis

   Competing interests

   No competing interests declared

9. Mohsin Raza

   Department of Genetics, Cell Biology and Anatomy, College of Medicine, University of Nebraska Medical Center, Omaha, United States

   Contribution

   Data curation, Formal analysis

   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

    Contribution

    Resources, Project administration

    Competing interests

    No competing interests declared

11. Michael S Toss

    Department of Histopathology, Nottingham University Hospital NHS Trust, City Hospital Campus, Nottingham, United Kingdom

    Contribution

    Data curation, Formal analysis

    Competing interests

    No competing interests declared

12. Jane L Meza

    1. Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, United States
    2. Department of Biostatistics, College of Public Health, University of Nebraska Medical Center, Omaha, United States

    Contribution

    Formal analysis, Validation

    Competing interests

    No competing interests declared

13. Wallace B Thoreson

    Stanley M. Truhlsen Eye Institute, University of Nebraska Medical Center, Omaha, United States

    Contribution

    Data curation, Formal analysis

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-7104-042X

14. Donald W Coulter

    1. Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, United States
    2. Department of Pediatrics, University of Nebraska Medical Center, Omaha, United States

    Contribution

    Formal analysis

    Competing interests

    No competing interests declared

15. Emad A Rakha

    Department of Histopathology, Nottingham University Hospital NHS Trust, City Hospital Campus, Nottingham, United Kingdom

    Contribution

    Formal analysis, Validation

    Competing interests

    No competing interests declared

16. Vimla Band

    1. Department of Genetics, Cell Biology and Anatomy, College of Medicine, University of Nebraska Medical Center, Omaha, United States
    2. Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, United States

    Contribution

    Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing – review and editing

    For correspondence

    vband@unmc.edu

    Competing interests

    received funding from Nimbus Therapeutics for an unrelated project

    "This ORCID iD identifies the author of this article:" 0000-0003-2014-7205

17. Hamid Band

    1. Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, United States
    2. Department of Genetics, Cell Biology and Anatomy, College of Medicine, University of Nebraska Medical Center, Omaha, United States
    3. Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, United States
    4. Department of Pathology & Microbiology, College of Medicine, University of Nebraska Medical Center, Omaha, United States

    Contribution

    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

    For correspondence

    hband@unmc.edu

    Competing interests

    received funding from Nimbus Therapeutics for an unrelated project

    "This ORCID iD identifies the author of this article:" 0000-0002-4996-9002

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