Regulation of ERα Stability and Estrogen Signaling in Breast Cancer by HOIL-1

Estrogen receptor α (ERα) is the major driver for breast tumor carcinogenesis and progression, while ERα positive breast cancer is the major subtype in breast malignancies, which account for 70% breast cancers in patients. The success of endocrine therapy such as tamoxifen is one of the biggest breakthroughs in breast cancer treatments. However, the endocrine therapy resistance is a headache problem in breast cancer. Further mechanisms need to be identified to the effect of ERα signaling in controlling breast cancer progression and drug resistance. HOIL-1 was firstly identified as the ERα transcriptional co-activator in modulating estrogen signaling in breast cancer. In our current study, we showed that HOIL-1, which was elevated in breast cancer, related to good prognosis in ERα positive breast cancer, but correlated with poor outcome in endocrine-treated patients. HOIL-1 was required for ERα positive breast cancer proliferation and clone formation, which effect could be rescued by further ERα overexpression. Further mechanism studies showed that HOIL-1 is required for ERα signaling activity in breast cancer cells. HOIL-1 could interact with ERα in the cytosol and modulate ERα stability via inhibiting ERα K48-linked poly-ubiquitination. Thus, our study demonstrated a novel post-translational modification in ERα signaling, which could provide novel strategy for ERα-driven breast cancer therapy.


INTRODUCTION
The ubiquitination process functions to modulate the protein disposal and function in eukaryotic cell hemostasis (1,2). E3 ubiquitin ligases promote the transfer of ubiquitin from the E2 ubiquitin conjugating enzymes to target protein substrates via their lysine residues (3). The ubiquitination can be classifies as several types through the lysine residues on the ubiquitin proteins, including K63, K48, linear ubiquitination and mono-ubiquitination (2). The ubiquitination process was firstly discovered as a target for proteins degradations (4). However, the studies in recent years showed that the ubiquitin systems play important role in protein functions in a group of regulatory pathways, such as signaling transduction, DNA damage response and endocytosis (5,6).
The ubiquitin process involves the coordinated reactions of E1 ubiquitin-activation enzyme, E2 ubiquitin enzymes and E3 ubiquitin ligases (4). It has been show that the E3 ubiquitin ligases are the key factors, which specifically interact with certain substrates and E2 enzymes for ubiquitin transferring. According to the functional domains of E3 ubiquitin ligases, they can be separated into four groups: HECT (Homologous to the E6-AP Carboxyl Terminus), RING, U-box and PHD-finger family (7). The RING family proteins are composed of more than 700 different proteins, most of which are not well investigated (8). Based on current understanding of RING proteins, the RING family members are involved in several cell physiological functions, including cell proliferation, protein trafficking and DNA repair (9,10). One of the most thoroughly studied proteins is BRCA1 (RNF53), which participates in DNA repair, gene expression and protein ubiquitination (11)(12)(13). In clinics, BRCA1 mutations are proved to be the major driver for familial breast cancer and ovarian cancer (14).
Recent studies showed that RING family proteins play important role in tumor carcinogenesis and progression (15). Several atypical ubiquitination manners, which modified quite a few nuclear receptors, exhibited regulatory functions in cancer signaling transductions. For example, RNF31 and RNF8 function ubiquitin ligases, which promote the monoubiquitination of ERa, enhance ERa protein stability and estrogen signaling activity in breast cancer cancers (16,17). Our previous studies also showed TRIM56, one of the RING family members, promotes ERa K48-linked ubiquitination and estrogen signaling in breast malignancies (18). In the current study, we identify HOIL-1 (Haem-oxidized IRP2 Ubiquitin Ligase-1) functions as an ubiquitin ligase to modulate ERa protein stability. HOIL-1 is composed of 510 amino acids, which is mainly localized in the cytoplasm in breast cancer cells. HOIL-1 associates with ERa AF1 domain via its RING domain and prolongs ERa protein stability, which subsequently enhances ERa target gene expression and breast cancer cell proliferation.

RNA Extraction and qPCR Analysis
RNeasy plus mini kits were used to extract total RNA (Qiagen). Real-time PCR was performed as previously described (18).

Quantification of Cell Viability
MCF-7 and T47D cells were transfected with siHOIL-1 or siControl in 24-well plates. Twenty-four hours after transfection, the cells number was countered and 4,000 cells were seeded into 96-well plates. The relative cell viability was measured at indicated time points. Cell numbers were determined using the WST-1 cell proliferation reagent as previously described (22).

Luciferase Assay
The luciferase activity of estrogen signaling activity was performed using the Dual-Luciferase Reporter kit (Promega, Germany). The ERE luciferase reporter was transfected together with the Renilla plasmid into the cells. Luciferase activity was measured after 24 h.

Co-Immunoprecipitation Assay
Immunoprecipitation was performed as described in previous study. The MCF-7 total cell lysls were pre-cleared with rabbit IgG for 2 h and subsequently immunoprecipitated with ERa antibody (D8H8, #8644) over night, while rabbit IgG (Santa Cruz) was used as the negative control. The bounded protein was analyzed by Anti-HOIL-1 antibody (Ab108479). For the overexpression experiment, HEK293 cells were transfected with 5 ug FLAG-HOIL-1 (Full length or deletion domains) and ERa plasmid (Full length or deletion domains) in 10 cm dish. Cell lysates were pre-cleared with IgG and subsequently incubate with Flag (Ab49763) antibody, while rabbit IgG was used as the negative control. The bound proteins were analyzed by western blotting.

Poly-Ubiquitination Detection Assay
To directly detect the enriched overall ubiquitinated or K63ubiqutinated ERa from the cell extracts, HEK293 cells were transfected with 4 ug Ub or 4 ug K63 Ubi plasmid, 2 ug ERa together with 0.5 ug Flag-HOIL-1 or Flag-vector. After 48 h, total protein was extracted and pre-cleared with 20 ul protein A (santa cruz, SC-2001) for 2 h. The supernatant was collected and immunoprecipitated by ERa antibody. Western blot with HA antibody was performed to detect K48, K63 poly-ubiquitinated or mono-ubiquitinated ERa.
Immunofluorescence Assay MCF-7 cells were fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100 for 5 min, and blocked by 5% BSA in PBS for 1 h. A rabbit anti-HOIL-1 polyclonal antibody (Ab108479) and mouse anti-ERa monoclonal antibodies (SC-56833) were used, followed by Alexa Flour 647 (Invitrogen) anti-rabbit antibody and FITC-conjugated anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA). As negative controls, the samples were incubated with the secondary antibodies without primary antibodies. Images were acquired under conditions fulfilling the Nyquist criterion using Nikon A+ laser scanning confocal system with a 60× oil NA1.4 objective and pinhole size of 1.0 Airy Unit. The acquired pictures were further processed and assembled using ImageJ.

Statistics
Student's t-test, Pearson correlation coefficient, and Cox regression analysis were used for comparisons. A P-value of <0.05 was considered to be significant.

HOIL-1 Is Elevated in Breast Cancer and Relates to Short Endocrine Treatment Outcome in Human Breast Cancer Tumors
We firstly analyzed the HOIL-1 expression level from public available database. The TCGA database (https://tcga-data.nci.nih.gov) showed that HOIL-1 was increased in breast cancer compared with breast tissues (Fold change = 1.35; P <0.001) ( Figure 1A). In breast cancer subtype analysis, the data showed that HOIL-1 is all breast subtypes, including luminal type, HER2 positive type and triple negative type ( Figure 1B). When we analyzed the HOIL-1 effect on breast cancer patient survival from KMPLOT database (https:// kmplot.com), we observed that HOIL-1 related to longer progression survival in all patents and luminal type patients ( Figures 1C, D). HIOL-1 expression also correlated with good prognosis in triple negative breast cancers ( Figure S1A). However, HOIL specially correlated with poor survival in endocrine-treated patients ( Figure 1E). Besides, the gene expression analysis from the TCGA database showed that HOIL-1 was positively correlated with ERa target gene expression including GREB1 and TFF1 in breast tumors (P <0.01, R = 0.17; P = 0.008, R = 0.08 respectively) ( Figures  1F, G). These clinical data showed the consistent trend with previous reports that HOIL-1 might promote ERa signaling and endocrine resistance (23,24).

HOIL-1 Depletion Inhibits ERa Protein and ERa Signaling in Breast Cancer Cells
In order to uncover the role of HOIL-1 in ERa signaling in breast cancer cells, we depleted HOIP in MCF-7 cells. HOIL-1 depletion via two independent siRNA showed that HOIL-1 knocking-down decreases ERa protein level and ERa target gene expression, including PS2, GREB1 and PDZK1 (Figures 2A, B). Besides, HOIL-1 depletion could decrease ERa protein level in both vehicle and E2-treated condition in MCF-7 and T47D cells ( Figures 2C, D). Consistent with this, HOIL-1 depletion also decreased ERa target gene expression, such as PS2, GREB1 and PDZK1 in both MCF7 and T47D cells ( Figures 2E, F). In order to determine if HOIL-1 knocking down could affect ERa transcriptional activity, we measured estrogen response element (ERE) luciferase activity in both MCF-7 and T47D cells. It showed that HOIL-1 depletion decreases ERE luciferase activity in both MCF7 and T47D cells ( Figures 2G, H). All these data indicate HOIL-1 is required for ERa signaling in breast cancer cells. In addition, we also found HOIL-1 depletion facilitated P53 protein level and its target gene expression (Figures S1B, C). We further investigated the function of HOIL-1 in ER negative breast cancer cells. In MDAMB231 cells, HOIL-1 depletion inhibited cell invasion ( Figures S2A-C). Besides, HOIL-1 depletion also inhibited cell proliferation and migration in MDAMB231 cells ( Figures S2D, E).

HOIL-1 Is Mainly Localized in the Cytoplasm and Modulates ERa Stability
In order to investigate the role of HOIL-1 in breast cancer cells, we depleted HOIL-1 in both MCF-7 and T47D cells. WST assay showed that HOIL-1 depletion significantly decreased breast cancer cell proliferation in MCF-7 and T47D cells In MTT assay ( Figures 3A, B). Besides, the EdU (5-ethynyl-2doxyuridine) incorporation assay showed that HOIL-1 depletion significantly decreased the EdU positive cells in MCF-7 and T47D cells ( Figures 3C, D). In the wound-healing Cell lysates were prepared for Western blot analysis. The results are representative for three independent experiments. The ERa relative density was measured by Image J software. ** means the P value is less than 0.01, but more than 0.001. *** means P value is less than 0.001. assay, we found that HOIL-1 was required for breast cancer cell migration in MCF-7 cells ( Figure 3E).
Based on the significant impact in breast cancer cell phenotype by HOIL-1, we further investigated the potential mechanism. Nuclear and cytoplasm separation showed that HOIL-1 is mainly localized in the cytoplasm, while ERa is mainly localized in the nuclear ( Figure 4A). Immuno-staining showed the same trend that ERa is located mainly in the nuclear, while HOIL-1 is mainly in the cytoplasm ( Figure 4B). Then we investigated the potential role of HOIL-1 on ERa stability. Since ERa could regulate its own expression in MCF-7 cells, which make it difficult to identify the direct effect of HOIL-1 on ERa protein or mRNA (25). We utilize HEK293 cells to measure the protein stability of ERa via co-transfection with HOIL-1. In the protein stability assay, HOIL-1 could stabilize ERa. However, with the presence of a proteasome inhibitor MG132, the stabilization effect on ERa protein level could not further been increased ( Figure 4C). With the inhibition of protein synthesis cycloheximde, HOIL-1 could significantly increases ERa stability in HEK293 cells ( Figures  4D, E). All these data indicate that HOIL-1 could prolong ERa stability.

HOIL-1 Associates With ERa AF1 Domain Through Its RING Domain and Stabilizes ERa Possibly by Promoting Mono-Ubiquitination
ERa is composed of three functional domains: AF1 domain, DNA binding domain and AF2 domain ( Figure 5A). HOIL-1 is composed of three function domains, including UBL domain, NZF domain and RBR domain ( Figure 5B). Endogenous immuno-precipitation shows that HOIL-1 could associate with ERa in MCF-7 cells (Figures 5C, D). Then the full length of ERa or ERa deletion constructs is transfected together with HOIL-1 in HEK293 cells. Co-IP indicates that HOIL-1 associates with ERa AF1 domain (Figures 5E, F). Besides, the full length of HOIL-1 or HOIL-1 deletion constructs is transfected together with ERa full length. Co-IP shows that ERa associates with HOIL-1 RBR domain ( Figure 5G). Further experiments are carried out to measure ERa ubiquitination. Ubiquitin-based immuno-precipitation assay show that HOIL-1 could inhibit ERa overall ubiquitination ( Figure 6A). K48-linked ubiquitin assay shows that HOIL-1 could inhibit ERa K48-linked ubiquitination ( Figure 6B).

DISCUSSION
In this study, we identified the RING family E3 ubiquitin ligase HOIL-1, which was highly expressed in human breast cancer samples, facilitated ERa signaling and breast cancer progression via post-translational modification. HOIL-1 associated with ERa and inhibits ERa poly-ubiquitination and degradation ( Figure 6C). ERa was firstly cloned from MCF-7 cell in 1985 (26). About 70% of breast cancers are ERa positive, while the risk of breast caner is also correlated with the ERa expression level in breast tissue (27). Higher levels of ERa expression in breast cancer cell can lead to increased estrogen-independent activity of ERa (28). ER-positive cancer depends on ERa signaling for cell growth, which makes ERa a suitable target for breast cancer therapy. For ERa positive breast cancer patients, selective estrogen receptor modulators, such as tamoxifen, are standard endocrine treatment. However, endocrine resistance is one important issue in breast cancer therapy. Interesting, still most of the endocrine resistant breast cancer are ERa positive, which might indicate that ERa might play important role in mediating tamoxifen resistance. Modulating ERa protein stability could be one plausible strategy to overcome endocrine resistance.
There are about 500-1,000 different E3 ubiquitin ligases. Among these families, RING family is the largest. RING-In-Between-RING (RBR) E3 ligase is a subfamily of the RING family (29). One of the important functions of RBRs is the modulation of NFKB signaling and nuclear receptors (30)(31)(32). Recent studies reveal that several RBRs are necessary for ERa signaling activation and breast cancer proliferation. For example, RNF8 could associate with ERa and functions as a co-activator for ERa target genes. Besides, RNF8 could also monoubiquitinate ERa and promote ERa protein stability (17). Our previous work focused on several E3 ubiquitin ligases, which were able to enhance ERa signaling activity either via genomic regulation or post-translational modifications, including RNF31, RNF168 and SMURF1 (9,20). Here, we identifies HOIL-1, which is one interaction protein with RNF31 could modulate ERa stability via inhibiting ERa poly-ubiquitination.
HOIL-1 was firstly identified in a yeast two-hybrid screen as a PKC interaction protein (33). The C-terminal part of the protein contains the RBR domain, while the N-terminal contains UBL domain and RZF domain (34). The RBR domain was regarded as functional domain for ubiquitin ligation, while the UBL domain could interact with 26 proteasome (35). Previous studies showed that HOIL1-could be an important marker for poor tamoxifen response (24). Nina et al. showed HOIL-1 could promoter ERa gene expression and also co-located with ERa at ERa target gene promoter regions (23). However, our study confirms that HOIL-1 is a positive modulator for ERa signaling, but through different mechanisms. Our immuno-staining indicates that HOIL-1 is mainly localized in the cytosol, not in the nuclear in MCF-7 cells. Since ERa is mostly degraded in the cytosol, HOIL-1 could exert its dual function in ERa signaling. When HOIL-1 in the nuclear, it co-activates ERa gene expression, while HOIL-1 is in the cytosol, it associates with ERa and enhances ERa stability.
Our study identifies that the RBR family protein HOIL-1 could modulate ERa signaling and breast cancer progression through a post-translational manner. Our study strengthens the critical role of HOIL-1 in ERa signaling and improves the understanding of HOIL-1 in both genomic regulation and post-translational regulation of ERa pathway. As such an important regulator of ERa signaling, HOIL-1 could be an important target for ERa positive breast cancer therapy.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AUTHOR CONTRIBUTIONS
JD performed most of the bench work. PK supervised the process of the study and performed the manuscript writing. JD participated in western blot and real-time PCR work. PK performed the prognosis data analysis. All authors contributed to the article and approved the submitted version.

FUNDING
The project is supported by the Young Scientist grant of Nanchang University (PK).

ACKNOWLEDGMENTS
We thank all the members of the oncology center of the First Affiliated Hospital of Nanchang University, Medical College of Nanchang University for sharing valuable material and research support.