The deubiquitinating enzyme USP15 stabilizes ERα and promotes breast cancer progression

Breast cancer has the highest incidence and mortality in women worldwide. There are 70% of breast cancers considered as estrogen receptor α (ERα) positive. Therefore, the ERα-targeted therapy has become one of the most effective solution for patients with breast cancer. Whereas a better understanding of ERα regulation is critical to shape evolutional treatments for breast cancer. By exploring the regulatory mechanisms of ERα at levels of post-translational modifications, we identified the deubiquitinase USP15 as a novel protector for preventing ERα degradation and a critical driver for breast cancer progression. Specifically, we demonstrated that USP15 promoted the proliferation of ERα⁺, but not ERα^- breast cancer, in vivo and in vitro. Meanwhile, USP15 knockdown notably enhanced the antitumor activities of tamoxifen on breast cancer cells. Importantly, USP15 knockdown induced the downregulation of ERα protein via promoting its K48-linked ubiquitination, which is required for proliferative inhibition of breast cancer cells. These findings not only provide a novel treatment for overcoming resistance to endocrine therapy, but also represent a therapeutic strategy on ERα degradation by targeting USP15-ERα axis.Subject terms: Breast cancer, Translational research     

MINDY1 promotes breast cancer cell proliferation by stabilizing estrogen receptor α

Breast cancer is the most commonly diagnosed malignant tumor among females. Estrogen receptor α (ERα) is initially expressed in 70% of breast cancers and is a well-known target of endocrine therapy for ERα-positive breast cancer. In the present study, we identified MINDY1, a member belongs to the motif interacting with Ubcontaining novel DUB family (MINDY), as a potential deubiquitylase of ERα in breast cancer. There was a positive correlation between ERα and MINDY1 protein levels in human breast cancer tissues. We found that high expression of MINDY1 was associated with poor prognosis. MINDY1 interacted with ERα, thereby mediating the deubiquitination of ERα and increased its stability in a deubiquitylation activity-dependent manner. MINDY1 depletion significantly decreased the ERα protein level and ERα signaling activity in breast cancer cells. Specifically, MINDY1 associated with the N-terminal of ERα via its catalytic domain, thus inhibiting K48-specific poly-ubiquitination process on ERα protein. In addition, MINDY1 depletion led to growth inhibition and cell cycle arrest of ERα-positive breast cancer cells. Finally, overexpression of ERα could rescue the MINDY1 depletion-induced growth inhibition both in vitro and in vivo, suggesting that MINDY1 promotes breast carcinogenesis through increasing ERα stability. Overall, our study proposed a novel post-translational mechanism of ERα in supporting breast cancer progression. Targeting the MINDY1 may prove to be a promising strategy for patients with ERα-positive breast cancer.Subject terms: Cancer, Ubiquitylation     

An epigenomic approach to therapy for tamoxifen-resistant breast cancer

Tamoxifen has been a frontline treatment for estrogen receptor alpha (ERα)-positive breast tumors in premenopausal women. However, resistance to tamoxifen occurs in many patients. ER still plays a critical role in the growth of breast cancer cells with acquired tamoxifen resistance, suggesting that ERα remains a valid target for treatment of tamoxifen-resistant (Tam-R) breast cancer. In an effort to identify novel regulators of ERα signaling, through a small-scale siRNA screen against histone methyl modifiers, we found WHSC1, a histone H3K36 methyltransferase, as a positive regulator of ERα signaling in breast cancer cells. We demonstrated that WHSC1 is recruited to the ERα gene by the BET protein BRD3/4, and facilitates ERα gene expression. The small-molecule BET protein inhibitor JQ1 potently suppressed the classic ERα signaling pathway and the growth of Tam-R breast cancer cells in culture. Using a Tam-R breast cancer xenograft mouse model, we demonstrated in vivo anti-breast cancer activity by JQ1 and a strong long-lasting effect of combination therapy with JQ1 and the ER degrader fulvestrant. Taken together, we provide evidence that the epigenomic proteins BRD3/4 and WHSC1 are essential regulators of estrogen receptor signaling and are novel therapeutic targets for treatment of Tam-R breast cancer.     

Modification of ERα by UFM1 Increases Its Stability and Transactivity for Breast Cancer Development

The post-translational modification (e.g., phosphorylation) of estrogen receptor α (ERα) plays a role in controlling the expression and subcellular localization of ERα as well as its sensitivity to hormone response. Here, we show that ERα is also modified by UFM1 and this modification (ufmylation) plays a crucial role in promoting the stability and transactivity of ERα, which in turn promotes breast cancer development. The elevation of ufmylation via the knockdown of UFSP2 (the UFM1-deconjugating enzyme in humans) dramatically increases ERα stability by inhibiting ubiquitination. In contrast, ERα stability is decreased by the prevention of ufmylation via the silencing of UBA5 (the UFM1-activating E1 enzyme). Lys171 and Lys180 of ERα were identified as the major UFM1 acceptor sites, and the replacement of both Lys residues by Arg (2KR mutation) markedly reduced ERα stability. Moreover, the 2KR mutation abrogated the 17β-estradiol-induced transactivity of ERα and the expression of its downstream target genes, including pS2, cyclin D1, and c-Myc; this indicates that ERα ufmylation is required for its transactivation function. In addition, the 2KR mutation prevented anchorage-independent colony formation by MCF7 cells. Most notably, the expression of UFM1 and its conjugating machinery (i.e., UBA5, UFC1, UFL1, and UFBP1) were dramatically upregulated in ERα-positive breast cancer cell lines and tissues. Collectively, these findings implicate a critical role attributed to ERα ufmylation in breast cancer development by ameliorating its stability and transactivity.     

OTUD7B stabilizes estrogen receptor α and promotes breast cancer cell proliferation

Breast cancer is the most common malignancy in women worldwide. Estrogen receptor α (ERα) is expressed in ∼70% of breast cancer cases and promotes estrogen-dependent cancer progression. In the present study, we identified OTU domain-containing 7B (OTUD7B), a deubiquitylase belonging to A20 subgroup of ovarian tumor protein superfamily, as a bona fide deubiquitylase of ERα in breast cancer. OTUD7B expression was found to be positively correlated with ERα in breast cancer and associated with poor prognosis. OTUD7B could interact with, deubiquitylate, and stabilize ERα in a deubiquitylation activity-dependent manner. Depletion of OTUD7B decreased ERα protein level, the expression of ERα target genes, and the activity of estrogen response element in breast cancer cells. In addition, OTUD7B depletion significantly decreased ERα-positive breast cancer cell proliferation and migration. Finally, overexpression of ERα could rescue the suppressive effect induced by OTUD7B depletion, suggesting that the ERα status was essential to the function of OTUD7B in breast carcinogenesis. In conclusion, our study revealed an interesting post-translational mechanism between ERα and OTUD7B in ERα-positive breast cancer. Targeting the OTUD7B–ERα complex may prove to be a potential approach to treat patients with ERα-positive breast cancer.Subject terms: Breast cancer, Cell growth     

ERβ Isoforms Have Differential Clinical Significance in Breast Cancer Subtypes and Subgroups

Simple SummaryERβ, an ER subtype first identified in 1996, is significantly expressed in ERα-negative breast cancer (BCa) and TNBC. Many studies investigated mostly ERβ1 protein expression in the entire cohort of BCa, and the results are inconsistent. In this study, we simultaneously investigated both ERβ mRNA and three ERβ 1, 2, and 5 protein isoforms in various subtypes and subgroups of BCa. Each ERβ isoform’s mRNA and protein expression seemingly plays a significant role in BCa subtypes and subgroups, and ERβ2 mRNA expression is risk factor for poor outcome. Studies in a large cohort of BCa are needed to explore the potential usefulness of ERβ as a prognostic and predictive marker and a therapeutic target in BCa. Furthermore, the standardization of a ERβ testing protocol may be required for ERβ testing to be utilized in a clinical setting.AbstractERβ, an ER subtype first identified in 1996, is highly expressed in different types of BCa including ERα-negative BCa and TNBC. Many studies on ERβ expression investigated mostly on ERβ1 protein expression in ERα-positive and ERα-negative BCa combined. The results are conflicting. This may be due to the complexity of ERβ isoforms, subject heterogeneity, and various study designs targeting different ERβ isoforms and either ERβ protein or mRNA expression, as well as to the lack of a standardized testing protocol. Herein, we simultaneously investigated both mRNA and protein expression of ERβ isoforms 1, 2, and 5 in different BCa subtypes and clinical characteristics. Patient samples (138) and breast cancer cell lines (BCC) reflecting different types of BCa were tested for ERα and ERβ mRNA expression using quantitative real-time PCR, as well as for protein expression of ERα, ERβ1, ERβ2, and ERβ5 isoforms, PR, HER2/neu, Ki-67, CK 5/6, and p53 using immunohistochemistry. Associations of ERβ isoform expression with clinical characteristics and overall survival (OS) were analyzed. ERβ1, 2, and 5 isoforms are differentially expressed in different BCa subtypes including ERα-negative and TNBC. Each ERβ isoform seemingly plays a distinct role and is associated with clinical tumor characteristics and patient outcomes. ERβ isoform expression is significantly associated with >15% Ki-67 positivity and poor prognostic markers, and it predicts poorer OS, mostly in the subgroups. High ERβ2 and 5 isoform expression in ERα-negative BCa and TNBC is predictive of poor OS. Further investigation of ERβ isoforms in a larger cohort of BCa subgroups is needed to evaluate the role of ERβ for the potential usefulness of ERβ as a prognostic and predictive marker and for therapeutic use. The inconsistent outcomes of ERβ isoform mRNA or protein expression in many studies suggest that the standardization of ERβ testing would facilitate the use of ERβ in a clinical setting.     

LKB1 Catalytic Activity Contributes to Estrogen Receptor α Signaling

The tumor suppressor serine-threonine kinase LKB1 is mutated in Peutz-Jeghers syndrome (PJS) and in epithelial cancers, including hormone-sensitive organs such as breast, ovaries, testes, and prostate. Clinical studies in breast cancer patients show low LKB1 expression is related to poor prognosis, whereas in PJS, the risk of breast cancer is similar to the risk from germline mutations in breast cancer (BRCA) 1/BRCA2. In this study, we investigate the role of LKB1 in estrogen receptor α (ERα) signaling. We demonstrate for the first time that LKB1 binds to ERα in the cell nucleus in which it is recruited to the promoter of ERα-responsive genes. Furthermore, LKB1 catalytic activity enhances ERα transactivation compared with LKB1 catalytically deficient mutants. The significance of our discovery is that we demonstrate for the first time a novel functional link between LKB1 and ERα. Our discovery places LKB1 in a coactivator role for ERα signaling, broadening the scientific scope of this tumor suppressor kinase and laying the groundwork for the use of LKB1 as a target for the development of new therapies against breast cancer.     

Immunoreactivities of AR,ERα, ERβ and aromatase in the nuptial pad of Chinese brown frog (Rana dybowskii) during pre-hibernation and the breeding period

There is a prominent local raised pad called nuptial pad on the forelimb of Chinese brown frog (Rana dybowskii), which is hypothetically concluded as an enhancement of the grip and a spreader of pheromone during the amplexus. In this study, we investigated the immunolocalization and protein expression levels of androgen receptors (AR), estrogen receptor α (ERα), ERβ and aromatase in the nuptial pad of R. dybowskii during pre-hibernation and the breeding period. Histologically, the annual development of the nuptial pad in R. dybowskii is manifested as the larger area of specialized mucous gland and the longer length of papillary epidermal projection during the breeding period. AR, ERα, ERβ and aromatase are present in the stratum granulosum, stratum spinosum, stratum basale and the secretory portion of specialized mucous glands during both periods. Western blotting results confirmed that AR, ERα and ERβ protein levels are higher during pre-hibernation than those during the breeding season. These results suggest that nuptial pad is the direct target organ of androgen and estrogen. Androgen may participate in the regulation of annual development and glandular function of nuptial pad, and estrogen may play an endocrine, autocrine or paracrine role during pre-hibernation and the breeding period.Key words: Androgen receptor, aromatase, estrogen receptor, nuptial pad, Rana dybowskii.     

Aptamer-Assisted Detection of the Altered Expression of Estrogen Receptor Alpha in Human Breast Cancer

An increase in the expression of estrogen receptors (ER) and the expanded population of ER-positive cells are two common phenotypes of breast cancer. Detection of the aberrantly expressed ERα in breast cancer is carried out using ERα-antibodies and radiolabelled ligands to make decisions about cancer treatment and targeted therapy. Capitalizing on the beneficial advantages of aptamer over the conventional antibody or radiolabelled ligand, we have identified a DNA aptamer that selectively binds and facilitates the detection of ERα in human breast cancer tissue sections. The aptamer is identified using the high throughput sequencing assisted SELEX screening. Biophysical characterization confirms the binding and formation of a thermodynamically stable complex between the identified DNA aptamer (ERaptD4) and ERα (Ka = 1.55±0.298×10⁸ M^-1; ΔH = 4.32×10⁴±801.1 cal/mol; ΔS = -108 cal/mol/deg). Interestingly, the specificity measurements suggest that the ERaptD4 internalizes into ERα-positive breast cancer cells in a target-selective manner and localizes specifically in the nuclear region. To harness these characteristics of ERaptD4 for detection of ERα expression in breast cancer samples, we performed the aptamer-assisted histochemical analysis of ERα in tissue samples from breast cancer patients. The results were validated by performing the immunohistochemistry on same samples with an ERα-antibody. We found that the two methods agree strongly in assay output (kappa value = 0.930, p-value <0.05 for strong ERα positive and the ERα negative samples; kappa value = 0.823, p-value <0.05 for the weak/moderate ER+ve samples, n = 20). Further, the aptamer stain the ERα-positive cells in breast tissues without cross-reacting to ERα-deficient fibroblasts, adipocytes, or the inflammatory cells. Our results demonstrate a significant consistency in the aptamer-assisted detection of ERα in strong ERα positive, moderate ERα positive and ERα negative breast cancer tissues. We anticipate that the ERaptD4 aptamer targeting ERα may potentially be used for an efficient grading of ERα expression in cancer tissues.     

Extracellular ATP promotes breast cancer chemoresistance via HIF-1α signaling

We have previously demonstrated that extracellular adenosine 5''-triphosphate (ATP) promotes breast cancer cell chemoresistance. However, the underlying mechanism remains unclear. Using a cDNA microarray, we demonstrated that extracellular ATP can stimulate hypoxia-inducible factor (HIF) signaling. In this study, we report that hypoxia-inducible factor 1α (HIF-1α) was upregulated after ATP treatment and mediated the ATP-driven chemoresistance process. We aimed to investigate the mechanisms and identify potential clinically relevant targets that are involved. Using mass spectrometry, we found that aldolase A (ALDOA) interacts with HIF-1α and increases HIF-1α expression. We then demonstrated that STAT3-ALDOA mediates ATP-HIF-1α signaling and upregulates the HIF-1 target genes adrenomedullin (ADM) and phosphoinositide-dependent kinase-1 (PDK1). Moreover, we show that PI3K/AKT acts upstream of HIF-1α in ATP signaling and contributes to chemoresistance in breast cancer cells. In addition, HIF-1α-knockdown or treatment with direct HIF inhibitors combined with the ATP hydrolase apyrase in MDA-MB-231 cells induced enhanced drug sensitivity in nude BALB/c mice. We then used in vitro spheroid formation assays to demonstrate the significance of ATP-HIF-1α in mediating chemoresistance. Furthermore, considering that indirect HIF inhibitors are effective in clinical cancer therapy, we treated tumor-bearing BALB/c mice with STAT3 and PI3K/AKT inhibitors and found that the dual-targeting strategy sensitized breast cancer to cisplatin. Finally, using breast cancer tissue microarrays, we found that ATP-HIF-1α signaling is associated with cancer progression, poor prognosis, and resistance to chemotherapy. Taken together, we suggest that HIF-1α signaling is vital in ATP-driven chemoresistance and may serve as a potential target for breast cancer therapies.Subject terms: Breast cancer, Cell signalling     

Expression of Estrogen-Related Gene Markers in Breast Cancer Tissue Predicts Aromatase Inhibitor Responsiveness

Aromatase inhibitors (AIs) are the most effective class of drugs in the endocrine treatment of breast cancer, with an approximate 50% treatment response rate. Our objective was to determine whether intratumoral expression levels of estrogen-related genes are predictive of AI responsiveness in postmenopausal women with breast cancer. Primary breast carcinomas were obtained from 112 women who received AI therapy after failing adjuvant tamoxifen therapy and developing recurrent breast cancer. Tumor ERα and PR protein expression were analyzed by immunohistochemistry (IHC). Messenger RNA (mRNA) levels of 5 estrogen-related genes–AKR1C3, aromatase, ERα, and 2 estradiol/ERα target genes, BRCA1 and PR–were measured by real-time PCR. Tumor protein and mRNA levels were compared with breast cancer progression rates to determine predictive accuracy. Responsiveness to AI therapy–defined as the combined complete response, partial response, and stable disease rates for at least 6 months–was 51%; rates were 56% in ERα-IHC-positive and 14% in ERα-IHC-negative tumors. Levels of ERα, PR, or BRCA1 mRNA were independently predictive for responsiveness to AI. In cross-validated analyses, a combined measurement of tumor ERα and PR mRNA levels yielded a more superior specificity (36%) and identical sensitivity (96%) to the current clinical practice (ERα/PR-IHC). In patients with ERα/PR-IHC-negative tumors, analysis of mRNA expression revealed either non-significant trends or statistically significant positive predictive values for AI responsiveness. In conclusion, expression levels of estrogen-related mRNAs are predictive for AI responsiveness in postmenopausal women with breast cancer, and mRNA expression analysis may improve patient selection.