Lupus susceptibility gene Esrrg modulates regulatory T cells through mitochondrial metabolism.

Estrogen-related receptor γ (Esrrg) is a murine lupus susceptibility gene associated with T cell activation. Here, we report that Esrrg controls Tregs through mitochondria homeostasis. Esrrg deficiency impaired the maintenance and function of Tregs, leading to global T cell activation and autoimmunity in aged mice. Further, Esrrg-deficient Tregs presented an impaired differentiation into follicular Tregs that enhanced follicular helper T cells' responses. Mechanistically, Esrrg-deficient Tregs presented with dysregulated mitochondria with decreased oxygen consumption as well as ATP and NAD+ production. In addition, Esrrg-deficient Tregs exhibited decreased phosphatidylinositol and TGF-ß signaling pathways and increased mTOR complex 1 activation. We found that the expression of human ESRRG, which is high in Tregs, was lower in CD4+ T cells from patients with lupus than in healthy controls. Finally, knocking down ESRRG in Jurkat T cells decreased their metabolism. Together, our results reveal a critical role of Esrrg in the maintenance and metabolism of Tregs, which may provide a genetic link between lupus pathogenesis and mitochondrial dysfunction in T cells.

High IRF8 expression correlates with CD8 T cell infiltration and is a predictive biomarker of therapy response in ER-negative breast cancer.

BACKGROUND: Characterization of breast cancer (BC) through the determination of conventional markers such as ER, PR, HER2, and Ki67 has been useful as a predictive and therapeutic tool. Also, assessment of tumor-infiltrating lymphocytes has been proposed as an important prognostic aspect to be considered in certain BC subtypes. However, there is still a need to identify additional biomarkers that could add precision in distinguishing therapeutic response of individual patients. To this end, we focused in the expression of interferon regulatory factor 8 (IRF8) in BC cells. IRF8 is a transcription factor which plays a well-determined role in myeloid cells and that seems to have multiple antitumoral roles: it has tumor suppressor functions; it acts downstream IFN/STAT1, required for the success of some therapeutic regimes, and its expression in neoplastic cells seems to depend on a cross talk between the immune contexture and the tumor cells. The goal of the present study was to examine the relationship between IRF8 with the therapeutic response and the immune contexture in BC, since its clinical significance in this type of cancer has not been thoroughly addressed. METHODS: We identified the relationship between IRF8 expression and the clinical outcome of BC patients and validated IRF8 as predictive biomarker by using public databases and then performed in silico analysis. To correlate the expression of IRF8 with the immune infiltrate in BC samples, we performed quantitative multiplex immunohistochemistry. RESULTS: IRF8 expression can precisely predict the complete pathological response to monoclonal antibody therapy or to select combinations of chemotherapy such as FAC (fluorouracil, adriamycin, and cytoxan) in ER-negative BC subtypes. Analysis of immune cell infiltration indicates there is a strong correlation between activated and effector CD8+ T cell infiltration and tumoral IRF8 expression. CONCLUSIONS: We propose IRF8 expression as a potent biomarker not only for prognosis, but also for predicting therapy response in ER-negative BC phenotypes. Its expression in neoplastic cells also correlates with CD8+ T cell activation and infiltration. Therefore, our results justify new efforts towards understanding mechanisms regulating IRF8 expression and how they can be therapeutically manipulated.

Tamoxifen suppresses brain metastasis of estrogen receptor-deficient breast cancer by skewing microglia polarization and enhancing their immune functions.

BACKGROUND: Brain metastasis of breast cancer exhibits exceedingly poor prognosis, and both triple negative (TN) and Her2+ subtypes have the highest incidence of brain metastasis. Although estrogen blockers are considered to be ineffective for their treatment, recent evidence indicates that estrogen blockade using tamoxifen showed certain efficacy. However, how estrogen affects brain metastasis of triple negative breast cancer (TNBC) remains elusive. METHODS: To examine the effect of estrogen on brain metastasis progression, nude mice were implanted with brain metastatic cells and treated with either estrogen supplement, tamoxifen, or ovariectomy for estrogen depletion. For clinical validation study, brain metastasis specimens from pre- and post-menopause breast cancer patients were examined for microglia polarization by immunohistochemistry. To examine the estrogen-induced M2 microglia polarization, microglia cells were treated with estrogen, and the M1/M2 microglia polarization was detected by qRT-PCR and FACS. The estrogen receptor-deficient brain metastatic cells, SkBrM and 231BrM, were treated with conditioned medium (CM) derived from microglia that were treated with estrogen in the presence or absence of tamoxifen. The effect of microglia-derived CM on tumor cells was examined by colony formation assay and sphere forming ability. RESULTS: We found that M2 microglia were abundantly infiltrated in brain metastasis of pre-menopausal breast cancer patients. A similar observation was made in vivo, when we treated mice systemically with estrogen. Blocking of estrogen signaling either by tamoxifen treatment or surgical resection of mice ovaries suppressed M2 microglial polarization and decreased the secretion of C-C motif chemokine ligand 5, resulting in suppression of brain metastasis. The estrogen modulation also suppressed stemness in TNBC cells in vitro. Importantly, estrogen enhanced the expression of signal regulatory protein α on microglia and restricted their phagocytic ability. CONCLUSIONS: Our results indicate that estrogen promotes brain metastasis by skewing polarity of M2 microglia and inhibiting their phagocytic ability, while tamoxifen suppresses brain metastasis by blocking the M2 polarization of microglia and increasing their anti-tumor phagocytic ability. Our results also highlight a potential therapeutic utility of tamoxifen for treating brain metastasis of hormone receptor-deficient breast cancer.

Gasdermin E deficiency attenuates acute kidney injury by inhibiting pyroptosis and inflammation.

Pyroptosis, one kind of inflammatory regulated cell death, is involved in various inflammatory diseases, including acute kidney injury (AKI). Besides Gasdermin D (GSDMD), GSDME is a newly identified mediator of pyroptosis via the cleavage of caspase-3 generating pyroptotic GSDME-N. Here, we investigated the role of GSDME in renal cellular pyroptosis and AKI pathogenesis employing GSDME-deficient mice and human tubular epithelial cells (TECs) with the interventions of pharmacological and genetic approaches. After cisplatin treatment, GSDME-mediated pyroptosis was induced as shown by the characteristic pyroptotic morphology in TECs, upregulated GSDME-N expression and enhanced release of IL-1ß and LDH, and decreased cell viability. Strikingly, silencing GSDME in mice attenuated acute kidney injury and inflammation. The pyroptotic role of GSDME was also verified in human TECs in vitro. Further investigation showed that inhibition of caspase-3 blocked GSDME-N cleavage and attenuated cisplatin-induced pyroptosis and kidney dysfunction. Moreover, deletion of GSDME also protected against kidney injury induced by ischemia-reperfusion. Taken together, the findings from current study demonstrated that caspase-3/GSDME-triggered pyroptosis and inflammation contributes to AKI, providing new insights into the understanding and treatment of this disease.

The Nuclear Receptor ESRRA Protects from Kidney Disease by Coupling Metabolism and Differentiation.

Kidney disease is poorly understood because of the organ's cellular diversity. We used single-cell RNA sequencing not only in resolving differences in injured kidney tissue cellular composition but also in cell-type-specific gene expression in mouse models of kidney disease. This analysis highlighted major changes in cellular diversity in kidney disease, which markedly impacted whole-kidney transcriptomics outputs. Cell-type-specific differential expression analysis identified proximal tubule (PT) cells as the key vulnerable cell type. Through unbiased cell trajectory analyses, we show that PT cell differentiation is altered in kidney disease. Metabolism (fatty acid oxidation and oxidative phosphorylation) in PT cells showed the strongest and most reproducible association with PT cell differentiation and disease. Coupling of cell differentiation and the metabolism was established by nuclear receptors (estrogen-related receptor alpha [ESRRA] and peroxisomal proliferation-activated receptor alpha [PPARA]) that directly control metabolic and PT-cell-specific gene expression in mice and patient samples while protecting from kidney disease in the mouse model.

Nutritional combinatorial impact on the gut microbiota and plasma short-chain fatty acids levels in the prevention of mammary cancer in Her2/neu estrogen receptor-negative transgenic mice.

Breast cancer is the second leading cause of cancer-related mortality in women. Various nutritional compounds possess anti-carcinogenic properties which may be mediated through their effects on the gut microbiota and its production of short-chain fatty acids (SCFAs) for the prevention of breast cancer. We evaluated the impact of broccoli sprouts (BSp), green tea polyphenols (GTPs) and their combination on the gut microbiota and SCFAs metabolism from the microbiota in Her2/neu transgenic mice that spontaneously develop estrogen receptor-negative [ER(-)] mammary tumors. The mice were grouped based on the dietary treatment: control, BSp, GTPs or their combination from beginning in early life (BE) or life-long from conception (LC). We found that the combination group showed the strongest inhibiting effect on tumor growth volume and a significant increase in tumor latency. BSp treatment was integrally more efficacious than the GTPs group when compared to the control group. There was similar clustering of microbiota of BSp-fed mice with combination-fed mice, and GTPs-fed mice with control-fed mice at pre-tumor in the BE group and at pre-tumor and post-tumor in the LC group. The mice on all dietary treatment groups incurred a significant increase of Adlercreutzia, Lactobacillus genus and Lachnospiraceae, S24-7 family in the both BE and LC groups. We found no change in SCFAs levels in the plasma of BSp-fed, GTPs-fed and combination-fed mice of the BE group. Marked changes were observed in the mice of the LC group consisting of significant increases in propionate and isobutyrate in GTPs-fed and combination-fed mice. These studies indicate that nutrients such as BSp and GTPs differentially affect the gut microbial composition in both the BE and LC groups and the key metabolites (SCFAs) levels in the LC group. The findings also suggest that temporal factors related to different time windows of consumption during the life-span can have a promising influence on the gut microbial composition, SCFAs profiles and ER(-) breast cancer prevention.

Downregulated METTL14 Expression Correlates with Breast Cancer Tumor Grade and Molecular Classification.

It is unclear whether the methyltransferase-like 14 (METTL14) protein promotes or suppresses cancer growth. We examined the association between METTL14 expression, cancer progression, and patient prognosis in a total of 398 breast cancer tissue specimens. Significantly fewer cancer tissue specimens compared with normal breast tissue expressed high levels of METTL14 (52.8% vs. 75.0%). METTL14 expression was negatively associated with tumor grade and positively associated with patient age, estrogen, and progesterone receptor status. High METTL14 expression was more common in luminal A and luminal B tissue (75.9% and 60.8%, respectively), compared with human epidermal growth factor receptor 2- (HER2-) enriched and triple-negative breast cancer (TNBC) samples (38.2% and 18.6%, respectively). In multiple logistic regression analysis, independent predictors of METTL14 expression in breast cancer included higher tumor grade (odds ratio (OR) = 0.494, 95% confidence interval (CI): 0.289-0.844; P = 0.010), TNBC subtype (OR = 0.109, 95% CI: 0.054-0.222; P < 0.001), and HER2-enriched subtype (OR = 0.298, 95% CI: 0.156-0.567; P < 0.001). No clear relationship was observed between patient prognosis and METTL14 expression. It appears that downregulated METTL14 expression in breast cancer is associated with tumor grade and molecular classification.

A hypertension patient-derived iPSC model demonstrates a role for G protein-coupled estrogen receptor in hypertension risk and development.

Hypertension (HTN) is a polyfactorial disease that can manifest severe cardiovascular pathologies such as heart failure or stroke. Genome-wide association studies (GWAS) of HTN indicate that single-nucleotide polymorphisms (SNPs) contribute to increased risk for HTN and resistance to some HTN drug regimens (Hiltunen TP et al., J Am Heart Assoc 4: e001521, 2015; Le MT et al., PLoS One 8: e52062, 2013; McDonough CW et al., J Hypertens 31: 698-704, 2013; Vandell AG et al., Hypertension 60: 957-964, 2012). However, cellular mechanistic insights of such SNPs remain largely unknown. Using a bank of induced pluripotent stem cells (iPSCs) derived from patients with HTN and CRISPR/Cas9-mediated gene-editing approach, we investigated the effects of a female HTN risk-associated SNP (rs1154431) of the G protein-coupled estrogen receptor (GPER) (Bassuk SS, Manson JE., Clin Chem 60: 68-77, 2014) in vascular endothelial cells. Although GPER1 deletion reduced endothelial nitric oxide synthase (eNOS) activation in iPSC-derived endothelial cells (iECs), the polymorphism itself did not significantly affect eNOS and NO production in a comparison of isogenic hemizygous iECs expressing either normal (P16) or HTN-associated (L16) GPER. Interestingly, we demonstrate for the first time that GPER plays a role in regulation of adhesion molecule expression and monocyte adhesion to iECs. Moreover, the L16 iECs had higher expression of inflammation genes than P16 iECs, implying that the risk variant may affect carrier individuals through increased inflammatory activity. This study further indicates that iPSCs are a useful platform for exploring mechanistic insights underlying hypertension GWAS endeavors.

Evidence for G-Protein-Coupled Estrogen Receptor as a Pronatriuretic Factor.

Background The novel estrogen receptor, G-protein-coupled estrogen receptor (GPER), is responsible for rapid estrogen signaling. GPER activation elicits cardiovascular and nephroprotective effects against salt-induced complications, yet there is no direct evidence for GPER control of renal Na+ handling. We hypothesized that GPER activation in the renal medulla facilitates Na+ excretion. Methods and Results Herein, we show that infusion of the GPER agonist, G1, to the renal medulla increased Na+ excretion in female Sprague Dawley rats, but not male rats. We found that GPER mRNA expression and protein abundance were markedly higher in outer medullary tissues from females relative to males. Blockade of GPER in the renal medulla attenuated Na+ excretion in females. Given that medullary endothelin 1 is a well-established natriuretic factor that is regulated by sex and sex steroids, we hypothesized that GPER activation promotes natriuresis via an endothelin 1-dependent pathway. To test this mechanism, we determined the effect of medullary infusion of G1 after blockade of endothelin receptors. Dual endothelin receptor subtype A and endothelin receptor subtype B antagonism attenuated G1-induced natriuresis in females. Unlike males, female mice with genetic deletion of GPER had reduced endothelin 1, endothelin receptor subtype A, and endothelin receptor subtype B mRNA expression compared with wild-type controls. More important, we found that systemic GPER activation ameliorates the increase in mean arterial pressure induced by ovariectomy. Conclusions Our data uncover a novel role for renal medullary GPER in promoting Na+ excretion via an endothelin 1-dependent pathway in female rats, but not in males. These results highlight GPER as a potential therapeutic target for salt-sensitive hypertension in postmenopausal women.

Scaffold attachment factor B: distribution and interaction with ERα in the rat brain.

Scaffold attachment factor (SAFB) 1 and its homologue SAFB2 are multifunctional proteins that are involved in various cellular mechanisms, including chromatin organization and transcriptional regulation, and are also corepressors of estrogen receptor alpha (ERα). Both SAFBs are expressed at high levels in the brain. However, the distributions of SAFB1 and SAFB2 have yet to be characterized in detail and it is unclear whether both proteins interact with ERα in the brain. In this study, we investigated the expression and distribution of both SAFBs and their interaction with ERα in adult male rat brain. Immunohistochemical staining showed that SAFB1 and SAFB2 have a similar distribution pattern and are widely expressed throughout the brain. Double-fluorescence immunohistochemical and immunocytochemical analyses in primary cultures showed that the two SAFB proteins are localized in nuclei of neurons, astrocytes, and oligodendrocytes. Of note, SAFB2 was also found in cytoplasmic regions in these cell lineages. Both SAFB proteins were also expressed in ERα-positive cells in the medial preoptic area (MPOA) and arcuate and ventromedial hypothalamic nuclei. Co-immunoprecipitation experiments revealed that both SAFB proteins from the MPOA reciprocally interact with endogenous ERα. These results indicate that, in addition to a role in basal cellular function in the brain, the SAFB proteins may serve as ERα corepressors in hormone-sensitive regions.

ER-depletion lowering the 'hypothalamus-uterus-kidney' axis functions by perturbing the renal ERß/Ptgds signalling pathway.

Researchers have long assumed that systematic estrogen fading might contribute to the sustained progression of menopausal degenerate syndromes, although definitive evidence has not been presented. Whether such findings represent a causal contribution or are the result of opportunistic messengers sent from the reproductive system to the brain is also a vital question. We constructed a multiscale network of the ovariectomy (OVX) induced estrogen receptors depletion (ER-depletion) model and integrated targeted proteomic, targeted lipidomic, cytochemical, and histopathological data across three tissues from the ovariectomy rodent model. We found that compared to control rats, OVX rats showed increased renal and uterine prostaglandin D2 synthase (Ptgds) expression and decreased hypothalamic Ptgds expression, abnormal Ptgds metabolites, the degenerate renal function profiles and decreased cognitive ability (learning and memory) in Morris water maze test. Importantly, we observed a regulatory relationship among ER (particularly ERß), the degree of the pathological phenotype, learning behavior test and the 'hypothalamus-uterus-kidney (HUK) axis functions. Collectively, this study elucidates that ER depletion promoted HUK aging is mostly attributed to a renal ERß/Ptgds signalling imbalance.

Competitive ELISA method for novel estrogen-negative breast cancer biomarker quantitation.

Estrogen-negative (ER-) breast cancer, is recognized as an aggressive subtype, more difficult to treat, with poor survival and prognosis. They are hormonally unresponsive, with no readily effective and specific target therapy. We have previously identified Nw-hydroxy L-Arginine (NOHA) as a blood-based biomarker to distinguish between ER- and ER+ breast cancer tumors based upon disease burden, progression and molecular phenotype (U.S. Utility Patent 10,073,099). In this study we have demonstrated a competitive ELISA based assay for NOHA measurement using a proprietary monoclonal antibody (mAb) specific for NOHA (U.S. provisional patent 62/754,053). The ELISA assay was evaluated on sensitivity, selectivity, precision, dilution linearity and percent recovery parameters. The assay showed sensitivity at ≥60 pg/ml NOHA antigen with 1 ng/ml NOHA mAb, and maintained NOHA antigen specificity even in the presence of other closely related cationic amino acids (i.e. L-Arginine, D-Arginine, l-Lysine, d-Lysine, L-Ornithine, and L-Citrulline). The reliability of the ELISA protocol was confirmed with the low percent-covariance, for all tested parameters of sensitivity (≤8.2%), selectivity (≤8.6%), precision (≤12.6%), dilution linearity (≤11.2%) and recovery (≤6.7%). Additionally, we can demonstrate NOHA quantification by this ELISA assay to complement the sensitivity achievable with LC-MS (in both assay buffer and with patient plasma samples), thus suggesting it's utility as a simple yet sensitive methodology that might help in ER- breast cancer prognosis, and disease progression monitoring without the need for expensive analytical equipment (such as LC-MS), large lab space, or specialized technical training.

A modular PROTAC design for target destruction using a degradation signal based on a single amino acid.

Proteolysis targeting chimeras (PROTACs) are bivalent molecules that bring a cellular protein to a ubiquitin ligase E3 for ubiquitination and subsequent degradation. Although PROTAC has emerged as a promising therapeutic means for cancers as it rewires the ubiquitin pathway to destroy key cancer regulators, the degradation signals/pathways for PROTACs remain underdeveloped. Here we append single amino acids, the simplest degradation signal, to a ligand specific for estrogen-related receptor α (ERRα) and demonstrate their utility in ERRα knockdown via the N-end rule pathway and also their efficiency in the growth inhibition of breast cancer cells. The modular design described offers unique advantages including smaller molecular size with shortest degradation sequences and degradation speed modulation with different amino acids. Our study expands the repertoire of limited ubiquitin pathways currently available for PROTACs and could be easily adapted for broad use in targeted protein degradation.

Dynamics of breast-cancer relapse reveal late-recurring ER-positive genomic subgroups.

The rates and routes of lethal systemic spread in breast cancer are poorly understood owing to a lack of molecularly characterized patient cohorts with long-term, detailed follow-up data. Long-term follow-up is especially important for those with oestrogen-receptor (ER)-positive breast cancers, which can recur up to two decades after initial diagnosis1-6. It is therefore essential to identify patients who have a high risk of late relapse7-9. Here we present a statistical framework that models distinct disease stages (locoregional recurrence, distant recurrence, breast-cancer-related death and death from other causes) and competing risks of mortality from breast cancer, while yielding individual risk-of-recurrence predictions. We apply this model to 3,240 patients with breast cancer, including 1,980 for whom molecular data are available, and delineate spatiotemporal patterns of relapse across different categories of molecular information (namely immunohistochemical subtypes; PAM50 subtypes, which are based on gene-expression patterns10,11; and integrative or IntClust subtypes, which are based on patterns of genomic copy-number alterations and gene expression12,13). We identify four late-recurring integrative subtypes, comprising about one quarter (26%) of tumours that are both positive for ER and negative for human epidermal growth factor receptor 2, each with characteristic tumour-driving alterations in genomic copy number and a high risk of recurrence (mean 47-62%) up to 20 years after diagnosis. We also define a subgroup of triple-negative breast cancers in which cancer rarely recurs after five years, and a separate subgroup in which patients remain at risk. Use of the integrative subtypes improves the prediction of late, distant relapse beyond what is possible with clinical covariates (nodal status, tumour size, tumour grade and immunohistochemical subtype). These findings highlight opportunities for improved patient stratification and biomarker-driven clinical trials.

TBCRC026: Phase II Trial Correlating Standardized Uptake Value With Pathologic Complete Response to Pertuzumab and Trastuzumab in Breast Cancer.

PURPOSE: Predictive biomarkers to identify patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer who may benefit from targeted therapy alone are required. We hypothesized that early measurements of tumor maximum standardized uptake values corrected for lean body mass (SULmax) on [18F]fluorodeoxyglucose positron emission tomography/computed tomography would predict pathologic complete response (pCR) to neoadjuvant pertuzumab and trastuzumab (PT). PATIENTS AND METHODS: Patients with stage II/III, estrogen receptor-negative, HER2-positive breast cancer received four cycles of neoadjuvant PT. [18F]Fluorodeoxyglucose positron emission tomography/computed tomography was performed at baseline and 15 days after PT initiation (C1D15). Eighty evaluable patients were required to test the null hypothesis that the area under the curve of percentage of change in SULmax by C1D15 predicting pCR is less than or equal to 0.65, with a one-sided type I error rate of 10%. RESULTS: Eighty-eight women were enrolled (83 evaluable), and 85% (75 of 88) completed all four cycles of PT. pCR after PT alone was 34%. Receiver operating characteristic analysis yielded an area under the curve of 0.76 (90% CI, 0.67 to 0.85), which rejected the null hypothesis. Between patients who obtained pCR versus not, a significant difference in median percent reduction in SULmax by C1D15 was observed (63.8% v 33.5%; P < .001), an SULmax reduction greater than or equal to 40% was more prevalent (86% v 46%; P < .001; negative predictive value, 88%; positive predictive value, 49%), and a significant difference in median C1D15 SULmax (1.6 v 3.9; P < .001) and higher proportion of C1D15 SULmax less than or equal to 3 (93% v 38%; P < .001; negative predictive value, 94%; positive predictive value, 55%) were observed. CONCLUSION: Early changes in SULmax predict response to four cycles of PT in estrogen receptor-negative, HER2-positive breast cancer. Once optimized, this quantitative imaging strategy may facilitate a more tailored approach to therapy in this setting.

Effect of 17ß-estradiol on estrogen receptor negative breast cancer cells in an osteolytic mouse model.

17ß-Estradiol (E2) promotes metastasis of triple negative breast cancer cells to bone. Recent studies show many triple negative breast cancer cell lines lacking the 66 kDa estrogen receptor (ER) alpha (ERα66) or its splice variant ERα46, express another splice variant, ERα36 associated with membrane-mediated rapid actions of the hormone. qPCR and western blot confirmed that MCF7 cells possessed ERα splice variants ERα66, ERα46 and ERα36, while ER-negative breast cancer cells MDA-MB-231 possessed only ERα36. MDA-MB-231 breast cancer cells were implanted into medullary canals of ovariectomized female athymic nude mice femurs (N = 8 mice/treatment). To examine the effect of E2 on osteolysis, mice were treated with 0.72 mg E2 or placebo via 60 day release osmotic pumps implanted subcutaneously. Legs were examined by Faxitron through the course of the study, and by microCT and histology after 8 weeks. Greater occurrence of osteolysis and pathologic fracture was observed in E2-treated animals compared to placebo, and microCT demonstrated less bone volume remaining in MDA-MB-231 treated legs compared to contralateral control legs, as well as E2-treated animals compared to placebo. E2-treated animals had significantly greater tumor volume compared to placebo. Large nests of anaplastic tumor cells with eroded cortical margin were observed in E2-treated animals compared to placebo. MDA MB 231 breast cancer cells positive for ERα36 but negative for ERα46/66 had enhanced osteolysis, pathologic fractures, and tumor volume in an in vivo osteolytic mouse model when treated with 17ß-estradiol compared to placebo, demonstrating a role for ERα36 in bone tumor progression.

Compensatory role of insulin-like growth factor 1 receptor in estrogen receptor signaling pathway and possible therapeutic target for hormone therapy-resistant breast cancer.

BACKGROUND: Hormone therapy targeting the estrogen receptor (ER) pathway is the most common treatment used for ER-positive breast cancer. However, some patients experience de novo or acquired resistance, which becomes a critical problem. Activation of the insulin-like growth factor (IGF) pathway allows breast cancer cells to proliferate and is associated with the ER pathway. Little is known about the role of the IGF pathway in hormone therapy and resistance; therefore, we investigated whether the inhibition of this pathway may represent a novel therapeutic target for overcoming hormone therapy resistance in ER-positive breast cancers. METHODS: Crosstalk between the ER and IGF pathways was analyzed in breast cancer cell lines by inhibiting or stimulating either one or both pathways. We studied the effect of insulin-like growth factor one receptor (IGF1R) inhibition in aromatase inhibitor-resistant breast cancer cell lines and fulvestrant-resistant cell lines which were uniquely established in our laboratory. RESULTS: Under normal conditions, IGF signaling is controlled by ER signaling to promote cell growth. Temporary disruption of the estrogen supply results in attenuated ER signaling, and IGF-1 dramatically increased relative growth compared with normal conditions. In addition, IGF1R inhibitor strongly suppressd cell growth in hormone-resistant breast cancer cells where ER remains than cells where ER decreased or was almost lost. CONCLUSIONS: Our study suggests that inhibition of the IGF pathway may be an effective strategy for ER-positive breast cancer therapy, even in hormone therapy-resistant cases.

AR-PDEF pathway promotes tumour proliferation and upregulates MYC-mediated gene transcription by promoting MAD1 degradation in ER-negative breast cancer.

BACKGROUND: Androgen receptor (AR) is expressed in 60%~ 70% oestrogen receptor (ER)-negative breast cancer (BC) cases and promotes the growth of this cancer subtype. Expression of prostate-derived Ets factor (PDEF), a transcription factor, is highly restricted to epithelial cells in hormone-regulated tissues. MYC and its negative regulator MAD1 play an important role in BC progression. Previously, we found that PDEF expression is strongly correlated with AR expression. However, the relationship between AR and PDEF and the function of PDEF in ER-negative BC proliferation are unclear. METHODS: AR and PDEF expression in ER-negative BC tissues and cell lines was determined by performing immunohistochemistry or western blotting. Protein expression levels and location were analysed by performing western blotting, RT-qPCR and immunofluorescence staining. Co-immunoprecipitation and chromatin immunoprecipitation assays were performed to validate the regulation of AR-PDEF-MAD1-MYC axis. Moreover, the effect of AR and PDEF on BC progression was investigated both in vitro and in vivo. RESULTS: We found that PDEF was overexpressed in ER-negative BC tissues and cell lines and appeared to function as an oncogene. PDEF expression levels were strongly correlated with AR expression in ER-negative BC, and PDEF transcription was positively regulated by AR. PDEF upregulated MYC-mediated gene transcription by promoting MAD1 degradation in ER-negative BC. Finally, we found that compared with the inhibition of AR expression alone, simultaneous inhibition of AR and PDEF expression further suppressed tumour proliferation both in vitro and in vivo. CONCLUSIONS: Our data highlight the role of the AR-PDEF-MAD1-MYC axis in BC progression and suggest that PDEF can be used as a new clinical therapeutic target for treating ER-negative BC.

Estrogen-related receptor α is essential for maintaining mitochondrial integrity in cisplatin-induced acute kidney injury.

Acute kidney injury (AKI) has been associated with not only higher in-hospital mortality but also the subsequent development of chronic kidney disease (CKD). Recent evidence has suggested the involvement of mitochondrial dysfunction and impaired dynamics in the pathogenesis of AKI. Estrogen-related receptor α (ERRα) is an orphan nuclear receptor that acts as a transcription factor to regulate the transcription of genes required for mitochondrial biogenesis and oxidative phosphorylation. In the present study, we examined the effects of ERRα deficiency on the progression of AKI induced by cisplatin. Male C57BL/6 J wild-type and ERRα-/- mice received a single intraperitoneal injection of 20 mg/kg cisplatin. Seventy-two hours after the injection, kidney function and morphology were evaluated. ERRα expression was observed in renal tubules, and cisplatin inhibited its translocation into nuclei. ERRα deficiency exacerbated cisplatin-induced renal dysfunction and tubular injury, as well as oxidative stress and apoptosis. ERRα-/- mice kidneys revealed lower mitochondrial DNA content and swollen mitochondria with reduced cristae. In addition, these mice had lower expression of the mitochondrial fusion protein mitofusin-2. The cisplatin-induced decrease in mitochondrial DNA and altered mitochondrial structure were more severe in ERRα-/- mice. In cultured mouse proximal tubular epithelial cells, the ERRα inverse agonist XCT-790 significantly inhibited mitofusin-2 expression and induced mitochondrial fragmentation. Taken together, our findings suggest the involvement of ERRα in the progression of cisplatin-induced AKI probably through impaired mitochondrial dynamics.

The Application of Non-Invasive Apoptosis Detection Sensor (NIADS) on Histone Deacetylation Inhibitor (HDACi)-Induced Breast Cancer Cell Death.

Breast cancer is the most common malignancy in women and the second leading cause of cancer death in women. Triple negative breast cancer (TNBC) subtype is a breast cancer subset without ER (estrogen receptor), PR (progesterone receptor) and HER2 (human epidermal growth factor receptor 2) expression, limiting treatment options and presenting a poorer survival rate. Thus, we investigated whether histone deacetylation inhibitor (HDACi) could be used as potential anti-cancer therapy on breast cancer cells. In this study, we found TNBC and HER2-enriched breast cancers are extremely sensitive to Panobinostat, Belinostat of HDACi via experiments of cell viability assay, apoptotic marker identification and flow cytometry measurement. On the other hand, we developed a bioluminescence-based live cell non-invasive apoptosis detection sensor (NIADS) detection system to evaluate the quantitative and kinetic analyses of apoptotic cell death by HDAC treatment on breast cancer cells. In addition, the use of HDACi may also contribute a synergic anti-cancer effect with co-treatment of chemotherapeutic agent such as doxorubicin on TNBC cells (MDA-MB-231), but not in breast normal epithelia cells (MCF-10A), providing therapeutic benefits against breast tumor in the clinic.