Gao, Huanyao

Abstract Background: Androgen receptor (AR) is widely expressed in ER+ breast cancers. Accumulated evidence showed that AR is a tumor suppressor in ERα+ breast cancers. Enzalutamide (ENZ), an AR antagonist, blocks AR signalling, and was reported to inhibit estrogen-driven tumor growth as effectively as tamoxifen. Hence, this study was performed to further examine the mechanism of action of ENZ in ERα+ breast cancers. Methods: The AR gene was knocked out using the CRISPR-Cas9 system. Four crRNAs targeting AR and trans-activating crRNA were co-transfected to Cas9-positive MCF7 and T47D cells, two ERα+ breast cancer cell lines with relatively low AR levels based on RNA seq. ESR1 knock down was performed using a lentivirus system. MCF7 and T47D cells were infected with the ESR1-shRNA lentivirus and selected by puromycin for 2 weeks. Single clones of knockdown or knockout cells were tested using western blot. MCF7 and T47D cells were collected for RNA-seq and ChIP-seq after treated with DMSO or 10µM ENZ for 6 hours in complete medium. All sequences were aligned to the Human Reference Genome (hg38, UCSC). Differential gene expression was performed using limma package and upstream analysis was done in Ingenuity Pathway Analysis software. ChIP peaks were called using MACS2 and overlapping was analyzed using ChIPpeakAnno package in R. Tritium labeled ENZ or estradiol were incubated with ERα overnight. The bound radioactivity was measured using a Beckmann LS 6500 liquidscintillation counter. Results: We found the cell growth inhibition effect of ENZ was little impaired by AR gene knock out in MCF7 and T47D cells as was expected. As ENZ has been shown to inhibit estrogen-driven tumor growth, we performed studies to understand the mechanism of ENZ.We performed RNA-seq and identified 193 differential expressed genes (76 up and 117 down) between DMSO and ENZ-treated MCF7 cells (FC>2 or <0.5, FDR<0.05). Upstream analysis showed that ESR1 might be the upstream regulator of those differentially expressed genes and ESR1 signaling was predicted to be inhibited after ENZ treatment (P=1.69×10-10). To examine the alteration of ERα genomic binding, we performed global ERα ChIP-seq and found 39.58% (8,681/21,933) of ERα-bound sites were abolished by ENZ and the binding intensity was significantly decreased upon ENZ treatment in 12,868 shared peaks. Moreover, radiolabled binding assays showed that ENZ displaced estrogen binding to ERα and directly bound to ERα with a KD= 399.60nM. We further observed a reduction of proliferation inhibition of ENZ in ESR1-KD cells compared with control cells. This suggests that ENZ inhibits ER+ cell growth by displacing E2 from ERα binding sites and subsequently diminishes ERα genomic binding and blocks ERα signaling in ER+ cancers. Conclusion: We found that enzalutamide inhibits cell growth not through AR but by antagonizing ERα activity in MCF7 and T47D ER+ cells. These results reveal ER is the main factor affecting enzalutamide efficacy in these two ERα+ breast cancer cell lines. Further clinical trials are needed to investigate AR antagonists in ERα+ breast cancers. Citation Format: Lixuan Wei, Jia Yu, Huanyao Gao, Huan Zhang, Thanh Nguyen, Yayun Gu, Richard M. Weinshilboum, James N. Ingle, Liewei Wang. Estrogen receptor is a target of enzalutamide in ER+ breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD1-03.

Abstract Our previous matched case–control study of postmenopausal women with resected early-stage breast cancer revealed that only anastrozole, but not exemestane or letrozole, showed a significant association between the 6-month estrogen concentrations and risk of breast cancer. Anastrozole, but not exemestane or letrozole, is a ligand for estrogen receptor α. The mechanisms of endocrine resistance are heterogenous and with the new mechanism of anastrozole, we have found that treatment of anastrozole maintains fatty acid synthase (FASN) protein level by limiting the ubiquitin-mediated FASN degradation, leading to increased breast cancer cell growth. Mechanistically, anastrozole decreases the guided entry of tail-anchored proteins factor 4 (GET4) expression, resulting in decreased BCL2-associated athanogene cochaperone 6 (BAG6) complex activity, which in turn, prevents RNF126-mediated degradation of FASN. Increased FASN protein level can induce a negative feedback loop mediated by the MAPK pathway. High levels of FASN are associated with poor outcome only in patients with anastrozole-treated breast cancer, but not in patients treated with exemestane or letrozole. Repressing FASN causes regression of breast cancer cell growth. The anastrozole-FASN signaling pathway is eminently targetable in endocrine-resistant breast cancer.

Background: Trastuzumab (Herceptin) is the key systemic therapy for HER2-positive breast cancer. However, the initial response rate is limited to approximately 50% in patients. Moreover, most patients, especially at an advanced stage, eventually develop acquired resistance. Understanding the mechanisms of trastuzumab resistance is crucial for achieving better treatment outcome in this group of patients. Methods: A trastuzumab-resistant (TR) cell line was developed using the BT474 HER2-positive breast cancer cell line. Whole-transcriptome expression array was performed and the TR-related gene NDUFA4L2 was identified by differential expression analysis between BT474 and BT474-TR. Mitochondrial localization of NDUFA4L2 was confirmed by immunofluorescence and western blotting using mitochondrial fractionation. Mitochondrial function and energy metabolism were evaluated using Seahorse, ATP production, and lactate production assays, and cellular reactive oxygen species (ROS) levels were determined using DCFDA. NDUFA4L2 expression in patients was evaluated by immunohistochemistry, and relapse-free survival was analyzed using the Kaplan–Meier method. Results: NDUFA4L2 was highly expressed in the TR HER2-positive breast cancer cell line. High expression level of NDUFA4L2 was associated with shorter relapse-free intervals in trastuzumab-treated HER2-positive breast cancer patients. Overexpression of NDUFA4L2 enhanced Warburg effects, enhanced aerobic glycolysis, reduced oxygen consumption, and lowered ROS production. Mechanistically, overexpression of NDUFA4L2 facilitated mitochondrial relocalization of HER2 and suppressed ROS production, thus rendering cancer cells more resistant to trastuzumab treatment. Conclusions: We identified NDUFA4L2 as a new biomarker and potential therapeutic target for TR HER2-positive breast cancer.

Aromatase inhibitors (AIs) are the treatment of choice for hormone receptor–positive early breast cancer in postmenopausal women. None of the third‐generation AIs are superior to the others in terms of efficacy. We attempted to identify genetic factors that could differentiate between the effectiveness of adjuvant anastrozole and exemestane by examining single‐nucleotide polymorphism (SNP)‐treatment interaction in 4,465 patients. A group of SNPs were found to be differentially associated between anastrozole and exemestane regarding outcomes. However, they showed no association with outcome in the combined analysis. We followed up common SNPs near LY75 and GPR160 that could differentiate anastrozole from exemestane efficacy. LY75 and GPR160 participate in epithelial‐to‐mesenchymal transition and growth pathways, in both cases with SNP‐dependent variation in regulation. Collectively, these studies identified SNPs that differentiate the efficacy of anastrozole and exemestane and they suggest additional genetic biomarkers for possible use in selecting an AI for a given patient.

Objectives We previously discovered that the single nucleotide polymorphisms (SNP) rs9940645 in the ZNF423 gene regulate ZNF423 expression and serve as a potential biomarker for response to selective estrogen receptor modulators (SERMs). Here we explored pathways involved in ZNF423-mediated SERMs response and drugs that potentially sensitize SERMs. Methods RNA sequencing and label-free quantitative proteomics were performed to identify genes and pathways that are regulated by ZNF423 and the ZNF423 SNP. Both cultured cells and mouse xenograft models with different ZNF423 SNP genotypes were used to study the cellular responses to metformin. Results We identified ribosome and AMP-activated protein kinase (AMPK) signaling as potential pathways regulated by ZNF423 or ZNF423 rs9940645 SNP. Moreover, using clustered regularly interspaced short palindromic repeats/Cas9-engineered ZR75-1 breast cancer cells with different ZNF423 SNP genotypes, striking differences in cellular responses to metformin, either alone or in the combination of tamoxifen, were observed in both cell culture and the mouse xenograft model. Conclusions We found that AMPK signaling is modulated by the ZNF423 rs9940645 SNP in estrogen and SERM-dependent fashion. The ZNF423 rs9940645 SNP affects metformin response in breast cancer and could be a potential biomarker for tailoring the metformin treatment.

Abstract Background: Androgen receptor (AR) is expressed in 60%-90% of breast cancers. The role of AR in breast cancer largely depends on estrogen receptor (ER) status and remains controversial. In ER+ cancers, AR expression is associated with improved prognosis. Enzalutamide (ENZ), an AR antagonist, impairs AR signaling, inhibits ER+/AR+ breast cancer cell proliferation and has been shown to mitigate resistance to anti-estrogen therapies. A recent study identified that RAD140, an AR agonist, suppressed the growth of ER+/AR+ breast cancer via stimulating AR signaling, resulting in down regulation of ERα. Both AR agonists and antagonists are effective in treating ER+ breast cancers. Identifying the subgroup of patients who most likely will benefit from an individual drug is important in clinical practice. Here, we sought to characterize the relative AR to ER levels and their relationships to drug response in ER+ breast cancers.Methods: We evaluated AR and ER expression levels among 68 samples with ER+ breast cancers. On each tumor, IHC staining for AR and ER were performed and results were scored by multiplying the percentage of positive cells by the intensity. TCGA-RPPA (reverse phase protein array) dataset was used to verify AR protein distribution in ER+ cancers. We created MCF7 and T47D cells with doxycycline-inducible AR and ER expression system to manipulate the relative AR to ER ratios and determined the antitumor activity of ENZ and RAD140 by cell proliferation assays. The levels of AR and ER in cells were measured by western blot. Linear regression was used to test the association between dose-response area under curve (AUC) and AR, ER levels. Drug preference was modeled against AR/ER expression levels (AR/ER ratios) using logistic regression.Results: Among 68 ER+ breast cancers, 69.12% were AR-positive. More than 2/3 cases (48 of 68) had more ER than AR expressed. The AR to ER ratios varied from 0 to 6. In the TCGA cohort, 84.15% of 347 ER+ patients had AR/ER ratios less than 1.The range of AR to ER ratios in TCGA dataset were comparable with our data (0.004 to 4.210). In our cell line models, the AR/ER ratios were controlled between 0.19 to 4.05. We found that the AUC of RAD140 was negatively associated with AR protein levels (P=0.008) and AR/ER ratios (P=0.013), and not significantly associated with ER expression levels. On the other hand, the AUC of ENZ was significantly negatively associated with ER protein (P=0.0016) but postiviely associated with AR/ER ratio (p=0.037). Preferred treatment comparing efficacy of the two drug can be best determined at extremes of AR/ER ratios. Using clinically relevant dosages, our model predicted that ENZ (10µM) would be a preferred treatment choice and have a better treatment efficacy compared with RAD140 when the AR/ER ratio was ≤ 0.42, whereas RAD140 (1µM) would be the preferred choice with a better treatment efficacy compared with ENZ when AR/ER ratios were ≥ 3.1 The efficacy preference of the two drugs are equivocal for AR/ER ratios between 0.42 and 3.10. Conclusion: We developed preclinical models using AR and ER expression levels to predict AR-targeting drug response. The results support the use of RAD140 in AR high patients and those with an AR/ER ratio >3.10, and enzalutamide in AR low patients and those with an AR/ER ratio <0.42. Equipoise on choice of drug was found for AR/ER ratios of 0.42-3.10. RAD140 and enzalutamide are compelling candidates for monotherapy or combination with anti-estrogen therapies in ER+/AR+ breast cancer. Future clinical validation of the models and therapeutic effect is warranted. Citation Format: Lixuan Wei, Huanyao Gao, Jia Yu, Duan Liu, Huan Zhang, Thanh Nguyen, Marie R Passow, Jodi M Carter, Richard M Weinshilboum, James N Ingle, Liewei Wang. Computational modeling of androgen receptor (AR) and estrogen receptor as predictive biomarkers of response to AR agonists and antagonists [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-36.