Overexpression of BQ323636.1 Modulated AR/IL-8/CXCR1 Axis to Confer Tamoxifen Resistance in ER-Positive Breast Cancer

NCOR2 is a co-repressor for estrogen receptor (ER) and androgen receptor (AR). Our group previously identified a novel splice variant of NCOR2, BQ323636.1 (BQ), that mediates tamoxifen resistance via interference of NCOR2 repression on ER. Luciferase reporter assay showed BQ overexpression could enhance the transcriptional activity of androgen response element (ARE). We proposed that BQ employs both AR and ER to confer tamoxifen resistance. Through in silico analysis, we identified interleukin-8 (IL-8) as the sole ERE and ARE containing gene responsiveness to ER and AR activation. We confirmed that BQ overexpression enhanced the expression of IL-8 in ER+ve breast cancer cells, and AR inhibition reduced IL-8 expression in the BQ overexpressing cell lines, suggesting that AR was involved in the modulation of IL-8 expression by BQ. Moreover, we demonstrated that IL-8 could activate both AKT and ERK1/2 via CXCR1 to confer tamoxifen resistance. Targeting CXCR1/2 by a small inhibitor repertaxin reversed tamoxifen resistance of BQ overexpressing breast cancer cells in vitro and in vivo. In conclusion, BQ overexpression in ER+ve breast cancer can enhance IL-8 mediated signaling to modulate tamoxifen resistance. Targeting IL-8 signaling is a promising approach to overcome tamoxifen resistance in ER+ve breast cancer.

miR-489 Confines Uncontrolled Estrogen Signaling Through a Negative Feedback Mechanism and Regulates Tamoxifen Resistance in Breast Cancer

Background: Approximately 75% of diagnosed breast cancer tumors are estrogen-receptor (ER) positive tumors and are associated with better prognosis due to their response to hormonal therapies. However, around 40% of patients relapse after hormonal therapies. In the current study, we aim to evaluate miR-489 as a novel molecular target to combat tamoxifen resistance. Methods: Genomic analysis of gene expression profiles in primary breast cancers and tamoxifen resistant cell lines unveiled the potential role of miR-489 in regulation of estrogen signaling and development of tamoxifen resistance. We manipulated miR-489 expression in breast cancer cell lines by transient transfection of a miR-489 mimic or establishment of knockout cell lines using the CRISPR/Cas9 system to study the reciprocal regulation of miR-489 and estrogen/ER signaling pathways. Cell proliferation assays, Sphere-formation assays and flow cytometry analysis were conducted to investigate the role of miR-489 in estrogen-induced cell proliferation, cancer stem cell expansion and development of tamoxifen resistance. Results: miR-489 expression was significantly downregulated in tamoxifen-resistant cell lines. Low levels of miR-489 were associated with poor clinical outcomes in patients with hormone treatment. In vitro analysis showed that loss of miR-489 expression promoted tamoxifen resistance while overexpression of miR-489 in tamoxifen-resistant cells restored tamoxifen sensitivity. Mechanistically, we found that miR-489 is an estrogen regulated miRNA that negatively regulated estrogen receptor signaling by using at least the following two mechanisms: i) modulation of ER phosphorylation status by inhibiting MAPK and AKT kinase activities and downregulating SHP2 expression; ii) regulation of nucleus to cytosol translocation of estrogen receptor α (ERα) by decreasing p38 expression and consequently ER phosphorylation. In addition, miR-489 could break the positive feed-forward loop between the estrogen-ERα axis and p38 MAPK in breast cancer cells, which was necessary for its function as a transcription factor. Conclusion: Our study unveiled the underlying molecular mechanism by which miR-489 regulates estrogen signaling pathway through a negative feedback loop and uncovered its role in both the development of and overcoming of tamoxifen resistance in breast cancers.

Mps1 contributes to tamoxifen resistance in Breast Cancer through phosphorylation of ERα

Overexpression of mitotic kinase monopolar spindle 1 (Mps1) has been identified in many tumor types and targeting Mps1 for tumor therapy has been shown great promise in multiple preclinical cancer models. However, the role of Mps1 in tamoxifen resistance in breast cancer has never been reported. Here in this study, we report that Mps1 determined the sensitivity of breast cancer cells to tamoxifen treatment. Mps1 overexpression rendered breast cancer cells more resistant to tamoxifen, while Mps1 inhibitor or siMps1 oligos could overcome tamoxifen resistance. Mechanistically, Mps1 interacted with ERα and stimulated its transcriptional activity in kinase activity-dependent manner. Mps1 was responsible for ERα phosphorylation at S559 and T224 sites. Importantly, Mps1 failed to enhance the transcriptional activity of ERα in the presence of ERα S559A or T224A mutant. Collectively, our findings suggest that Mps1 contributes to tamoxifen resistance in breast cancer and is a potential therapeutic to overcome tamoxifen resistance in breast cancer.

Corylin sensitizes breast cancer cells to overcome tamoxifen resistance by regulating OAS1/miR-22-3p/SIRT1 axis

Breast cancer (BCa) is one of the leading causes of cancer-related death among women worldwide. At present, the clinical treatment with tamoxifen (TAM) is challenged by the development of drug resistance. To investigate the effect of corylin on TAM resistance in BCa cells, this study investigated the molecular mechanisms involving miRNA-mRNA targets modulated by corylin. The TAM-resistant MCF-7TR and T47DTR cell lines were generated, and it was found that corylin treatment reduced the cell viability of these cells significantly. Furthermore, OAS1 was validated to be highly expressed in TAM-resistant cells, while OAS1 knockdown sensitized MCF-7TR and T47DTR cells to TAM treatment. Meanwhile, OAS1 was also repressed by corylin treatment, indicating that OAS1 was a key regulator of corylin function. Through bioinformatic analysis, the tumor suppressive miRNA miR-22-3p was identified to directly target and inhibit OAS1. Moreover, corylin treatment up-regulated miR-22-3p expression, which thus down-regulated the OAS1 expression. Interestingly, OAS1 itself functioned as a miR-22-3p sponge to repress miR-22-3p expression. Further, SIRT1 was identified to be up-regulated in TAM-resistant cells and participated in the OAS1/miR-22-3p regulatory axis via the miR-22-3p direct target. In conclusion, corylin sensitized TAM-resistant cells to TAM treatment by inhibiting OAS1 expression and modulating the OAS1/miR-22-3p/SIRT1 axis.

Lnc-DC promotes estrogen independent growth and tamoxifen resistance in breast cancer

Selective estrogen receptor modulators (SERMs) such as tamoxifen have proven to be effective in the treatment of estrogen receptor (ER) positive breast cancer. However, a major obstacle for such endocrine therapy is estrogen independent growth, leading to resistance, and the underlying mechanism is not fully understood. The purpose of this study was to determine whether long non-coding RNAs (lncRNAs) are involved in regulation of estrogen independent growth and tamoxifen resistance in ER positive breast cancer. Using a CRISPR/Cas9-based SAM (synergistic activation mediator) library against a focus group of lncRNAs, we identify Lnc-DC as a candidate lncRNA. Further analysis suggests that Lnc-DC is able to reduce tamoxifen-induced apoptosis by upregulation of anti-apoptotic genes such as Bcl2 and Bcl-xL. Furthermore, Lnc-DC activates STAT3 by phosphorylation (pSTAT3Y705), and the activated STAT3 subsequently induces expression of cytokines which in turn activate STAT3, forming an autocrine loop. Clinically, upregulation of Lnc-DC is associated with poor prognosis. In particular, analysis of a tamoxifen-treated patient cohort indicates that Lnc-DC expression can predict the response to tamoxifen. Together, this study demonstrates a previously uncharacterized function of Lnc-DC/STAT3/cytokine axis in estrogen independent growth and tamoxifen resistance, and Lnc-DC may serve as a potential predictor for tamoxifen response.

A combination approach of pseudotime analysis and mathematical modeling for understanding drug-resistant mechanisms

Cancer cells acquire drug resistance through the following stages: nonresistant, pre-resistant, and resistant. Although the molecular mechanism of drug resistance is well investigated, the process of drug resistance acquisition remains largely unknown. Here we elucidate the molecular mechanisms underlying the process of drug resistance acquisition by sequential analysis of gene expression patterns in tamoxifen-treated breast cancer cells. Single-cell RNA-sequencing indicates that tamoxifen-resistant cells can be subgrouped into two, one showing altered gene expression related to metabolic regulation and another showing high expression levels of adhesion-related molecules and histone-modifying enzymes. Pseudotime analysis showed a cell transition trajectory to the two resistant subgroups that stem from a shared pre-resistant state. An ordinary differential equation model based on the trajectory fitted well with the experimental results of cell growth. Based on the established model, it was predicted and experimentally validated that inhibition of transition to both resistant subtypes would prevent the appearance of tamoxifen resistance.