Chemoresistance In Breast Cancer Research Articles

Abstract PO2-23-10: Obesity May Modulate the Relationship Between High Mammographic Breast Density and Neoadjuvant Chemoresistance in Early Breast Cancer

Abstract Background - An inverse association has been described in a number of studies between mammographic breast density (MBD) and pathologic complete response (pCR) following neoadjuvant chemotherapy (NAC) for early breast cancer (BC), implicating high MBD in chemotherapy resistance. However, this effect has not been seen in all there is a paucity of data exploring whether this relationship extends the relationship beyond pCR to overall survival. This study aims to validate the relationship between MBD and pCR in patients undergoing NAC for early BC as well as to assess the relation of MBD with clinical complete response (cCR), relapse-free (RFS) and BC-specific survival (BCSS). Further, this study examines these associations stratified by body mass index (BMI). Methods - MBD was measured in contralateral mammograms in 127 women before NAC, using Cumulus software. As the study spanned the transition period from film to digital mammography, film mammograms were digitised using a high-powered scanner and processed full-field digital mammograms were extracted from the imaging storage facility. Basic patient, tumour and treatment demographics were collated including BMI. Percent dense area (PDA) was correlated with patient and tumour characteristics, short-term (pCR/cCR incidences), and long-term chemotherapy outcomes (RFS and BCSS). Results - Overall rates of cCR and pCR of 49% and 21% were observed across the whole cohort. Mean PDA was higher in those not undergoing cCR (p=0.041) and relapsing patients (p=0.041) but did not significantly vary by pCR or BC-deaths. As a dichotomous variable, high PDA relative to low PDA corresponded to lower cCR (40 v 58%, p=0.027), but no significant difference was seen for pCR (17.5 v 25.0%, p=0.15), BC relapse (38 v 30%, p=0.15) or BC-death (32 v 25%, p=0.20). High PDA (relative to low PDA) in patients with obesity (BMI >30kg/m2) was associated with lower pCR (0% v 28.1%, p=0.036) and numerically higher relapse rates (56% v 28%, p=0.063) and breast cancer deaths (56 v 28%, p=0.071. No such relationship was observed in patients with a BMI< 30kg/m2. Conclusion - Higher MBD associated with lower cCR and higher relapse but not with pCR or BC death. The inferior outcomes with high MBD were confined to patients with obesity in our study. Further analysis of the relationship between MBD and outcomes, including by BMI, is warranted. Citation Format: Andrew Redfern, Veenoo Agarwal, Ellie Darcey, Lisa Spalding, Hilary Martin, Jennifer Stone. Obesity May Modulate the Relationship Between High Mammographic Breast Density and Neoadjuvant Chemoresistance in Early Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-23-10.

The regulation of microRNAs on chemoresistance in triple-negative breast cancer: a recent update.

Triple-negative breast cancer (TNBC) has negative expressions of ER, PR and HER2. Due to the insensitivity to both endocrine therapy and HER2-targeted therapy, the main treatment method for TNBC is cytotoxic chemotherapy. However, the curative effect of chemotherapy is limited because of the existence of acquired or intrinsic multidrug resistance. MicroRNAs (miRNAs) are frequently dysregulated in malignant tumors and involved in tumor occurrence and progression. Interestingly, growing studies show that miRNAs are involved in chemoresistance in TNBC. Thus, targeting dysregulated miRNAs could be a plausible way for better treatment of TNBC. Here, we present the updated knowledge of miRNAs associated with chemoresistance in TNBC, which may be helpful for the early diagnosis, prognosis and treatment of this life-threatening disease.

CCAAT enhancer binding protein delta activates vesicle associated membrane protein 3 transcription to enhance chemoresistance and extracellular PD-L1 expression in triple-negative breast cancer

BackgroundChemoresistance and immunosuppression are two major obstacles in the current anti-cancer treatments. This study investigates the involvements of a CCAAT enhancer binding protein delta (CEBPD)/vesicle associated membrane protein 3 (VAMP3) axis in paclitaxel (PTX) resistance and immune evasion in triple-negative breast cancer (TNBC).MethodsPTX resistance-related genes were screened by bioinformatics. CEBPD and VAMP3 expression in clinical TNBC samples was examined by immunohistochemistry. Three PTX-resistant TNBC cell lines (MDA-MB-231/PTX, MDA-MB-468/PTX and MDA-MB-453/PTX) were generated, and their drug resistance was analyzed. Autophagy of cells was analyzed by immunofluorescence staining. Interaction between CEBPD and VAMP3 promoter was identified by immunoprecipitation and luciferase assays. The extracellular expression of programmed cell death-ligand 1 (PD-L1) in TNBC cells was detected. Extracellular vesicles (EVs) from TNBC cells were isolated to examine their effects on CD8+ T cell exhaustion.ResultsCEBPD and VAMP3 were upregulated in chemo-resistant tissue samples and in PTX-resistant TNBC cells. The CEBPD downregulation enhanced PTX sensitivity of cells. However, further upregulation of VAMP3 in cells restored PTX resistance, which was likely due to the activation of autophagy, as the autophagy antagonist chloroquine enhanced PTX sensitivity of cells. CEBPD was found to bind to the VAMP3 promoter to activate its transcription. The CEBPD/VAMP3 axis also increased the PD-L1 expression in the conditioned medium of TNBC cells. The TNBC cell-derived EVs increased the exhaustion of co-cultured CD8+ T cells.ConclusionThis study provides novel evidence that CEBPD plays a key role in enhancing PTX resistance in TNBC cells across various subtypes through VAMP3-mediated autophagy activation. Additionally, the CEBPD/VAMP3 axis also increases extracellular PD-L1 level, delivered by cancer cell-derived EVs, to suppress CD8+ T cell-mediated anti-tumor immune response. These significant observations may provide new insights into the treatment of TNBC, suggesting CEBPD and VAMP3 as promising targets to overcome treatment resistance.

In silico identification and in vitro validation of alpha-hederin as a potent inhibitor of Wnt/β-catenin signaling pathway in breast cancer stem cells.

Cancer stem cells (CSCs) play a vital role in metastasis, recurrence and chemoresistance in breast cancer. β-catenin, which is a frequently over activated protein in CSCs, binds to T-cell factor/lymphoid enhancer factor (Tcf/Lef) family transcription factors leading to ectopic expression of Wnt pathway responsive genes necessary for the maintenance and action of CSCs. With the aim of identifying a small molecules that can effectively eliminate CSCs, molecular docking studies were performed against the Tcf/Lef binding hotspot on β-catenin using a library of 100 natural or synthetic small molecules. Small molecule ligands giving docking energy better than -7kcal/mol were further investigated by binding interactions analysis and molecular dynamics (MD) simulations. These compounds were then investigated in vitro, for cytotoxicity against CSCs isolated from MDA-MB-231 triple negative breast cancer cells. Alpha-hederin (AH) was identified as the only compound in the selected library that has cytotoxicity against breast CSCs. AH was further investigated for it's ability to regulate Wnt pathway target genes (Cyclin D1 and CD44)and the tumor suppressor p53by real-time quantitative PCR. Absorption, distribution, metabolism, excretion and toxicity properties of the AH was predicted in silico. AH significantly down regulated the transcription of Cyclin D1 and CD44 while up-regulating the transcription of p53. AH was predicted to have acceptable drug likeness. Although AH is currently known to inhibit the growth of various cancer cells in vitro, present study demonstrated for the first time that it is a potent inhibitor of Wnt/β-catenin signaling pathway and induce apoptosis in breast CSCs.

Abstract 4556: USP51-GRP78-ABCB1 axis promotes chemoresistance of triple negative breast cancer by decreasing the accumulation of doxorubicin in cells

Abstract Background: Resistance to chemotherapy remains an obstacle for triple-negative breast cancer (TNBC) patients. The core components of the ubiquitin-proteasome system have been demonstrated to regulate chemoresistance of TNBC. As a novel deubiquitinase (DUB), ubiquitin-specific peptidase 51 (USP51) plays a pivotal role in chemotherapeutic resistance in multiple malignancies. Herein, we sought to better understand how USP51 performs a cell-intrinsic role in mediating chemotherapeutic resistance in TNBC. Methods: Western blotting, RNA-sequencing and CCK8 assays were used to identify the DUBs for chemoresistance of breast cancer. Coimmunoprecipitation, GST-pulldown, protein deubiquitination and immunohistochemistry assays were then used to determine the biological phenotypes of ectopic USP51 in TNBC chemoresistance and associated signaling pathways in several different cell lines, mouse models and patient tissue samples. Ubiquitin-7-amido-4-methylcoumarin (Ub-AMC)-based deubiquitinase activity, cellular thermal shift and surface plasmon resonance (SPR) analyses were performed to investigate the activity of USP51 inhibitors. Results: To identify the critical DUBs involved in breast cancer chemoresistance, we established doxorubicin-resistant Cal51 and MDA-MB-231 TNBC cell lines by multiple dose exposure. We found ectopic USP51 in doxorubicin-resistant cells compared with their parental cells. Moreover, USP51-interfered chemoresistant tumor cells exhibited impaired cell viability as well as increased DNA damage and cell apoptosis upon treatment with doxorubicin. On the contrary, overexpression of USP51 performed the opposite effects to enhance cell viability but decrease DNA damage and apoptosis in response to doxorubicin; however, these outcomes were not shown for its catalytically inactive mutant USP51C372S. At the molecular level, GRP78 was identified as a bona fide substrate of USP51. Importantly, knockdown USP51 increased doxorubicin-induced DNA damage and apoptosis, which was rescued by overexpression of GRP78 in vitro and in vivo. In addition, the activity of ABCB1, the main efflux pump of doxorubicin, was enhanced by GRP78. Consistently, the expression of USP51, GRP78 and ABCB1 were correlated with chemoresistant phenotypes in TNBC patients. Of note, we also explored a small molecular inhibitor of USP51, WCY-4-1, which conferred chemosensitization in TNBC. Conclusion: These findings collectively indicated that ectopic USP51 enhances the activity of ABCB1 by deubiquitinating and stabilizing GRP78, which leads to decreased accumulation of doxorubicin as well as decreased DNA damage and cell apoptosis in TNBC. Our results also revealed that specific inhibition of USP51 significantly impairs doxorubicin-resistance in TNBC. Citation Format: Yang Ou, Kun Zhang, Qiuying Shuai, Chenyang Wang, Huayu Hu, Lixia Cao, Chunchun Qi, Min Guo, Zhaoxian Li, Jie Shi, Yuxin Liu, Siyu Zuo, Xiao Chen, Yanjing Wang, Mengdan Feng, Hang Wang, Yi Shi, Guang Yang, Shuang Yang. USP51-GRP78-ABCB1 axis promotes chemoresistance of triple negative breast cancer by decreasing the accumulation of doxorubicin in cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4556.

E2F8-CENPL pathway contributes to homologous recombination repair and chemoresistance in breast cancer

Chemoresistance poses a significant obstacle to the treatment of breast cancer patients. The increased capacity of DNA damage repair is one of the mechanisms underlying chemoresistance. Bioinformatic analyses showed that E2F8 was associated with cell cycle progression and homologous recombination (HR) repair of DNA double-strand breaks (DSBs) in breast cancer. E2F8 knockdown suppressed cell growth and attenuated HR repair. Accordingly, E2F8 knockdown sensitized cancer cells to Adriamycin and Cisplatin. Centromere protein L (CENPL) is a transcriptional target by E2F8. CENPL overexpression in E2F8-knockdowned cells recovered at least in part the effect of E2F8 on DNA damage repair and chemotherapy sensitivity. Consistently, CENPL knockdown impaired DNA damage repair and sensitized cancer cells to DNA-damaging drugs. These findings demonstrate that targeting E2F8-CENPL pathway is a potential approach to overcoming chemoresistance.

LINC00115 promotes chemoresistant breast cancer stem-like cell stemness and metastasis through SETDB1/PLK3/HIF1α signaling

BackgroundCancer stem-like cell is a key barrier for therapeutic resistance and metastasis in various cancers, including breast cancer, yet the underlying mechanisms are still elusive. Through a genome-wide lncRNA expression profiling, we identified that LINC00115 is robustly upregulated in chemoresistant breast cancer stem-like cells (BCSCs).MethodsLncRNA microarray assay was performed to document abundance changes of lncRNAs in paclitaxel (PTX)-resistant MDA-MB-231 BCSC (ALDH+) and non-BCSC (ALDH−). RNA pull-down and RNA immunoprecipitation (RIP) assays were performed to determine the binding proteins of LINC00115. The clinical significance of the LINC00115 pathway was examined in TNBC metastatic lymph node tissues. The biological function of LINC00115 was investigated through gain- and loss-of-function studies. The molecular mechanism was explored through RNA sequencing, mass spectrometry, and the CRISPR/Cas9-knockout system. The therapeutic potential of LINC00115 was examined through xenograft animal models.ResultsLINC00115 functions as a scaffold lncRNA to link SETDB1 and PLK3, leading to enhanced SETDB1 methylation of PLK3 at both K106 and K200 in drug-resistant BCSC. PLK3 methylation decreases PLK3 phosphorylation of HIF1α and thereby increases HIF1α stability. HIF1α, in turn, upregulates ALKBH5 to reduce m6A modification of LINC00115, resulting in attenuated degradation of YTHDF2-dependent m6A-modified RNA and enhanced LINC00115 stability. Thus, this positive feedback loop provokes BCSC phenotypes and enhances chemoresistance and metastasis in triple-negative breast cancer. SETDB1 inhibitor TTD-IN with LINC00115 ASO sensitizes PTX-resistant cell response to chemotherapy in a xenograft animal model. Correlative expression of LINC00115, methylation PLK3, SETDB1, and HIF1α are prognostic for clinical triple-negative breast cancers.ConclusionsOur findings uncover LINC00115 as a critical regulator of BCSC and highlight targeting LINC00115 and SETDB1 as a potential therapeutic strategy for chemotherapeutic resistant breast cancer.

LncRNA MCM3AP-AS1 promotes chemoresistance in triple-negative breast cancer through the miR-524-5p/RBM39 axis.

Triple-negative breast cancer (TNBC) is the most lethal subtype of BC, with unfavorable treatment outcomes. Evidence suggests the engagement of lncRNA MCM3AP-AS1 in BC development. This study investigated the action of MCM3AP-AS1 in chemoresistance of TNBC cells. Drug-resistant TNBC cell lines SUM159PTR and MDA-MB-231R were constructed by exposure to increasing concentrations of doxorubicin/docetaxel (DOX/DXL). MCM3AP-AS1 and miR-524-5p expression levels were determined by RT-qPCR. RNA binding motif 39 (RBM39) level was measured using Western blot. Cell viability and apoptosis were assessed by CCK-8 assay and flow cytometry. The targeted binding of miR-524-5p with MCM3AP-AS1 or RBM39 was predicted by ECORI database and validated by dual-luciferase assays. The gain-and-loss of function assays were conducted in cells to investigate the interactions among MCM3AP-AS1, miR-524-5p, and RBM39. TNBC xenograft mouse models were established through subcutaneous injection of MCM3AP-AS1-silencing MDA-MB-231R cells and intraperitoneally administrated with DOX/DXL to verify the role of MCM3AP-AS1 in vivo. MCM3AP-AS1 was upregulated in drug-resistant TNBC cells, and MCM3AP-AS1 silencing could sensitize drug-resistant TNBC cells to chemotherapeutic drugs by promoting apoptosis. MCM3AP-AS1 targeted miR-524-5p. After DOX/DXL treatment, miR-524-5p inhibition partially reversed the effect of MCM3AP-AS1 silencing on inhibiting chemoresistance and promoting apoptosis of drug-resistant TNBC cells. miR-524-5p targeted RBM39. Silencing MCM3AP-AS1 promoted apoptosis via the miR-524-5p/RBM39 axis, thereby enhancing chemosensitivity of drug-resistant TNBC cells. MCM3AP-AS1 knockdown upregulated miR-524-5p, downregulated RBM39, and restrained tumor development in vivo. MCM3AP-AS1 silencing potentiates apoptosis of drug-resistant TNBC cells by upregulating miR-524-5p and downregulating RBM39, thereby suppressing chemoresistance in TNBC.

CYR61 confers chemoresistance by upregulating survivin expression in triple-negative breast cancer.

Cysteine-rich angiogenic inducer 61 (CYR61) is a protein from the CCN family of matricellular proteins that play diverse regulatory roles in the extracellular matrix. CYR61 is involved in cell adhesion, migration, proliferation, differentiation, apoptosis, and senescence. Here, we show that CYR61 induces chemoresistance in triple-negative breast cancer (TNBC). We observed that CYR61 is overexpressed in TNBC patients, and CYR61 expression correlates negatively with the survival of patients who receive chemotherapy. CYR61 knockdown reduced cell migration, sphere formation, and the cancer stem cell (CSC) population and increased the chemosensitivity of TNBC cells. Mechanistically, CYR61 activated Wnt/β-catenin signaling and increased survivin expression, which are associated with chemoresistance, the epithelial-mesenchymal transition, and CSC-like phenotypes. Altogether, our study demonstrates a novel function of CYR61 in chemotherapy resistance in breast cancer.

Microbiota enterotoxigenic Bacteroides fragilis-secreted BFT-1 promotes breast cancer cell stemness and chemoresistance through its functional receptor NOD1.

Tumor-resident microbiota in breast cancer promote cancer initiation and malignant progression. However, targeting microbiota to improve the effects of breast cancer therapy has not been investigated in detail. Here, we evaluated the microbiota composition of breast tumors and found that enterotoxigenic Bacteroides fragilis (ETBF) was highly enriched in the tumors of patients who did not respond to taxane-based neoadjuvant chemotherapy. ETBF, albeit at low biomass, secreted the toxic protein BFT-1 to promote breast cancer cell stemness and chemoresistance. Mechanistic studies showed that BFT-1 directly bound to NOD1 and stabilized NOD1 protein. NOD1 was highly expressed on ALDH+ breast cancer stem cells (BCSCs) and cooperated with GAK to phosphorylate NUMB and promote its lysosomal degradation, thereby activating the NOTCH1-HEY1 signaling pathway to increase BCSCs. NOD1 inhibition and ETBF clearance increases the chemosensitivity of breast cancer by impairing BCSCs.

PRMT1 Sustains De Novo Fatty Acid Synthesis by Methylating PHGDH to Drive Chemoresistance in Triple-Negative Breast Cancer.

PRMT1 promotes chemoresistance in TNBC by methylating metabolic enzymes PFKFB3, PKM2, and PHGDH to augment de novo fatty acid synthesis, indicating that targeting this axis is a potential treatment strategy.

Increased PIEZO1 Expression Is Associated with Worse Clinical Outcomes in Hormone-Receptor-Negative Breast Cancer Patients.

PIEZO1 plays a crucial role in the human body as a mechanosensory ion channel. It has been demonstrated that PIEZO1 is important in tissue development and regulating many essential physiological processes. Studies have suggested that the PIEZO1 ion channel plays a role in invasion and progression in cancer; elevated levels of PIEZO1 have been correlated with increased migration in breast cancer cells, chemo-resistance and invasion in gastric cancer cells, and increased invasion of osteosarcoma cells. In addition, high PIEZO1 expression levels were correlated with a worse prognosis in glioma patients. On the other hand, studies in lung cancer have attributed high PIEZO1 levels to better patient outcomes. However, the clinical impact of PIEZO1 in breast cancer is not well characterized. Therefore, our goal was to determine the clinical relevance of PIEZO1 in breast cancer. An analysis of breast cancer data from The Cancer Genome Atlas (TCGA) was conducted to investigate PIEZO1 expression levels and correlation to survival, followed by validation in an independent dataset, GSE3494. We also performed gene set enrichment analysis (GSEA) and pathway enrichment analysis. We also analyzed the immune cell composition in breast tumors from TCGA through a CIBERSORT algorithm. Our results demonstrated that the PIEZO1 expression levels are higher in hormone-receptor (HR)-negative than in HR-positive cohorts. High PIEZO1 expression is correlated with a significant decrease in survival in HR-negative cohorts, especially in triple-negative breast cancer (TNBC), suggesting that PIEZO1 could be utilized as a prognostic biomarker in HR-negative breast cancer. GSEA showed that various signaling pathways associated with more invasive phenotypes and resistance to treatments, including epithelial-mesenchymal transition (EMT), hypoxia, and multiple signaling pathways, are enriched in high-PIEZO1 HR-negative tumors. Our results also demonstrated a decrease in CD8+ and CD4+ T cell infiltration in high-PIEZO1 HR-negative tumors. Further investigations are necessary to elucidate the mechanistic roles of PIEZO1 in HR-negative breast cancer.

DNAJC12 causes breast cancer chemotherapy resistance by repressing doxorubicin-induced ferroptosis and apoptosis via activation of AKT

BackgroundChemotherapy is a primary treatment for breast cancer (BC), yet many patients develop resistance over time. This study aims to identify critical factors contributing to chemoresistance and their underlying molecular mechanisms, with a focus on reversing this resistance. MethodsWe utilized samples from the Gene Expression Omnibus (GEO) and West China Hospital to identify and validate genes associated with chemoresistance. Functional studies were conducted using MDA-MB-231 and MCF-7 cell lines, involving gain-of-function and loss-of-function approaches. RNA sequencing (RNA-seq) identified potential mechanisms. We examined interactions between DNAJC12, HSP70, and AKT using co-immunoprecipitation (Co-IP) assays and established cell line-derived xenograft (CDX) models for in vivo validations. ResultsBoruta analysis of four GEO datasets identified DNAJC12 as highly significant. Patients with high DNAJC12 expression showed an 8 % pathological complete response (pCR) rate, compared to 38 % in the low expression group. DNAJC12 inhibited doxorubicin (DOX)-induced cell death through both ferroptosis and apoptosis. Combining apoptosis and ferroptosis inhibitors completely reversed DOX resistance caused by DNAJC12 overexpression. RNA-seq suggested that DNAJC12 overexpression activated the PI3K-AKT pathway. Inhibition of AKT reversed the DOX resistance induced by DNAJC12, including reduced apoptosis and ferroptosis, restoration of cleaved caspase 3, and decreased GPX4 and SLC7A11 levels. Additionally, DNAJC12 was found to increase AKT phosphorylation in an HSP70-dependent manner, and inhibiting HSP70 also reversed the DOX resistance. In vivo studies confirmed that AKT inhibition reversed DNAJC12-induced DOX resistance in the CDX model. ConclusionDNAJC12 expression is closely linked to chemoresistance in BC. The DNAJC12-HSP70-AKT signaling axis is crucial in mediating resistance to chemotherapy by suppressing DOX-induced ferroptosis and apoptosis. Our findings suggest that targeting AKT and HSP70 activities may offer new therapeutic strategies to overcome chemoresistance in BC.

RUNX2 prompts triple negative breast cancer drug resistance through TGF-β pathway regulating breast cancer stem cells

Triple-negative breast cancer (TNBC) stands out as the most aggressive subtype within the spectrum of breast cancer. The current clinical guidelines propose treatment strategies involving cytotoxic agents like epirubicin or paclitaxel. However, the emergence of acquired resistance frequently precipitates secondary tumor recurrence or the spread of metastasis. In recent times, significant attention has been directed toward the transcription factor RUNX2, due to its pivotal role in both tumorigenesis and the progression of cancer. Previous researches suggest that RUNX2 might be intricately linked to the development of resistance against chemotherapy, with its mechanism of action possibly intertwined with the signaling of TGF-β. Nevertheless, the precise interplay between their effects and the exact molecular mechanisms underpinning chemoresistance in TNBC remain elusive. Therefore, we have taken a multifaceted approach from in vitro and in vivo experiments to validate the relationship between RUNX2 and TGF-β and to search for their pathogenic mechanisms in chemoresistance. In conclusion, we found that RUNX2 affects chemoresistance by regulating cancer cell stemness through direct binding to TGF-β, and that TGF-β dually regulates RUNX2 expression. The important finding will provide a new reference for clinical reversal of the development of chemoresistance in breast cancer.

The Sensitization of Triple-Negative Breast Cancers to Poly ADP Ribose Polymerase Inhibition Independent of BRCA1/2 Mutation Status by Chemically Modified microRNA-489.

Chemoresistance and inefficient therapeutic efficacies in triple-negative breast cancers (TNBCs) are among the major clinical problems in breast cancers. A potential new method to sensitize these tumors to current treatment options is, therefore, urgent and necessary. Our previous studies demonstrated that miR-489 serves as one of the top tumor-suppressing miRs and features downregulated expression in metastatic TNBCs and that the restoration of miR-489 expression in TNBCs effectively inhibits the metastatic potentials of TNBCs both in vitro and in vivo. The chemical modification of miR-489 (CMM489) through the replacement of uracil with 5-FU further enhances the therapeutic potential of miR-489. In the present study, we tested the effects of CMM489 in synergizing DNA damage response (DDR) inhibitors such as PARP inhibitors. CMM489 is particularly effective in sensitizing TNBC cell lines with inherent resistance to PARP inhibitors regardless of BRCA mutation status. One of the anti-cancer mechanisms through which CMM489 synergizes with PARP inhibitors is the blockade of homologous recombination (HR) in TNBC cells upon DNA damage. The results of this study highlight the potential use of CMM489 in combination treatments with PARP inhibitors in TNBCs.

Exploring the Regulatory Roles of miR-21, miR-15, and miR-let-7 in ABC Transporter-Mediated Chemoresistance: Implications for Breast Cancer Etiology and Treatment.

Breast cancer, a prevalent and aggressive malignancy among females worldwide, poses a significant challenge due to resistance to chemotherapy and tyrosine kinase inhibitors. In breast cancer, ABC transporters play a pivotal role by contributing to chemoresistance and drug efflux, a phenomenon observed also in various cancers. This study aims to elucidate the role of oncomiRs miR-15, miR-21, and miR-let-7 in breast cancer etiology and their impact on chemotherapy-resistant oncogenes ABCA1, ABCB1, and ABCC1. Blood samples from female breast cancer patients were analyzed to assess the expression levels of miRNAs and oncogenes by qPCR. Significantly, miR-21 exhibited a positive correlation with ABCA1 in newly diagnosed patients, while miR-15 and miR-let-7 displayed a positive correlation with ABCA1 in the metastasis group. Additionally, miR-let-7 demonstrated a negative correlation with ABCC1 in newly diagnosed patients. This study's findings provide valuable insights into the cancer etiology of these miRNAs and their interactions with ABCA1, ABCB1, and ABCC1. Targeting these interactions holds promise for mitigating drug efflux and chemoresistance in breast cancer, potentially enhancing current treatments and improving patient outcomes.

M6A demethylase ALKBH5 regulates FOXO1 mRNA stability and chemoresistance in triple-negative breast cancer

Resistance to chemotherapy is the main reason for treatment failure and poor prognosis in patients with triple-negative breast cancer (TNBC). Although the association of RNA N6-methyladenosine (m6A) modifications with therapy resistance is noticed, its role in the development of therapeutic resistance in TNBC is not well documented. This study aimed to investigate the potential mechanisms underlying reactive oxygen species (ROS) regulation in doxorubicin (DOX)-resistant TNBC. Here, we found that DOX-resistant TNBC cells displayed low ROS levels because of increased expression of superoxide dismutase (SOD2), thus maintaining cancer stem cells (CSCs) characteristics and DOX resistance. FOXO1 is a master regulator that reduces cellular ROS in DOX-resistant TNBC cells, and knockdown of FOXO1 significantly increased ROS levels by inhibiting SOD2 expression. Moreover, the m6A demethylase ALKBH5 promoted m6A demethylation of FOXO1 mRNA and increased FOXO1 mRNA stability in DOX-resistant TNBC cells. The analysis of clinical samples revealed that the increased expression levels of ALKBH5, FOXO1, and SOD2 were significantly positively correlated with chemoresistance and poor prognosis in patients with TNBC. To our knowledge, this is the first study to highlight that ALKBH5-mediated FOXO1 mRNA demethylation contributes to CSCs characteristics and DOX resistance in TNBC cells. Furthermore, pharmacological targeting of FOXO1 profoundly restored the response of DOX-resistant TNBC cells, both in vitro and in vivo. In conclusion, we demonstrated a critical function of ALKBH5-mediated m6A demethylation of FOXO1 mRNA in restoring redox balance, which in turn promoting CSCs characteristics and DOX resistance in TNBC, and suggested that targeting the ALKBH5/FOXO1 axis has therapeutic potential for patients with TNBC refractory to chemotherapy.

Differential impact of cytoplasmic vs. nuclear RAD51 expression on breast cancer progression and patient prognosis.

RAD51 recombinase is one of the DNA damage repair proteins associated with breast cancer risk. Apart from its function to maintain genomic integrity within the cell nucleus, RAD51 localized to the cytoplasm has also been implicated in breast malignancy. However, limited information exists on the roles of cytoplasmic vs. nuclear RAD51 in breast cancer progression and patient prognosis. In the present study, the association of cytoplasmic and nuclear RAD51 with clinical outcomes of patients with breast cancer was analyzed, revealing that elevated cytoplasmic RAD51 expression was associated with breast cancer progression, including increased cancer stage, grade, tumor size, lymph node metastasis and chemoresistance, along with reduced patient survival. By contrast, elevated nuclear RAD51 expression largely had the inverse effect. Results from invitro investigations supported the cancer‑promoting effect of RAD51, showing that overexpression of RAD51 promoted breast cancer cell growth, chemoresistance and metastatic ability, while knockdown of RAD51 repressed these malignant behaviors. The current data suggest that differential expression of subcellular RAD51 had a distinct impact on breast cancer progression and patient survival. Specifically, cytoplasmic RAD51 in contrast to nuclear RAD51 was potentially an adverse marker in breast cancer.

177P The immune-microenvironment confers chemoresistance in breast cancer through activation of VEGFR2/STAT3/BIRC5 signaling

177P The immune-microenvironment confers chemoresistance in breast cancer through activation of VEGFR2/STAT3/BIRC5 signaling

Abstract B085: Beyond endocrine resistance: estrogen receptor (ESR1) activating mutations mediate chemotherapy resistance through the JNK/c-Jun MDR1 pathway in breast cancer

Abstract Purpose: All patients with metastatic breast cancer (BC) expressing estrogen receptor α (ESR1) will eventually develop resistance to endocrine therapies. In up to 40% of cases, this resistance is caused by activating mutations in the ligand-binding domain (LBD) of ESR1. Clinical evidence suggests adverse outcomes for these patients. Our hypothesis was that ESR1 mutations may confer resistance to subsequent treatment lines, specifically chemotherapy. Experimental Design: MCF-7 cells harboring ESR1 mutations (Y537S and D538G) were used to characterize proliferation, apoptosis, and tumor response to chemotherapy in vivo. JNK/c-Jun/MDR1 pathway was studied using qRT-PCR, western-blot, gene-reporter, ChIP assays and pharmacological inhibition. Results: Our results revealed that the mutated-ER (mut-ER) cells displayed relative chemoresistance, demonstrated by higher viability and reduced apoptosis. In vivo mouse model confirmed these findings. To elucidate the underlying mechanism, we examined MDR1 expression and JNK pathway activity. We observed elevated MDR1 expression in mut-ER cells, which was also reflected in clinical BC samples. Additionally, increased occupancy of the MDR1 promoter by c-Jun was observed in mut-ER cells. Importantly, inhibition of the JNK pathway resulted in decreased MDR1 expression, particularly in D538G cells. Moreover, JNK inhibition increased apoptosis and reduced viability in all cells, with a more pronounced effect in D538G cells. Conclusions: Taken together, our data indicate that ESR1 mutations confer chemoresistance in BC through the activation of the JNK/MDR1 axis. These findings suggest that targeting this pathway could be a potential strategy for novel treatments aimed at restoring chemotherapy sensitivity and improving outcomes for patients with metastatic BC. Citation Format: Marwa Taya, Keren Merenbakh-Lamin, Asia Zubkov, Zohar Honig, Ori Mayer, Noam Shomron, Ido Wolf, Tami Rubinek. Beyond endocrine resistance: estrogen receptor (ESR1) activating mutations mediate chemotherapy resistance through the JNK/c-Jun MDR1 pathway in breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B085.