Chemoresistance In Triple-negative Breast Cancer Research Articles

Abstract 1780: Functional inhibition of RNA-binding protein HuR reverses chemotherapeutic resistance in triple-negative breast cancer

Abstract The 5-year relative survival rate of patients with triple-negative breast cancer (TNBC) is lower than the overall patients with breast cancer. The primary systemic treatment for TNBC remains to be chemotherapy. But chemoresistance frequently develops with the conventional usage of chemotherapy drugs, which results in poorer prognosis and higher recurrence of TNBC than other subtypes of breast cancer. Therefore, to improve the treatment for TNBC, overcoming chemoresistance is a critical challenge to conquer. The RNA-binding protein Hu antigen R (HuR) is a posttranscriptional regulator. It can stabilize target mRNAs through binding to U- or AU- rich elements mainly in 3’ untranslated region (UTR) of mRNA and upregulate their translation level in most cases. The encoded proteins of HuR target mRNAs are implicated in multiple cancer hallmarks, including chemotherapeutic sensitivity. The cytoplasmic accumulation of HuR is reported to contribute to chemoresistance in multiple cancer cells, and HuR inhibition sensitizes cancer cells to chemodrugs. We hypothesize that inhibition of HuR function by disrupting its interaction with mRNA can accelerate the decay of target mRNAs and thus reduce the translation level of proteins responsible for chemoresistance. Recently, our lab identified a small molecule HuR inhibitor, KH-3, which potently inhibits HuR function by disrupting HuR-mRNA interactions. In this study, KH-3 is used as a tool compound to investigate the roles HuR plays in chemoresistance development and evaluate whether HuR inhibition can enhance the efficacy of chemotherapy for TNBC cells. Two MDA-MB-231 cell sublines resistant to docetaxel (231-TR) or doxorubicin (231-DR) were generated in our lab. Compared to the parental cell line, two sub-lines exhibit similar sensitivity to KH-3, and KH-3 re-sensitizes chemoresistant cells to docetaxel or doxorubicin in the MTT-based cytotoxicity assay and the colony formation assay, indicating that HuR inhibition can overcome the acquired chemoresistance. The in vivo efficacy studies in orthotopic xenograft mouse models of human TNBC confirm that KH-3 synergizes docetaxel treatment. Regarding to mechanisms of action, several HuR direct target mRNAs implicated in chemoresistance were found upregulated in the resistant cells, which were reversed by KH-3 treatment. Detailed molecular mechanisms are now under investigation. This study suggests that HuR inhibition is a promising strategy to overcome the challenge of chemoresistance of TNBC. Citation Format: Lanjing Wei, Qi Zhang, Cuncong Zhong, Jeffrey Aubé, Danny R. Welch, Xiaoqing Wu, Liang Xu. Functional inhibition of RNA-binding protein HuR reverses chemotherapeutic resistance in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1780.

Abstract 2465: Optical imaging captures chemo-induced metabolic reprogramming during disease regression

Abstract Less than 20% of Triple Negative Breast Cancer (TNBC) patients experience long-term responses to mainstay chemotherapy, as tumors develop chemo-resistance. Prediction of chemo-resistance will guide treatment planning, thus improving response while reducing toxicity and cost associated with ineffective therapies. Spatial and temporal metabolic reprogramming holds promise as a biomarker of therapy response as resistant tumor subpopulations utilize alternate metabolic pathways to escape therapy, enter dormancy and recur. Currently, there are no tools to temporally evaluate heterogenous changes along distinct metabolic axes in vivo at a spatial resolution capable of resolving vulnerabilities of residual tumor subpopulations. We developed an optical-imaging based platform to correlate long-term treatment outcomes to early chemo-induced metabolic changes along three relevant axes of TNBC chemoresistance (Glycolysis, Oxidative Phosphorylation and Fatty Acid Oxidation) at high spatial resolutions. Here, we validated our system’s ability to capture metabolic signatures of disease regression that accompany chemo-resistance by assessing metabolic reprogramming and tumor kinetics in MDA-MB-231 TNBC xenografts. Xenografts were established by orthotopic cell injection and mice were treated with Paclitaxel (PTX), a commonly used chemotherapeutic drug in TNBC treatment, under a conventional maximum dose density regimen. Mice were monitored for drug response via caliper measurements. Xenografts were partially sensitive to PTX: tumor kinetics showed an initial response to PTX, a dormancy period, and a resurgence in tumor volume at ~60 days post drug withdrawal (n=3). A separate cohort of mice were implanted with window chambers on the primary tumor and imaged every two days with previously validated fluorophores 2-NBDG, TMRE and Bodipy to directly report on glucose uptake, mitochondrial membrane potential or fatty acid uptake, respectively. Wide field fluorescence imaging showed a significant increase in TMRE as early as two days after the 3rd PTX dose (n=5, p<0.05) and a significant decrease in 2-NBDG as early as two days after 5th PTX dose (n=5, p<0.05). No significant changes in Bodipy were seen. Decreased 2-NBDG and increased TMRE remained up to 40 days after drug withdrawal. Consistent with the literature, our results point towards a metabolic switch from glycolysis to mitochondrial respiration and suggests decreased use of glucose as a metabolic substrate. For this cell line, no altered uptake of fatty acids was detected, possibly suggesting amino acid catabolism as a fuel for mitochondrial respiration. This study confirms our platform’s ability to capture the resulting metabolic changes following chemotherapy and during disease regression. We aim to use this system to visualize and exploit in vivo metabolic vulnerabilities of disease regression that correlate to local and distal recurrences. Citation Format: Enakshi Devi Sunassee, Elizabeth Maydew, Sultan Erturk, Megan Madonna, Brian Crouch, Nirmala Ramanujam. Optical imaging captures chemo-induced metabolic reprogramming during disease regression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2465.

Triple-negative breast cancer drug resistance, durable efficacy, and cure: How advanced biological insights and emerging drug modalities could transform progress

Introduction Triple-negative breast cancer (TNBC) is a heterogeneous disease characterized by the lack of estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2) and often associated with poor survival outcomes. The backbone of current treatments for TNBC relies on chemotherapy; however, resistance to cytotoxic agents is a commonly encountered hurdle to overcome. Areas covered : Current understanding on the mechanisms involved in TNBC chemoresistance is evaluated and novel potential actionable targets and recently explored modalities for carrying and delivering chemotherapeutics are highlighted. Expert opinion : A comprehensive identification of both genomic and functional TNBC signatures is required for a more definite categorization of the patients in order to prevent insensitivity to chemotherapy and therefore realize the full potential of precision-medicine approaches. In this scenario, cell-line-derived xenografts (CDX), patient-derived xenografts (PDX), patient-derived orthotopic xenografts (PDOX) and patient-derived organoids (PDO) are indispensable experimental models for evaluating the efficacy of drug candidates and predicting the therapeutic response. The combination of increasingly sensitive “omics” technologies, computational algorithms and innovative drug modalities may accelerate the successful translation of novel candidate TNBC targets from basic research to clinical settings, thus contributing to reach optimal clinical output, with lower side effects and reduced resistance.

Thymoquinone Alterations of the Apoptotic Gene Expressions and Cell Cycle Arrest in Genetically Distinct Triple-Negative Breast Cancer Cells.

Breast cancer (BC) is the most common cancer in women worldwide, and it is one of the leading causes of cancer death in women. triple-negative breast Cancer (TNBC), a subtype of BC, is typically associated with the highest pathogenic grade and incidence in premenopausal and young African American (AA) women. Chemotherapy, the most common treatment for TNBC today, can lead to acquired resistance and ineffective treatment. Therefore, novel therapeutic approaches are needed to combat medication resistance and ineffectiveness in TNBC patients. Thymoquinone (TQ) is shown to have a cytotoxic effect on human cancer cells in vitro. However, TQ’s mode of action and precise mechanism in TNBC disease in vitro have not been adequately investigated. Therefore, TQ’s effects on the genetically different MDA-MB-468 and MDA-MB-231 human breast cancer cell lines were assessed. The data obtained show that TQ displayed cytotoxic effects on MDA-MB-468 and MDA-MB-231 cells in a time- and concentration-dependent manner after 24 h, with IC50 values of 25.37 µM and 27.39 µM, respectively. Moreover, MDA-MB-231 and MDA-MB-468 cells in a scratched wound-healing assay displayed poor wound closure, inhibiting invasion and migration via cell cycle blocking after 24 h. TQ arrested the cell cycle phase in MDA-MB-231 and MDA-MB-468 cells. The three cell cycle stages in MDA-MB-468 cells were significantly affected at 15 and 20 µM for G0/G1 and S phases, as well as all TQ concentrations for G2/M phases. In MDA-MB-468 cells, there was a significant decrease in G0/G1 phases with a substantial increase in the S phase and G2/M phases. In contrast, MDA-MB-231 showed a significant effect only during the two cell cycle stages (S and G2/M), at concentrations of 15 and 20 µM for S phases and all TQ values for G2/M phases. The TQ effect on the apoptotic gene profiles indicated that TQ upregulated 15 apoptotic genes in MDA-MB-231 TNBC cells, including caspases, GADD45A, TP53, DFFA, DIABLO, BNIP3, TRAF2/3, and TNFRSF10A. In MDA-MB-468 cells, 16 apoptotic genes were upregulated, including TNFRSF10A, TNF, TNFRSF11B, FADD TNFRSF10B, CASP2, and TRAF2, all of which are important for the apoptotic pathway andsuppress the expression of one anti-apoptotic gene, BIRC5, in MDA-MB-231 cells. Compared to MDA-MB-231 cells, elevated levels of TNF and their receptor proteins may contribute to their increased sensitivity to TQ-induced apoptosis. It was concluded from this study that TQ targets the MDA-MB-231 and MDA-MB-468 cells differently. Additionally, due to the aggressive nature of TNBC and the lack of specific therapies in chemoresistant TNBC, our findings related to the identified apoptotic gene profile may point to TQ as a potential agent for TNBC therapy.

Abstract B030: Mimicking the evolution of doxorubicin resistance in triple negative breast cancer

Abstract Chemotherapy resistance presents a major challenge in the treatment of triple negative breast cancer (TNBC), which is characterized by low or no expression of the estrogen receptor (ER), the progesterone receptor (PR), and the human epidermal growth factor receptor-2 (HER2). Because TNBC tumors do not express these receptors, there has been little to no advancement in the establishment of drug therapies to specifically target this breast cancer subtype. Additionally, unfavorable side effects and drug resistance reveal an urgent need for safer, more effective therapeutic options. In this study, we aim to mimic cancer recurrence by establishing doxorubicin (DOX) resistance in TNBC cell lines, HCC1806 and HCC70 and investigate the effect on cell survival, cell migration, and changes in gene expression. Here we show that TNBC cell growth was sustained after pulse treatments with increasing concentrations of DOX. HCC1806 and HCC70 cells treated with DOX titrants (0.5 µM-2.0 µM) demonstrate cell growth sustainability after 3h treatments. The DOX-treated TNBC cells also demonstrate anti-migratory potential. RNA sequencing analysis show that IL6, TP53, and TNFSF12 are uniquely expressed after 24h exposure to 0.5uM DOX. We plan to further investigate the roles these genes play in chemoresistance. Understanding changes in the gene network, as a result of doxorubicin-resistance, may help to establish biomarkers that could represent targets for drug therapies to overcome chemoresistance in TNBC. Citation Format: Kashenya Pearson, Terrence Everett, Checo J. Rorie. Mimicking the evolution of doxorubicin resistance in triple negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr B030.

Fatty acid oxidation protects cancer cells from apoptosis by increasing mitochondrial membrane lipids.

Overcoming resistance to chemotherapies remains a major unmet need for cancers, such as triple-negative breast cancer (TNBC). Therefore, mechanistic studies to provide insight for drug development are urgently needed to overcome TNBC therapy resistance. Recently, an important role of fatty acid β-oxidation (FAO) in chemoresistance has been shown. But how FAO might mitigate tumor cell apoptosis by chemotherapy is unclear. Here, we show that elevated FAO activates STAT3 by acetylation via elevated acetyl-coenzyme A (CoA). Acetylated STAT3 upregulates expression of long-chain acyl-CoA synthetase 4 (ACSL4), resulting in increased phospholipid synthesis. Elevating phospholipids in mitochondrial membranes leads to heightened mitochondrial integrity, which in turn overcomes chemotherapy-induced tumor cell apoptosis. Conversely, in both cultured tumor cells and xenograft tumors, enhanced cancer cell apoptosis by inhibiting ASCL4 or specifically targeting acetylated-STAT3 is associated with a reduction in phospholipids within mitochondrial membranes. This study demonstrates a critical mechanism underlying tumor cell chemoresistance.

Reversal chemotherapeutic resistance of triple‐negative breast cancer via functionally inhibiting RNA‐binding protein HuR

Chemotherapy remains the primary option for the systemic treatment of triple‐negative breast cancer (TNBC). However, chemoresistance frequently occurs with the conventional use of chemotherapeutic drugs, resulting in a poorer prognosis and a higher recurrence rate of TNBC than other subtypes of breast cancer. Therefore, overcoming chemoresistance is a critical challenge to conquer for the successful treatment of TNBC. The RNA‐binding protein Hu antigen R (HuR) is a posttranscriptional regulator. It can stabilize mRNA by binding to U‐ or AU‐rich elements (ARE) mainly in 3’ untranslated region (UTR) of mRNA and therefore upregulate the translation most of the time. The encoded proteins of HuR target mRNAs are implicated in multiple cancer hallmarks, including therapeutic resistance. The cytoplasmic accumulation of HuR is reported to contribute to chemoresistance in several types of cancer cells, and HuR inhibition sensitizes cells to chemodrugs. We hypothesize that inhibition of HuR function by disrupting its interaction with mRNA can accelerate the decay of target mRNA and thus reduce the translation level of proteins responsible for chemoresistance.Recently, our lab reported a small molecule HuR inhibitor, KH‐3, which potently inhibits HuR function by disrupting HuR‐mRNA interactions. In this study, we investigate the roles HuR played in the chemoresistance of TNBC and evaluate whether HuR inhibition by KH‐3 can enhances the chemotherapy efficacy. To determine whether HuR inhibition overcomes acquired chemoresistance of TNBC, two MDA‐MB‐231 cell sub‐lines resistant to docetaxel (231‐TR) or doxorubicin (231‐DR) were generated. Compared to the parental cell line, two sub‐lines exhibit similar sensitivity to KH‐3, and KH‐3 re‐sensitizes chemoresistant cells to docetaxel or doxorubicin in the MTT‐based cytotoxicity assay and the colony formation assay, indicating that HuR inhibition can overcome the acquired chemoresistance. The combination index suggests that the combination of KH‐3 with docetaxel or doxorubicin has a synergistic effect. The in vivo efficacy studies in both MDA‐MB‐231 and 231‐TR orthotopic xenograft mouse models confirm that KH‐3 synergizes docetaxel treatment. Mechanistically, several HuR direct target mRNAs implicated in chemoresistance were found upregulated in the resistant cells, which were reversed by KH‐3 treatment. The detailed molecular mechanisms of how KH‐3 sensitized TNBC cells to chemodrugs are being investigated. This research suggests that HuR inhibition is a promising strategy for overcoming chemoresistance in TNBC.

CircINTS4 Facilitates Chemoresistance of TNBC by Competitively Binding miR-129-5p/POM121 Axis

Objectives To detect the expression of circular RNA (circRNA) circINTS4 in triple-negative breast cancer (TNBC) and to analyze the relationship between the expression of circRNA circINTS4 and the clinicopathological characteristics and chemotherapy resistance of patients with TNBC. Methods Bioinformatics was used to predict that circINTS4 and POM121 could bind to miR-129-5p, and dual luciferase reporter genes proved that circINTS4 could bind to miR-129-5p and miR-129-5p could bind to POM121. RNA immunoprecipitation (RIP) and RNA pull-down experiments confirmed that circINTS4 binds to miR-129-5p. The correlation among circINTS4, miR-129-5p, and POM121 was detected by qRT-PCR. Results In ADR-resistant TNB cells, circINTS4 was significantly up-regulated, miR-129-5p was down-regulated, and POM121 protein expression was significantly up-regulated. Experimental results showed that circINTS4 knockdown inhibited proliferation, migration, invasion, and autophagy. Knocking down miR-129-5p or overexpression of POM121 reversed the inhibitory effect of sh-circints4 on the development of ADR-resistant TNBC cells. In addition, CIRCINTS4 regulates POM121 expression by sponge-adsorbed miR-129-5p. CIRCINTS4 knockdown prevents ADR-resistant tumor growth by regulating the miR-129-5p/POM121 axis in vivo. Conclusions CircRNA circINTS4 may act as the ceRNA of miR-129-5p to regulate the expression of target gene POM121, thereby promoting the progress of TNBC molecular mechanism and providing scientific basis for circINTS4 as a new molecular target for clinical diagnosis and drug resistance therapy of TNBC.

Identification of a novel mechanism for reversal of doxorubicin-induced chemotherapy resistance by TXNIP in triple-negative breast cancer via promoting reactive oxygen-mediated DNA damage

Given that triple-negative breast cancer (TNBC) lacks specific receptors (estrogen and progesterone receptors and human epidermal growth factor receptor 2) and cannot be treated with endocrine therapy, chemotherapy has remained the mainstay of treatment. Drug resistance is reportedly the main obstacle to the clinical use of doxorubicin (DOX) in this patient population. Accordingly, screening molecules related to chemoresistance and studying their specific mechanisms has clinical significance for improving the efficacy of chemotherapy in TNBC patients. Thioredoxin-interacting protein (TXNIP) is a metabolism-related protein that plays a tumor suppressor role in various malignant tumors; however, the specific role of TXNIP in tumor chemoresistance has not been reported. In the present study, we explored the potential molecular mechanism of TXNIP in the chemoresistance of TNBC for the first time. The results showed that TXNIP inhibited the proliferation of TNBC drug-resistant cells and promoted apoptosis in vitro and in vivo. Furthermore, TXNIP promoted the synthesis of reactive oxygen species (ROS) and the accumulation of DNA damage caused by DOX and increased γ-H2AX levels in a time and dose-dependent manner. Moreover, ROS scavenger pretreatment could block DNA damage induced by TXNIP and restore the resistance of TNBC resistant cells to DOX to a certain extent. In addition, we found that the small molecule c-Myc inhibitor 10058-F4 promoted TXNIP expression, increased ROS synthesis in cells, and could enhance the cytotoxicity of chemotherapy drugs in vitro and in vivo when combined with DOX. These results indicated that c-Myc inhibitor 10058-F4 could induce TXNIP upregulation in TNBC drug-resistant cells, and the upregulated TXNIP increased the accumulation of ROS-dependent DNA damage, thereby decreasing chemotherapy resistance of TNBC. Our findings reveal a new mechanism of mediating drug resistance and provide a new drug combination strategy to overcome DOX resistance in TNBC.

Targeting HIF1-alpha/miR-326/ITGA5 axis potentiates chemotherapy response in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer that is frequently treated with chemotherapy. However, many patients exhibit either de novo chemoresistance or ultimately develop resistance to chemotherapy, leading to significantly high mortality rates. Therefore, increasing the efficacy of chemotherapy has potential to improve patient outcomes. Here, we performed whole transcriptome sequencing (both RNA and small RNA-sequencing), coupled with network simulations and patient survival data analyses to build a novel miRNA-mRNA interaction network governing chemoresistance in TNBC. We performed cell proliferation assay, Western blotting, RNAi/miRNA mimic experiments, FN coating, 3D cultures, and ChIP assays to validate the interactions in the network, and their functional roles in chemoresistance. We developed xenograft models to test the therapeutic potential of the identified key miRNA/proteins in potentiating chemoresponse in vivo. We also analyzed several patient datasets to evaluate the clinical relevance of our findings. We identified fibronectin (FN1) as a central chemoresistance driver gene. Overexpressing miR-326 reversed FN1-driven chemoresistance by targeting FN1 receptor, ITGA5. miR-326 was downregulated by increased hypoxia/HIF1A and ECM stiffness in chemoresistant tumors, leading to upregulation of ITGA5 and activation of the downstream FAK/Src signaling pathways. Overexpression of miR-326 or inhibition of ITGA5 overcame FN1-driven chemotherapy resistance in vitro by inhibiting FAK/Src pathway and potentiated the efficacy of chemotherapy in vivo. Importantly, lower expression of miR-326 or higher levels of predicted miR-326 target genes was significantly associated with worse overall survival in chemotherapy-treated TNBC patients. FN1 is central in chemoresistance. In chemoresistant tumors, hypoxia and resulting ECM stiffness repress the expression of the tumor suppressor miRNA, miR-326. Hence, re-expression of miR-326 or inhibition of its target ITGA5 reverses FN1-driven chemoresistance making them attractive therapeutic approaches to enhance chemotherapy response in TNBCs.

38P New tetraamine compounds targeting BCSCs from chemoresistant basal-like TNBC subtypes

Being a highly aggressive breast cancer subtype and lacking the expression of prognostic markers that allow the use of targeted therapies, triple-negative breast cancer (TNBC) possesses a high mortality rate. Recently, Lehmann classified TNBC into six subtypes, dividing basal subtypes into groups named Basal-Like 1 (BL1) and 2 (BL2). While current chemotherapy deals with tumour bulk, the lack of therapeutic alternatives targeting cells displaying stem-like properties allow for relapse. Thus, there is an urgent need for the development of breast cancer stem cell (BCSC) specific drugs.

Abstract P4-01-08: Morphological and functional plasticity of mitochondria in chemoresistant triple negative breast cancer

Abstract BACKGROUND: Neoadjuvant chemotherapy (NACT) used for triple negative breast cancer (TNBC) eradicates tumors in only 45% of patients. TNBC patients with substantial residual cancer burden have poor metastasis free and overall survival rates. Therefore, understanding the mechanisms of resistance to standard chemotherapy is essential. Our previous studies found mitochondrial oxidative phosphorylation (OXPHOS) was elevated and was a unique therapeutic dependency of residual tumor cells that survived after NACT. Mitochondria are morphologically plastic and dynamic organelles that continuously cycle between fission and fusion to maintain mitochondrial integrity and metabolic homeostasis. Mitochondrial fusion is thought to support OXPHOS through maintenance of the mitochondrial genome (mtDNA) which encodes 13 subunits of electron transport chain complexes. On the other hand, mitochondrial fission can be the precursor to mitophagy, the selective degradation of damaged mitochondria. We are investigating how mitochondrial structure dynamics impact metabolic states driving chemoresistance in TNBC. Methods We used a previously characterized orthotopic patient-derived xenograft (PDX) mouse model derived from the primary tumor of a treatment-naïve metastatic TNBC patient. After treatment with a standard NACT regimen, combined Adriamycin (aka doxorubicin) and cyclophosphamide (AC), we observed initial partial response followed by tumor regrowth. We collected PDX tumors at three time points: pre-treatment, post-AC residual (when tumors reached the volume nadir), and post-AC tumors that later regrew to the starting tumor volume. We used transmission electron microscopy (TEM) and immunohistochemical (IHC) analysis of human mitochondria to assess mitochondrial morphology and mass, respectively. For in vitro assays, we assessed mitochondria morphology, mtDNA content, and OXPHOS activity in TNBC cells treated with two different types of chemotherapeutic drugs, DNA-damaging agents (doxorubicin and carboplatin) and a microtubule-stabilizing agent (paclitaxel). To examine metabolic adaptations upon chemotherapy, we conducted 13C-glucose and glutamine flux metabolomics. To pharmaceutically perturb mitochondrial morphology, we used Mdivi-1 and Silibinin, known to induce mitochondrial fusion and fission, respectively. We assessed cell growth by Incucyte real-time imaging. Results: Analyses of residual PDX tumors after in vivo AC treatment revealed increased mitochondrial content, and increased average mitochondrial length in residual tumor cells. Metabolomic analysis of TNBC cells revealed all three chemotherapies induced glucose, but not glutamine, flux through the TCA cycle. Flux through glycolysis was not affected by chemotherapies. Treatment with doxorubicin and carboplatin, increased mitochondrial elongation, mtDNA content, and OXPHOS activity. Conversely, paclitaxel treatment reduced mitochondrial length and OXPHOS activity. We observed Mdivi-1 induced fusion concomitant with increased OXPHOS activity, while treatment with Silibinin reduced fusion and decreased OXPHOS. We next tested if induction of mitochondrial fission enhanced chemosensitivity. Sequential treatment of TNBC cells with DNA-damaging chemotherapeutics followed by Silibinin perturbed OXPHOS and resulted in enhanced cell killing. Conclusion: These findings establish a functional role for mitochondrial structure in chemotherapeutic response and metabolic reprogramming, which may confer a survival advantage to TNBC cells. Given the increased chemosensitivity we observed after pharmacologic inhibition of mitochondrial fusion, our study suggests perturbing mitochondrial structure adaptations may provide an opportunity to overcome TNBC chemoresistance in the neoadjuvant setting. Citation Format: Lily Mokryun Baek, Junegoo Lee, James P. Barrish, Bora Lim, Jeffrey T. Chang, Phillip L. Lorenzi, Weston Porter, Gloria V. Echeverria. Morphological and functional plasticity of mitochondria in chemoresistant triple negative 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 P4-01-08.

Abstract P4-01-16: Overcome chemoresistance of triple-negative breast cancer by inhibiting the RNA-binding protein HuR

Abstract Chemotherapy remains the primary systemic treatment for triple-negative breast cancer (TNBC). However, patients with TNBC often develop resistance to conventional chemotherapy, resulting in a poorer prognosis and a higher recurrence rate than those with other subtypes of breast cancer. The RNA-binding protein Hu antigen R (HuR) is a posttranscriptional regulator, which can stabilize target mRNAs and regulate the translation of encoded proteins implicated in several hallmarks of cancer, including drug resistance. The high cytoplasmic expression of HuR is associated with high-grade malignancy and poor clinical outcomes of breast cancer. The accumulated cytoplasmic HuR has also been reported to contribute to chemoresistance in several types of cancer cells and inhibition of HuR sensitizes cancer cells to chemotherapy. Therefore, HuR is a promising target to overcome chemoresistance. We hypothesize that inhibition of HuR function by disrupting its interaction with mRNA can accelerate the decay of target mRNAs and thus reduce the translation level of proteins responsible for chemoresistance.Recently, our lab reported a small molecule HuR inhibitor, KH-3, which potently inhibits HuR function by disrupting HuR-mRNA interactions. In this study, we aim to investigate the functions of HuR in TNBC chemoresistance formation and evaluate whether HuR inhibition by KH-3 can enhance the efficacy of chemotherapy for TNBC. In order to determine whether HuR inhibition overcomes acquired chemoresistance of TNBC, we generated two MDA-MB-231 cell sub-lines with acquired resistance to docetaxel (231-TR) or doxorubicin (231-DR), respectively. Compared to the parental cell line, the two resistant sub-lines exhibit similar sensitivity to KH-3, and KH-3 re-sensitizes chemoresistant cells to docetaxel and doxorubicin in the MTT-based cytotoxicity assay and the colony formation assay, indicating that HuR inhibition can overcome acquired chemoresistance. The combination index suggests that the combination KH-3 with docetaxel or. doxorubicin has a synergistic effect. Moreover, the in vivo efficacy studies confirm that KH-3 synergized docetaxel treatment in both MDA-MB-231 and 231-TR orthotopic xenograft models. Mechanistically, several HuR direct target mRNAs implicated in chemoresistance are upregulated in the resistant cells, and KH-3 treatment can reverse the enhanced mRNA levels. The bioinformatic analysis suggests that several pathways may involve in the acquired resistance to docetaxel and doxorubicin. However, detailed molecular mechanisms of how KH-3 sensitizes TNBC to chemotherapy are still under investigation. This study suggests that inhibition of HuR is a promising strategy for overcoming chemotherapy resistance of TNBC. Citation Format: Lanjing Wei, Qi Zhang, Cuncong Zhong, Jeffrey Aubé, Danny R. Welch, Xiaoqing Wu, Liang Xu. Overcome chemoresistance of triple-negative breast cancer by inhibiting the RNA-binding protein HuR [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 P4-01-16.

Abstract P5-12-06: Msc engulfing by triple negative breast cancer cells induces a hybrid cell population with senescence

Abstract Introduction: Triple negative breast cancer (TNBC) represents ~15% of all breast cancers. The TNBC subtype is characterized by the absence of estrogen and progesterone receptors and of ErbB2. Biologically, it is well recognized that TNBC has a more aggressive phenotype compared to other subtypes, offering limited treatment options and a worse clinical outcome. Tumor cells, through production of paracrine and endocrine molecules, can recruit mesenchymal stem/stromal cells (MSCs) from the bone marrow and adipose tissue to the tumor microenvironment. MSCs, reside mainly in the bone marrow and in adipose tissue. MSCs may exert a crucial role in tumor development and progression, by increasing stemness of tumor cells and promoting extracellular matrix remodeling, cell migration and invasion, neo-angiogenesis, and drug resistance. We have shown that breast cancer cells (BCCs) can engulf MSCs and induce an aggressive phenotype and increased metastasis. The mechanisms through which MSC engulfment occurs and the generated phenotypes need to be investigated. In this study, we tested the hypothesis that MSC engulfment by BCC cells may result in a hybrid tumor cell population with features of cell senescence, a well-recognized mechanism of tumor progression and recurrence. Methods: We employed MDA-MB-231 TNBC cells and patient-derived MSCs developed and validated in our lab. MDA-MB-231 were labeled with DsRED (231- DsRED) and MSCs were not labeled. Single culture of MDA-MB-231, MSCs, and MDA-MB-231/MSC co-cultures (1:1 ratio) were subjected to SA-β-galactosidase activity assay, flow cytometry, and immunofluorescence to detect and quantify the percentage of hybrid and senescence cells. SA-β-galactosidase activity assay was assessed using Senescence-associated β-galactosidase Staining Kit. For each sample of the single cultures and co-cultures, 10 randomly chosen fields were photographed using a camera-equipped bright field microscope (Olympus). Data was plotted as the percentage of cells with β-galactosidase activity (blue cells). We performed flow cytometry to detect β-galactosidase activity in MSCs and in DsRED-231 in single cultures or co-cultures. Immunofluorescence assay was performed to evaluate β-galactosidase activity in the same conditions previously described. Results: Our studies identified the emergence of a hybrid cell population in the co-cultures of MDA-MB-231 and MSCs cells. The hybrid population contained a significantly higher percentage of senenscent cells compared to 231 and MSCs (44%, 10.23%, and 14%, respectively, t test 0.001). These results were confirmed by immunofluorescence analysis for β-galactosidase, DsRed and DAPI in situ. Conclusion: These results highlight that MSCs engulfment by TNBC cells induces the formation of senescent hybrid breast cancer cells, which may explain their survival advantage. Studies are under way to investigate if the senescent phenotype of hybrid cells is influenced by EMT-MET states and its role in TNBC chemoresistance. Citation Format: Alessandro Paolì, Maria Elena Gonzalez, Loredana Mauro, Sebastiano Andò, Celina Graciela Kleer. Msc engulfing by triple negative breast cancer cells induces a hybrid cell population with senescence [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 P5-12-06.

Abstract P5-08-18: Disruption of redox balance in glutaminolytic triple negative breast cancer by inhibition of glutamate transport and glutaminase

Abstract OBJECTIVES: Upregulated glutamine metabolism is an important metabolic signature for aggressive and treatment-resistant cancers, including many triple negative breast cancers (TNBCs). Glutamate (Glu) produced from glutamine (Gln) via mitochondrial glutaminase (GLS) feeds into the TCA cycle to provide energy and biosynthetic precursors for tumor growth. Recent studies from our lab and others have revealed that Glu also plays an important role in maintaining redox homeostasis which is essential for survival of cancer cells. Glu is a component amino acid of glutathione, the most abundant cellular antioxidant. Furthermore, via the xCT antiporter (SLC7A11), Glu is exported for import of cystine, a rate-limiting step for glutathione synthesis. We assessed the effect of metabolic inhibition of GLS and/or xCT pathways on cellular reactive oxygen species (ROS) level and induction of apoptosis. To track cytosolic Glu transport cross plasma membrane in vivo, we tested (4S)-4-(3-[18F]fluoropropyl)-L-glutamic acid ([18F]FSPG), a non-metabolized structural analog of Glu. We hypothesize that mitochondrial Glu supply is necessary for maintaining cellular redox homeostasis and [18F]FSPG PET can serve as a tool to track Glu transport in glutaminolytic TNBC and guide treatment targeting GLS and redox balance leading to unmitigated oxidative stress and cell death. METHODS: A human glutaminolytic TNBC cell line (HCC1806) and a non-glutaminolytic estrogen-receptor positive cell line (MCF-7) were used to generate xenografts in athymic mice. Cells were exposed to culture media containing GLS inhibitor CB839 (1 µM), xCT inhibitor Erastin (ERA, 3 µM) and/or chemotherapy doxorubicin (DOX, 0.2 µM) for 24 h. ROS were measured by DHE and CellROX Green assays; early apoptosis was estimated by flow cytometry analysis of cells double-stained with Annexin V-FITC and TOR-PR-3. In vivo dynamic [18F]FSPG PET imaging (1 h) was performed on HCC1806 and MCF-7 xenograft models. Lipid peroxidation induced by treatments was examined using C11-Bodipy, a fluorescent sensor. RESULTS: ROS level in HCC1806 cells increased robustly with CB839 (20% over untreated cells), ERA (30%), or ERA plus CB839 (50%), whereas ROS responses in MCF-7 cells were more blunted. Compared to untreated cells, DOX treatment increased ROS level in HCC1806 cells by 40% whereas DOX combined with CB839 plus ERA led to a remarkable increase of cellular ROS by more than 200%. Combined treatment also led to 31% of cells undergoing early apoptosis, compared to 3% in DOX only and 2% in untreated cells. Time-activity curves (TACs) from [18F]FSPG PET of these xenograft models were fit by a reversible one-compartment model in keeping with the known biology of the tracer. The tumor-to-blood ratio (T/B) averaged from the last 6 points (30 min) of the TACs reveal a large difference between HCC1806 vs. MCF-7 by a factor > 2. C11-Bodipy signal in HCC1806 cells was not changed after CB839 but increased substantially after ERA only (45%) or DOX only (68%) treatment and after combined treatment of CB839, ERA and DOX (111%). DISCUSSIONS & CONCLUSION: Our preliminary data support the efficacy of dual targeting of glutaminolysis and Glu transport for sensitizing glutaminolytic TNBC to cytotoxic chemotherapy. T/B of [18F]FSPG PET appears to be distinct for glutaminolytic versus non-glutaminolytic breast cancers. Future work will focus on developing [18F]FSPG PET, complementing our prior work with a Gln analog as a measure of Gln metabolism. The two PET tracers may guide treatment targeting both Gln and Glu usage to overcome chemoresistant TNBC. SUPPORT: R01CA211337 and Komen SAC130060. Reference: 1. Gross MI, et al. Mol Cancer Ther 2014;13(4):890-901. 2. Viswanath V, et al. J Nucl Med 2020. 3. Zhou R, et al. Cancer Res 2017;77(6):1476-84. Citation Format: Hoon Choi, Hsiaoju Lee, Austin Pantel, Christopher Hensley, David Mankoff, Rong Zhou. Disruption of redox balance in glutaminolytic triple negative breast cancer by inhibition of glutamate transport and glutaminase [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 P5-08-18.

Persistent EGFR/K-RAS/SIAH pathway activation drives chemo-resistance and early tumor relapse in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. It disproportionately affects BRCA mutation carriers and young women, especially African American (AA) women. Chemoresistant TNBC is a heterogeneous and molecularly unstable disease that challenges our ability to apply personalized therapies. With the approval of immune checkpoint blockade (ICB) for TNBC, the addition of pembrolizumab to systemic chemotherapy has become standard of care (SOC) in neoadjuvant systemic therapy (NST) for high-risk early-stage TNBC. Pembrolizumab plus chemotherapy significantly increased the pathologic complete response (pCR) and improved event-free survival in TNBC. However, clinical uncertainties remain because similarly treated TNBC partial responders with comparable tumor responses to neoadjuvant therapy often experience disparate clinical outcomes. Current methods fall short in accurately predicting which high-risk patients will develop chemo-resistance and tumor relapse. Therefore, novel treatment strategies and innovative new research initiatives are needed. We propose that the EGFR-K-RAS-SIAH pathway activation is a major tumor driver in chemoresistant TNBC. Persistent high expression of SIAH in residual tumors following NACT/NST reflects that the EGFR/K-RAS pathway remains activated (ON), indicating an ineffective response to treatment. These chemoresistant tumor clones persist in expressing SIAH (SIAHHigh/ON) and are linked to early tumor relapse and poorer prognosis. Conversely, the loss of SIAH expression (SIAHLow/OFF) in residual tumors post-NACT/NST reflects EGFR/K-RAS pathway inactivation (OFF), indicating effective therapy and chemo-sensitive tumor cells. SIAHLow/OFF signal is linked to tumor remission and better prognosis post-NACT/NST. Therefore, SIAH is well-positioned to become a novel tumor-specific, therapy-responsive, and prognostic biomarker. Potentially, this new biomarker (SIAHHigh/ON) could be used to quantify therapy response, predict chemo-resistance, and identify those patients at the highest risk for tumor relapse and poor survival in TNBC.

Pseudo-Temporal Dynamics of Chemo-Resistant Triple-Negative Breast Cancer Cells Reveal EGFR/HER2 Inhibition as Synthetic Lethal During Mid-neoadjuvant Chemotherapy

Pseudo-Temporal Dynamics of Chemo-Resistant Triple-Negative Breast Cancer Cells Reveal EGFR/HER2 Inhibition as Synthetic Lethal During Mid-neoadjuvant Chemotherapy

The Role of Breast Cancer Stem Cells in Chemoresistance and Metastasis in Triple-Negative Breast Cancer.

Simple SummaryTriple negative breast cancer lacks targeted therapies and has poor prognosis; chemotherapy is currently the main treatment modality. Growing evidence has shown that breast cancer stem cells are associated with tumor initiation and metastasis and may play a critical role in chemoresistance. Multiple targets against breast cancer stem cells are now under investigation. Recent advances in the role of breast cancer stem cells in triple negative breast cancer and the identification of cancer stem cell biomarkers have paved the way for the development of new targeted therapies. The discovery of potential molecular signaling pathways targeting breast cancer stem cells to overcome chemoresistance and prevent metastasis will improve the overall survival of patients with triple negative breast cancer.Triple negative breast cancer (TNBC) remains an aggressive disease due to the lack of targeted therapies and low rate of response to chemotherapy that is currently the main treatment modality for TNBC. Breast cancer stem cells (BCSCs) are a small subpopulation of breast tumors and recognized as drivers of tumorigenesis. TNBC tumors are characterized as being enriched for BCSCs. Studies have demonstrated the role of BCSCs as the source of metastatic disease and chemoresistance in TNBC. Multiple targets against BCSCs are now under investigation, with the considerations of either selectively targeting BCSCs or co-targeting BCSCs and non-BCSCs (majority of tumor cells). This review article provides a comprehensive overview of recent advances in the role of BCSCs in TNBC and the identification of cancer stem cell biomarkers, paving the way for the development of new targeted therapies. The review also highlights the resultant discovery of cancer stem cell targets in TNBC and the ongoing clinical trials treating chemoresistant breast cancer. We aim to provide insights into better understanding the mutational landscape of BCSCs and exploring potential molecular signaling pathways targeting BCSCs to overcome chemoresistance and prevent metastasis in TNBC, ultimately to improve the overall survival of patients with this devastating disease.

Movement of Mitochondria with Mutant DNA through Extracellular Vesicles Helps Cancer Cells Acquire Chemoresistance.

Triple negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer with the worst prognosis after chemo- or radiation therapy. This is mainly due to the development of cancer chemoresistance accompanied by tumor recurrence. In this work, we investigated a new mechanism of acquired chemoresistance of TNBC cells. We showed that extracellular vehicles (EVs) of chemoresistant TNBC cells can transfer mitochondria to sensitive cancer cells, thus increasing their chemoresistance. Such transfer, but with less efficiency, can be carried out over short distances using tunneling nanotubes. In addition, we showed that exosome fractions carrying mitochondria from resistant TNBC cells contribute to acquired chemoresistance by increasing mtDNA levels with mutations in the mtND4 gene responsible for tumorigenesis. Blocking mitochondrial transport by exosome inhibitors, including GW4869, reduced acquired TNBC chemoresistance. These results could lead to the identification of new molecular targets necessary for more effective treatment of this type of cancer.

Noncoding RNAs in triple negative breast cancer: Mechanisms for chemoresistance.

Triple-negative breast cancer (TNBC) is the most aggressive subtype among breast cancers with high recurrence and this condition is partly due to chemoresistance. Therefore, fully understanding the mechanism of TNBC-resistance is the key to overcoming chemoresistance, which will be an effective strategy for TNBC therapy. Various potential mechanisms involved in the chemoresistance of TNBC have been investigated and indicated that noncoding RNAs (ncRNAs) especially microRNAs (miRNAs), long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) take part in most TNBC resistance. The ncRNA-induced chemoresistance process is involved in the alteration of many activities. here, we mainly summarize the mechanisms of ncRNAs in the chemoresistance of TNBC and discuss the potential clinical application of ncRNAs in the treatment of TNBC, indicating that targeting ncRNAs might be a promising strategy for resensitization to chemotherapies.