Drug Resistance In Triple-negative Breast Cancer Research Articles

YTHDF1 enhances stemness and chemoresistance in triple-negative breast cancer cells by upregulating SIAH2.

Triple-negative breast cancer (TNBC) is the most lethal and aggressive subtype of breast cancer, and chemoresistance is the major determinant of TNBC treatment failure. This study explores the molecular mechanism of TNBC chemoresistance.The Cancer Genome Atlas, breast cancer integrative platform, and GEPIA databases were used to analyze the expression and correlation of YTHDF1 and seven in absentia homology 2 (SIAH2) in breast cancer. Knockdown of YTHDF1 and SIAH2, or overexpression of SIAH2 in vitro and in vivo, was conducted to evaluate the impact of changes in YTHDF1 and SIAH2 expression on TNBC cell proliferation, apoptosis, stemness, drug resistance, and Hippo pathway gene expression.YTHDF1 and SIAH2 were highly expressed in breast cancer patients and TNBC cells. Knockdown of YTHDF1 and SIAH2 significantly inhibited proliferation and stemness and promoted apoptosis and chemosensitivity of TNBC cells. Mechanistically, the knockdown of YTHDF1 inhibited the expression of SIAH2, thereby downregulating the Hippo pathway, which inhibited proliferation and stemness and promoted apoptosis and chemosensitivity of TNBC cells.The current findings revealed the regulatory mechanism of YTHDF1 in TNBC and clarified the role of the YTHDF1/SIAH2 axis in TNBC drug resistance and stemness. This could provide new insights into the vital role of targeting YTHDF1/SIAH2 to suppress drug resistance and stemness in TNBC cells.

Integrin β4 promotes DNA damage-related drug resistance in triple-negative breast cancer via TNFAIP2/IQGAP1/RAC1

Anti-tumor drug resistance is a challenge for human triple-negative breast cancer (TNBC) treatment. Our previous work demonstrated that TNFAIP2 activates RAC1 to promote TNBC cell proliferation and migration. However, the mechanism by which TNFAIP2 activates RAC1 is unknown. In this study, we found that TNFAIP2 interacts with IQGAP1 and Integrin β4. Integrin β4 activates RAC1 through TNFAIP2 and IQGAP1 and confers DNA damage-related drug resistance in TNBC. These results indicate that the Integrin β4/TNFAIP2/IQGAP1/RAC1 axis provides potential therapeutic targets to overcome DNA damage-related drug resistance in TNBC.

Integrin β4 promotes DNA damage-related drug resistance in triple-negative breast cancer via TNFAIP2/IQGAP1/RAC1.

Anti-tumor drug resistance is a challenge for human triple-negative breast cancer (TNBC) treatment. Our previous work demonstrated that TNFAIP2 activates RAC1 to promote TNBC cell proliferation and migration. However, the mechanism by which TNFAIP2 activates RAC1 is unknown. In this study, we found that TNFAIP2 interacts with IQGAP1 and Integrin β4. Integrin β4 activates RAC1 through TNFAIP2 and IQGAP1 and confers DNA damage-related drug resistance in TNBC. These results indicate that the Integrin β4/TNFAIP2/IQGAP1/RAC1 axis provides potential therapeutic targets to overcome DNA damage-related drug resistance in TNBC.

TRAF6 promotes chemoresistance to paclitaxel of triple negative breast cancer via regulating PKM2-mediated glycolysis.

Ample evidence reveals that glycolysis is crucial to tumor progression; however, the underlying mechanism of its drug resistance is still worth being further explored. TRAF6, an E3 ubiquitin ligase, is well recognized to overexpress in various types of cancer, which predicts a poor prognosis. In our study, we discovered that TRAF6 was expressed more significantly in the case of triple-negative breast cancer (TNBC) than in other of breast cancers, promoting chemoresistance to paclitaxel; that inhibited TRAF6 expression in the chemoresistant TNBC (TNBC-CR) cells enhanced the sensitivity by decreasing glucose uptake and lactate production; that TRAF6 regulated glycolysis and facilitated chemoresistance via binding directly to PKM2; and that overexpressing PKM2 in the TNBC-CR cells with TRAF6 knocked down regained significantly TRAF6-dependent drug resistance and glycolysis. Additionally, we verified that TRAF6 could facilitate PKM2-mediated glycolysis and chemoresistance in animal models and clinical tumor tissues. Thus, we identified the novel function of TRAF6 to promote glycolysis and drug resistance in TNBC with the regulation of PKM2, which could provide a potential molecular target for TNBC treatment.

Molecular mechanisms of immunotherapy resistance in triple-negative breast cancer.

The emergence of immunotherapy has profoundly changed the treatment model for triple-negative breast cancer (TNBC). But the heterogeneity of this disease resulted in significant differences in immunotherapy efficacy, and only some patients are able to benefit from this therapeutic modality. With the recent explosion in studies on the mechanism of cancer immunotherapy drug resistance, this article will focus on the processes of the immune response; summarize the immune evasion mechanisms in TNBC into three categories: loss of tumor-specific antigen, antigen presentation deficiency, and failure to initiate an immune response; together with the aberrant activation of a series of immune-critical signaling pathways, we will discuss how these activities jointly shape the immunosuppressive landscape within the tumor microenvironment. This review will attempt to elucidate the molecular mechanism of drug resistance in TNBC, identify potential targets that may assist in reversing drug resistance, and lay a foundation for research on identifying biomarkers for predicting immune efficacy and selection of breast cancer populations that may benefit from immunotherapy.

Monoacylglycerol Lipase Inhibitor JJKK048 Ameliorates ABCG2 Transporter-Mediated Regorafenib Resistance Induced by Hypoxia in Triple Negative Breast Cancer Cells

Triple negative breast cancer (TNBC) is among the most aggressive and deadly cancer subtypes. Intra-tumoral hypoxia is associated with aggressiveness and drug resistance in TNBC. One of the underlying mechanisms of hypoxia-induced drug resistance is the elevated expression of efflux transporters such as breast cancer resistant protein (ABCG2). In the present study, we investigated the possibility of ameliorating ABCG2-mediated drug resistance in hypoxic TNBC cells by monoacylglycerol lipase (MAGL) inhibition and the consequent downregulation of ABCG2 expression.The effect of MAGL inhibition on ABCG2 expression, function, and efficacy of regorafenib, an ABCG2 substrate was investigated in cobalt dichloride (CoCl2) induced pseudohypoxic TNBC (MDA-MB-231) cells, using quantitative targeted absolute proteomics, qRT-PCR, anti-cancer drug accumulation in the cells, cell invasiveness and resazurin-based cell viability assays.Our results showed that hypoxia-induced ABCG2 expression led to low regorafenib intracellular concentrations, reduced the anti-invasiveness efficacy, and elevated half maximal inhibitory concentration (IC50) of regorafenib in vitro MDA-MB-231 cells. MAGL inhibitor, JJKK048, reduced ABCG2 expression, increased regorafenib cell accumulation, which led to higher regorafenib efficacy.In conclusion, hypoxia-induced regorafenib resistance due to ABCG2 over-expression in TNBC cells can be ameliorated by MAGL inhibition.

Identification of INFG/STAT1/NOTCH3 as γ-Mangostin's potential targets for overcoming doxorubicin resistance and reducing cancer-associated fibroblasts in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a very aggressive subtype of breast cancer characterized by drug resistance and distant metastasis. Cancer stem cells (CSCs) are considered a major contributor to TNBC's drug resistance. Thus targeting and eliminating CSCs have been vigorously researched. However, the precise targetable molecular networks responsible for CSC genesis remain unclear; this conundrum is mainly due to the high heterogeneity of the TNBC tumor microenvironment (TME). The cancer-associated fibroblasts (CAFs) are one of the most abundant cellular components of the TME. Emerging studies indicate that CAFs facilitate TNBC's progression by establishing a pro-tumor TME. Hence, identifying the molecular networks involved in CAF transformation and CAF-associated oncogenesis are essential areas to be explored. Through a bioinformatics approach, we identified INFG/STAT1/NOTCH3 as a molecular link between CSCs and CAF. DOX-resistant TNBC cell lines showed increased expression of INFG/STAT1/NOTCH3 and CD44 and were associated with increased self-renewal ability and CAF-transformative ability. Downregulation of STAT1 significantly reduced the tumorigenic properties of MDA-MB-231 and −468 cells and their CAF-transforming potential. Our molecular docking analysis suggested that gamma mangostin (gMG), a xanthone, formed complexes with INFG/STAT1/NOTCH3 better than celecoxib. We then demonstrated that gMG treatment reduced the tumorigenic properties similarly observed in STAT1-knocked down conditions. Finally, we utilized a DOX-resistant TNBC tumoroid-bearing mouse model to demonstrate that gMG treatment significantly delayed tumor growth, reduced CAF generation, and improved DOX sensitivity. Further investigations are warranted for clinical translation.

Abstract PD4-09: PD4-09 Single cell RNA-sequencing identifies intra-tumoral cellular heterogeneity and drug-induced subpopulation shifts in Triple Negative Breast Cancer mouse models

Abstract Introduction: Triple Negative Breast Cancer (TNBC) is an aggressive disease with a poor prognosis that accounts for 10-20% of breast cancer cases worldwide. Intra-tumoral heterogeneity and tumor cell plasticity are thought to contribute to drug resistance in TNBCs. Our work aims to: 1) precisely identify the intra-tumoral heterogeneity in cellular states present within TNBC, and 2) test whether drugs can block or initiate plasticity between subpopulations to improve drug sensitivity. We hypothesize that treatment(s) induce cellular plasticity, thus causing cells to shift into resistant states that persist until treatment is removed. We further hypothesize that these resistant subpopulations give rise to new tumor outgrowths once the treatment stops. Methods: To test this, we are treating TP53-/- Genetically Engineered Mouse Model (GEMM) syngeneic transplant tumors of the basal-like TNBC phenotype with the chemotherapeutic doublet of carboplatin/paclitaxel, and targeted agents implicated in plasticity including the MEK inhibitor trametinib, a chromatin remodeling inhibitor I-BET151, and the dihydroorotate dehydrogenase inhibitor brequinar. To identify cellular subpopulations and examine their response to treatment, we performed both in vivo and in vitro drug sensitivity testing, as well as gene expression profiling using single cell RNA-sequencing (scRNAseq). Results: We have identified clear intra-tumoral heterogeneity with at least 6 distinct cell states present, including basal, mesenchymal/claudin-low, and proliferative subpopulations in vivo in most TNBC GEMM models. We performed 18 individual scRNAseq experiments on the TP53-/- 2225L GEMM transplant line with the aforementioned treatments and untreated controls in triplicate and compared subpopulation frequencies in treated versus untreated tumors. Notably, treatment with trametinib and brequinar caused the rise of two rare subpopulations (i.e. 1% to 3-4% of total tumor cells) that express genes consistent with previously described drug-tolerant persisters (DTP), which we have called “Epithelial-DTP” (Tacstd2, Krt6a, and Cryab enriched) and “Mesenchymal-DTP” (Snai2 and Sca-1 enriched). A gene signature generated from the Epithelial-DTP subpopulation predicted poor patient outcomes in neoadjuvant chemotherapy treated TNBC patients. Further, in TNBC patient-derived xenografts (PDX), these two DTP subpopulations are also present and induced by treatment to an even greater frequency. Ongoing experiments include the use of fluorescence-activated cell sorting to isolate and functionally test the tumor-initiating capabilities of these two rare cell subpopulations. In addition, many experiments are underway to identify means to therapeutically target these DTP cells, with these results to be presented. Ultimately, identifying these rare drug resistant subpopulations, and identifying means to eradicate them, could vastly improve therapeutic regimens and outcomes for patients with TNBCs. Citation Format: Cherise R. Glodowski, Kevin R. Mott, Denis Okumu, Michael P. East, Timothy C. Elston, Gary L. Johnson, Charles M. Perou. PD4-09 Single cell RNA-sequencing identifies intra-tumoral cellular heterogeneity and drug-induced subpopulation shifts in Triple Negative Breast Cancer mouse models [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD4-09.

Abstract PD2-08: Targeting of eIF4A1 along with anti-PD-L1 therapy limits lung metastases efficaciously in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype of metastatic breast cancer with poor clinical prognosis. Currently used neoadjuvant chemotherapy for TNBC, such as taxanes, and/or anthracyclines demonstrate an initial treatment response but is often followed by drug resistance, tumor relapse, and high proclivity to develop metastases. There is an unmet need to develop effective combination therapies against metastatic TNBC (mTNBC). eIF4A1 is an mRNA helicase that help translate many oncogenic mRNAs including PD-L1 (through STAT1 translational regulation) that are involved in tumor growth, chemoresistance and metastases. Our prior studies pointed out that eIF4A1 is intricately linked to cancer stemness and drug resistance in TNBC. Emerging evidence has revealed that the tumor immune microenvironment (TIME) of TNBC do consist of mononuclear cells and lymphocytes. Combining chemotherapy with immune checkpoint inhibition had demonstrated a significant benefit for high-risk TNBC patients. In this study, we examine the efficacy of targeting eIF4A1 along with anti-PD-L1 therapy as an effective therapeutic strategy to limit cancer stemness, primary tumor progression and metastasis in an immunocompetent murine model of TNBC. Approach: Initially, we examined the human TNBC biospecimens and found that eIF4A1 protein level is upregulated in drug-resistant cases. STAT1 is an upstream regulator of PD-L1 and a downstream effector of eIF4A1. The protein level of STAT1 was significantly elevated as assessed by immunoblotting of human tumor lysates. In human TNBC cells, when eIF4A1 is pharmacologically or genetically targeted, PD-L1 was downregulated at the protein level. Based on this premise, we targeted eIF4A1 with the small molecule inhibitor, Rocaglamide A (RocA), along with anti-PD-L1 immunotherapy in orthotopically implanted 4T1 mouse TNBC tumors in BALB/c mice. The treatment arms were 1) Control (vehicle for RocA + control IgG) 2) RocA 3) RocA + anti-PD-L1 antibodies 4) Control IgG only 5) Anti-PD-L1 antibodies only with 10 mice in each group. Results: The combination treatment comprising of RocA and anti-PD-L1 antibodies worked out efficaciously as the primary tumor volume, wet tumor weight, bioluminescent tumor imaging and quantitation were significantly lower than the combination vehicle control. Furthermore, metastasis to the lungs were significantly impaired (1 out of 10 mice only had metastasis and that too it was small). On the contrary, ‘RocA single arm’ group had 6 out of 10 mice with lung metastasis. The lung metastases were observed in 4 out of 10 mice in the PD-L1 single arm and 6 out of 10 mice in control IgG single arm group. The RocA was tolerated well at 0.6 mg/kg with no significant change in the body weight and the liver and kidney function test values were within the normal range. Importantly, the combination treatment with RocA and PD-L1 was also tolerated well. Conclusions: The combination therapy involving RocA and PD-L1 had the significant impact in reducing lung metastases and may pave the way for phase I/II clinical trials. Citation Format: Dharmindra Dulal, Andrew Boring, Dayanidhi Raman. Targeting of eIF4A1 along with anti-PD-L1 therapy limits lung metastases efficaciously in triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD2-08.

Nucleolin aptamer conjugated MSNPs-PLR-PEG multifunctional nanoconstructs for targeted co-delivery of anticancer drug and siRNA to counter drug resistance in TNBC

The emergence of drug resistance in cancer cells is among the major challenges for treating cancer. In the last few years, the co-delivery of drug and siRNA has shown promising results against drug-resistant cancers. In the present study, we developed mesoporous silica-based multifunctional nanocarrier for co-delivery against drug-resistant triple-negative breast cancer (TNBC) cells. We synthesized the nanocarrier by modifying mesoporous silica nanoparticles with poly-L-arginine, polyethylene glycol and AS1411 aptamer to impart siRNA binding ability, biocompatibility, and cancer cell specificity, respectively. We optimized the loading of doxorubicin (DOX) within the developed nanocarrier to avoid interference with siRNA binding. We ascertained the target specificity by performing a receptor blockade assay during cellular uptake studies. The cytotoxic efficacy of DOX and siRNA co-delivered using the developed nanocarrier was assessed using DOX-resistant MDA-MB-231 TNBC cells. The nanocarrier exhibited >10-fold and 40-fold reduction in the IC50 values of DOX due to co-delivery with BCl-xL and BCL-2 siRNA, respectively. The results were further validated using a 3-D in vitro cell culture system. This study demonstrates that the targeted co-delivery of drug and siRNA has a strong potential to overcome drug resistance in TNBC cells.

FBXL2 promotes E47 protein instability to inhibit breast cancer stemness and paclitaxel resistance.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with a high risk of metastasis and recurrence. Although chemotherapy has greatly improved the clinical outcome of TNBC patients, acquired drug resistance remains a huge challenge for TNBC treatment. Breast cancer stem cells (BCSCs) play a critical role in breast cancer development, metastasis, recurrence, and chemotherapy resistance. Thus, it is of great importance to decipher the underlying molecular mechanism of BCSCs regulation for TNBC drug resistance. In this study, we demonstrate that the F-box protein FBXL2 is a critical negative regulator of BCSCs stemness and that downregulation of FBXL2 plays a causal role in TNBC drug resistance. We show that expression levels of FBXL2 significantly influence CD44high/CD24low subpopulation and the mammosphere formation ability of TNBC cells. Ectopic expression of FBXL2 inhibits initiation of TNBC and overcomes paclitaxel resistance in vivo. In addition, activation of FBXL2 by nebivolol, a clinically used small-molecule inhibitor of the beta-1 receptor, markedly overcomes BCSCs-induced paclitaxel resistance. Mechanistically, we show that FBXL2 targets transcriptional factor E47 for polyubiquitin- and proteasome-mediated degradation, resulting in inhibition of BCSC stemness. Clinical analyses indicate that low expression of FBXL2 correlates with high expression of E47 as well as with high stemness features, and is associated with poor clinical outcomes of breast cancer patients. Taken together, these results highlight that the FBXL2-E47 axis plays a critical role in the regulation of BCSC stemness and paclitaxel resistance. Thus, targeting FBXL2 might be a potential therapeutic strategy for drug-resistant TNBC.

Rutin induces endoplasmic reticulum stress-associated apoptosis in human triple-negative breast carcinoma MDA-MB-231 cells – In vitro and in silico docking studies

Rutin is a bioactive compound that possesses anti-tumor activities through triggering apoptosis. Triple-negative breast cancer (TNBC) is insensitive to targeted anti-tumoral drugs, and drug resistance in TNBC poses a challenge for a successful cure. The accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER) results in cellular stress that initiates a specialized response designated as the unfolded protein response. This study aimed to find potential ER stress targets in triple-negative breast cancer. The viability of cells was evaluated using an MTT assay. Cell migration and proliferation were done by wound scratch and colony formation assay. Cell cycle detection, measurement of ER stress, mitochondrial membrane potential disruption, and cell death identification was performed using flow cytometry. The interaction of rutin with ER stress proteins is predicted using in silico docking. The pattern of gene expression was determined by qRT-PCR. The elevated rate of cell viability, cell cycle arrest, ER stress, MMP, and apoptotic induction was observed in combination treatment. Rutin exhibited the highest glide score with ASK1 and JNK. The results of qRT-PCR showed that rutin induced apoptosis through upregulation of ASK1 and JNK. The present study provides strong evidence supporting an important role of the ER stress response in mediating rutin-induced apoptosis in triple-negative breast cancer.

Hypoxia induces docetaxel resistance in triple-negative breast cancer via the HIF-1α/miR-494/Survivin signaling pathway

Cytotoxic chemotherapy is the major strategy to prevent and reduce triple-negative breast cancer (TNBC) progression and metastasis. Hypoxia increases chemoresistance and is associated with a poor prognosis for patients with cancer. Based on accumulating evidence, microRNAs (miRNAs) play an important role in acquired drug resistance. However, the role of miRNAs in hypoxia-induced TNBC drug resistance remains to be clarified. Here, we found that hypoxia induced TNBC docetaxel resistance by decreasing the miR-494 level. Modulating miR-494 expression altered the sensitivity of TNBC cells to DTX under hypoxic conditions. Furthermore, we identified Survivin as a direct miR-494 target. Hypoxia upregulated survivin expression. In a clinical study, the HIF-1α/miR-494/Survivin signaling pathway was also active in primary human TNBC, and miR-494 expression negatively correlated with HIF-1α and survivin expression. Finally, in a xenograft model, both miR-494 overexpression and the HIF-1α inhibitor PX-478 increased the sensitivity of TNBC to DTX by suppressing the HIF-1α/miR-494/Survivin signaling pathway in vivo. In conclusion, treatments targeting the HIF-1α/miR-494/Survivin signaling pathway potentially reverse hypoxia-induced drug resistance in TNBC.

Long non-coding RNA ANRIL promotes chemoresistance in triple-negative breast cancer via enhancing aerobic glycolysis

AimslncRNA ANRIL expression is dysregulated in many human cancers and is thus a useful prognostic marker for cancer patients. However, whether ANRIL is involved in drug resistance in triple-negative breast cancer (TNBC) has not yet been investigated. Main methodsA luciferase reporter assay was conducted to verify the binding between miR-125a and ANRIL. RT-PCR and western blotting were performed to detect the expression of miR-125a, ANRIL, and ENO1. Glycolysis stress was assessed using the Seahorse extracellular flux analyzer. Functional studies were performed using both in vitro and in vivo xenograft models. Key findingsANRIL was markedly upregulated in both patients with TNBC and TNBC cell lines. Knockdown of ANRIL increased the cytotoxic effect of ADR and repressed cellular glycolytic activity in TNBC cells. Mechanistic analysis showed that ANRIL may act as a competing endogenous RNA of miR-125a to relieve the repressive effect of miR-125a on its target glycolytic enzyme enolase (ENO1). Notably, 2-deoxy-glucose attenuated ANRIL-induced increase in drug resistance in TNBC cells. SignificanceThese results indicate that knockdown of ANRIL plays an active role in overcoming drug resistance in TNBC by inhibiting glycolysis through the miR-125a/ENO1 pathway, which may be useful for the development of novel therapeutic targets for treating patients with TNBC, especially those with drug resistance.

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.

Clinical Identification of Dysregulated Circulating microRNAs and Their Implication in Drug Response in Triple Negative Breast Cancer (TNBC) by Target Gene Network and Meta-Analysis.

Resistance to therapy is a persistent problem that leads to mortality in breast cancer, particularly triple-negative breast cancer (TNBC). MiRNAs have become a focus of investigation as tissue-specific regulators of gene networks related to drug resistance. Circulating miRNAs are readily accessible non-invasive potential biomarkers for TNBC diagnosis, prognosis, and drug-response. Our aim was to use systems biology, meta-analysis, and network approaches to delineate the drug resistance pathways and clinical outcomes associated with circulating miRNAs in TNBC patients. MiRNA expression analysis was used to investigate differentially regulated circulating miRNAs in TNBC patients, and integrated pathway regulation, gene ontology, and pharmacogenomic network analyses were used to identify target genes, miRNAs, and drug interaction networks. Herein, we identified significant differentially expressed circulating miRNAs in TNBC patients (miR-19a/b-3p, miR-25-3p, miR-22-3p, miR-210-3p, miR-93-5p, and miR-199a-3p) that regulate several molecular pathways (PAM (PI3K/Akt/mTOR), HIF-1, TNF, FoxO, Wnt, and JAK/STAT, PD-1/PD-L1 pathways and EGFR tyrosine kinase inhibitor resistance (TKIs)) involved in drug resistance. Through meta-analysis, we demonstrated an association of upregulated miR-93, miR-210, miR-19a, and miR-19b with poor overall survival outcomes in TNBC patients. These results identify miRNA-regulated mechanisms of drug resistance and potential targets for combination with chemotherapy to overcome drug resistance in TNBC. We demonstrate that integrated analysis of multi-dimensional data can unravel mechanisms of drug-resistance related to circulating miRNAs, particularly in TNBC. These circulating miRNAs may be useful as markers of drug response and resistance in the guidance of personalized medicine for TNBC.

DNA N6-Methyladenine (6mA) Modification Regulates Drug Resistance in Triple Negative Breast Cancer.

Triple negative breast cancer (TNBC) is a subtype of breast cancer with strong aggressiveness and poor clinical treatment effect, accounting for about 10–20% of breast cancer cases. N(6)-methyldeoxyadenosine (6mA) is the most conservative DNA modification in prokaryotes and eukaryotes. It is widely found in bacteria and has such functions as DNA mismatch repair, chromosome separation and virulence regulation. We determined that 6mA was modified in TNBC cell line MDA-MB-231 and the TNBC tissue. Meanwhile, compared with normal tissues, the expression level of 6mA and its methylase N6AMT1 was significantly decreased in TNBC tissue. MDA-MB-231cells were cultured with 8μM Olaparib for 2 months to construct drug-resistant cell line 231-RO. It was found that the level of 6mA also increased significantly, and the expression of N6AMT1 or ALKBH1 could effectively influence the drug resistance. Subsequently, we found that LINP1 was highly expressed in 231-RO, which was involved in DNA repair, and the expression of LINP1 could be positively regulated by 6mA modification. LINP1 expression level is directly related to TNBC drug resistance. The above results indicate that 6mA may be a new biological marker of TNBC. Meanwhile, 6mA modification may be involved in the regulation of Olaparib resistance.

Mitochondria-targeted magnetic gold nanoheterostructure for multi-modal imaging guided photothermal and photodynamic therapy of triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a type of highly aggressive cancer that is hard to be cured by the commonly used chemotherapy, mainly due to the lack of effective tumor targeting ability of the current drugs and TNBC drug resistance. Therefore, development of novel strategies for precise and high-efficient therapy of TNBCs is urgently needed. In the present work, a multifunctional magnetic gold nanoheterostructure with photosensitizer Ce6 loading (MGN@Ce6) was designed and synthesized for synergistic photothermal and photodynamic therapy (PTT/PDT) of TNBC. To improve the tumor targeting ability, cRGD and TPP cationic molecule, which can specifically target αvβ3 integrin of cancer cell membrane and mitochondria, respectively, were functionalized on the nanosystem obtaining MGN@Ce6@RT. In vitro and in vivo fluorescent imaging demonstrated that cRGD and TPP functionalization largely enhanced the delivery-efficiency of MGN@Ce6@RT into TNBC cells and tumors. Under 880 nm and 660 nm laser irradiations, MGN@Ce6@RT exhibited strong hyperthermia effect and ROS generating ability, which exerted a synergistic anti-TNBC effect by completely suppressed the tumor growth of the nude mice model. Moreover, by integrating superparamagnetic and near infrared light absorption properties into a single nanocomposite, MGN@Ce6@RT was proved to be able to realize magnetic resonance, X-ray computed tomography and photoacoustic tri-modal imaging of in vivo tumors. In all, we provide a novel strategy for precise delivery of functional nanosystems to TNBC tumors to achieve more satisfying treating outcome.

Structure of LINC00511-siRNA-conjugated nanobubbles and improvement of cisplatin sensitivity on triple negative breast cancer.

The drug resistance of triple negative breast cancer (TNBC) is considered as a major obstacle for the curative effect of chemotherapy. Long intergenic noncoding RNA 00511 (LINC00511) has been considered as a target gene of drug resistance. A novel theranostic agent loaded with LINC00511-siRNA to deliver siRNA was structured, and the responses of drug sensitivity in TNBC were detected. Next-generation high-throughput RNA sequencing (RNA-Seq) was performed to accurately analyze the differential expression of mRNAs and lncRNA targets after LINC00511-siRNA transfection with low-frequency ultrasound (LFUS). The LINC00511-siRNA conjugated nanobubble complexes showed appropriate characterization, with a mean diameter of 516.1±24.7nm and a zeta potential of -38.05±0.24mV. The transfection efficiency of nanobubble complexes was approximately 50% with LFUS. By RNA-Seq, the differential expressions of lncRNA transcripts and mRNA transcripts were identified, and then analyzed. The GO and KEGG enrichment analyses revealed the TNBC drug resistance related target genes and pathways. The combination of LFUS irradiation and nanobubble complexes is regarded as an efficient and safe method for siRNA transfection. The TNBC drug resistance occurs as a result of synergistic reactions between a variety of genes and a variety of pathways.

Aspirin attenuates YAP and β-catenin expression by promoting β-TrCP to overcome docetaxel and vinorelbine resistance in triple-negative breast cancer.

e13099 Background: YAP and β-catenin play critical roles in the carcinogenesis of several cancers, but their functions in chemoresistance remain unclear in triple-negative breast cancer (TNBC). Herein, we examined the expression and function of the two proteins in the chemoresistance of TNBC. Methods: We examined the expression levels of YAP and β-catenin in TNBC samples by immunohistochemistry (IHC) and their roles in prognosis. Dynamic changes in the two proteins in TNBC tissues were determined by RT-PCR. CCK-8 assays, colony formation assays, flow cytometry, migration assays and nude mouse models were used to investigate the functions of YAP and β-catenin in drug-resistant TNBC cells. The involved signalling pathways were detected by western blot assays. Results: TNBC patients with high YAP and/or β-catenin expression had a higher risk of relapse or mortality than patients with low YAP and/or β-catenin expression, and changes in the expression of the two proteins might be a promising predictive biomarker for neoadjuvant chemotherapy sensitivity. Inhibition of YAP or β-catenin enhanced the cytotoxicity of the anti-microtubule agents docetaxel and vinorelbine against TNBC cells in vitro and in vivo. Moreover, YAP and β-catenin were upregulated in docetaxel- or vinorelbine-resistant TNBC cells, while inhibition of YAP or β-catenin enhanced the sensitivity of drug-resistant cells. Interestingly, aspirin, a kind of acetylsalicylic acid, reversed the drug resistance of cells and in combination with aspirin and docetaxel or vinorelbine significantly inhibited the growth of drug-resistant TNBC cells. The involved mechanism is that aspirin impaired YAP or β-catenin expression by upregulating the E3 ubiquitin ligase β-TrCP. Conclusions: Both YAP and β-catenin act as key biomarkers of prognosis and anti-microtubule drug resistance in TNBC, and the combined use of aspirin and anti-microtubule drugs inhibits YAP and β-catenin, presenting several promising therapeutic approaches for TNBC treatment.