Paclitaxel-resistant Breast Cancer Research Articles

MAP7 promotes proliferation and migration of breast cancer cells and reduces the sensitivity of breast cancer cells to paclitaxel

Breast cancer is a common malignancy that severely threatens women's mental health and lives. The paclitaxel-resistant breast cancer cells were established through a continuous stimulation with paclitaxel in a stepwise escalating concentration manner. The expression of MAP7 was detected by RT-P CR and western blot. The annexin V staining assay was used to measure the cell apoptosis ratio. The expression of cell invasive ability and apoptosis-related proteins was detected by western blot assay. The cellular motility was tested via transwell and wound healing assays. This study indicated that the MAP7 expression was upregulated in breast cancer cells and paclitaxel-resistant breast cancer cells. Moreover, downregulating MAP7 not only suppressed cell viability, motility and invasion, but also enhanced cellular apoptosis in paclitaxel-resistant breast cancer cells. In summary, this study investigated the effect of MAP7 protein on cell critical physiological function, which provided a novel potential target for treating paclitaxel-resistant breast cancer.

Knockdown of nuclear receptor binding SET domain-containing protein 1 (NSD1) inhibits proliferation and facilitates apoptosis in paclitaxel-resistant breast cancer cells via inactivating the Wnt/β-catenin signaling pathway

ABSTRACT The burden of breast cancer (BC) has exacerbated over decades. Paclitaxel resistance is responsible for increasing BC treatment burden. Nuclear receptor binding SET domain-containing protein 1 (NSD1) is positively correlated with a poor prognosis in patients with BC. This study investigates the function of NSD1 in paclitaxel-resistant (PR) BC cells. The high levels of NSD1 and Wnt10b in PR BC cell lines (MCF-7/PR) or MCF-7 parental cells were determined by RT-qPCR. Western blotting was conducted to measure the levels of NSD1 protein, apoptosis-associated proteins, Wnt10b protein, H3K36me2 protein, H3K27me3 protein, and signal pathway-associated proteins in MCF-7/PR cells or MCF-7 cells or in vivo subcutaneous xenografted tumor model, and the results demonstrated that NSD1 inhibited cell apoptosis and promoted cell proliferation and tumor growth via activating Wnt/β-catenin pathway. Cell apoptosis and viability were estimated using cell counting kit-8 assays and flow cytometry. Positive correlation between NSD1 and Wnt10b was identified by chromatin immunoprecipitation assay. The distribution of β-catenin was determined by immunofluorescence assays. We conclude that NSD1 knockdown inhibits the viability and promotes the apoptosis of paclitaxel-resistant BC cells by inactivating the NSD1/H3K27me3/Wnt10b/β-catenin signaling pathway.

Antiviral Drug 2-thio-6-azauridine Sensitizes Paclitaxel-Resistant Triple Negative Breast Cancer Cells by Targeting Mammosphere Formation and ABC Transporters

Background: 2-thio-6-azauridine (TAU) is a nucleoside analog and potential antiviral drug. The antiproliferative activity of TAU has been evaluated in limited cancer cell lines. The present study is aimed to evaluate the effect of TAU on drug sensitization mechanism in paclitaxel (PTX) resistant triple-negative breast cancer (TNBC) cells.Methods: The cell death mechanism was determined using MTT, BrdU incorporation, apoptosis, and DNA damage Western blot and RT-PCR assays. A specific ELISA method was used to determine the caspase-3 activity and expression levels of MRP1, MDR1, BCRP, and MRP8. Western blot analysis was used to assess the expression of CD151, MRP1, MDR1, and BCRP in CD151 overexpressing PTX-resistant TNBC cells.Results: The combination of TAU and PTX (10:20nM) synergistically inhibited the 50% viability of 12-fold PTX-resistant TNBC cells. Mechanistically, the combination inhibited the proliferation by arresting the cell cycle at the G2M phase and induced apoptosis by altering cell integrity and nuclear morphology as well as damaging DNA. The combination sensitized the PTX-resistant TNBC cells by increasing BAX and decreasing Bcl-2 expression, activating caspase-3, and reducing the expression of ABC transporters MRP1 and MDR1. The combination reduced the expression of MRP1 and MDR1 in CD151 overexpressing PTX-resistant TNBC cells, indicating the role of CD151in TAU mediated sensitization of PTX-resistant TNBC cells. The combination also reduced the mammosphere formation efficiency of PTX-resistant TNBC cells. Conclusion: Overall, the present study illustrated the promising ability of TAU in sensitizing drug-resistant TNBC cells to PTX.

Hypoxia-Induced Intracellular and Extracellular Heat Shock Protein gp96 Increases Paclitaxel-Resistance and Facilitates Immune Evasion in Breast Cancer.

BackgroundsHypoxia contributes to cancer progression, drug resistance and immune evasion in various cancers, including breast cancer (BC), but the molecular mechanisms have not been fully studied. Thus, the present study aimed to investigate this issue.MethodsThe paclitaxel-sensitive BC (PS-BC) cells were administered with continuous low-dose paclitaxel treatment to establish paclitaxel-resistant BC (PR-BC) cells. Exosomes were isolated/purified by using the commercial kit, which were observed by Transmission electron microscopy (TEM). Cell viability was measured by MTT assay, cell apoptosis was determined by flow cytometer (FCM). Gene expressions were respectively measured by Real-Time qPCR, Western Blot and immunofluorescence staining assay. The peripheral mononuclear cells (PBMCs) derived CD8+ T cells were obtained and co-cultured with gp96-containing exosomes, and cell proliferation was evaluated by EdU assay. ELISA was employed to measure cytokine secretion in CD8+ T cells’ supernatants.ResultsHSP gp96 was significantly upregulated in the cancer tissues and plasma exosomes collected from BC patients with paclitaxel-resistant properties. Also, continuous low-dose paclitaxel treatment increased gp96 levels in the descendent PR-BC cells and their exosomes, in contrast with the parental PS-BC cells. Upregulation of gp96 increased paclitaxel-resistance in PS-BC cells via degrading p53, while gp96 silence sensitized PR-BC cells to paclitaxel treatments. Moreover, PR-BC derived gp96 exosomes promoted paclitaxel-resistance in PS-BC cells and induced pyroptotic cell death in the CD8+ T cells isolated from human peripheral blood mononuclear cells (pPBMCs). Furthermore, we noticed that hypoxia promoted gp96 generation and secretion through upregulating hypoxia-inducible factor 1 (HIF-1), and hypoxia increased paclitaxel-resistance and accelerated epithelial-mesenchymal transition (EMT) in PS-BC cells.ConclusionsHypoxia induced upregulation of intracellular and extracellular gp96, which further degraded p53 to increase paclitaxel-sensitivity in BC cells and activated cell pyroptosis in CD8+ T cells to impair immune surveillance.

Nuclear receptor binding SET domain protein 1 promotes epithelial-mesenchymal transition in paclitaxel-resistant breast cancer cells via regulating nuclear factor kappa B and F-box and leucine-rich repeat protein 11

ABSTRACT Breast cancer (BC) is regarded as the major cause of cancer-associated deaths in women. Paclitaxel exerts a critical impact on the chemotherapy of BC, but the resistance to paclitaxel becomes a great obstacle in treating the disease. It is reported that noncoding RNA nuclear receptor binding SET domain protein 1 (NSD1) plays a significant role in drug resistance; however, the special role of NSD1 in paclitaxel-resistant BC is unclear. Human BC cell line MCF-7 was used to establish paclitaxel-resistant BC cells (MCF-7/PR). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) displayed that NSD1 and F-box and leucine-rich repeat protein 11 (FBXL11) were highly expressed in BC tissues. Western blotting was utilized for protein level assessment. Cell counting kit-8 (CCK-8), Transwell, wound healing assays, and animal experiments were conducted to examine the influence of NSD1 or FBXL11 on the malignant behaviors of BC in vitro and in vivo, respectively. Transfected MCF-7/PR cells were injected subcutaneously into BALB/c nude mice with or without treatment of paclitaxel. The nuclear factor kappa B (NF-kB) activity was evaluated by the luciferase reporter assay. Results showed that NSD1 knockdown inhibited the epithelial-mesenchymal transition (EMT), migration and invasiveness of BC in vitro, which was rescued by FBXL11 overexpression. Furthermore, NSD1 silencing promoted paclitaxel sensitivity of paclitaxel-resistant BC cells and suppressed tumor growth and paclitaxel resistance in vivo. NSD1 knockdown reduced NF-kB activity, while FBXL11 inhibition markedly increased NF-kB activity. Collectively, NSD1 facilitates the EMT, migration and invasion in paclitaxel-resistant BC cells via regulating NF-kB and FBXL11.

Carbonic anhydrase 12 gene silencing reverses the sensitivity of paclitaxel in drug-resistant breast cancer cells

ABSTRACT This study aimed to investigate the effects of carbonic anhydrase 12 (CA12)-siRNA on the paclitaxel sensitivity of breast cancer cells. Normal mammary glandular cell (MCF-10), breast cancer cell (MCF-7), and paclitaxel-resistant breast cancer cells (MCF-7 TaxR) were cultured in experimental control group. Western blot was adopted to detect the expressions of CA12 protein and apoptosis-related proteins in mitochondrial pathway of MCF-10, MCF-7, and MCF-7 TaxR cells. The methylthialazole tetrazolium (MTT) method was used to measure cell proliferation. The apoptosis of MCF-7 and MCF-7 TaxR cells was observed in phase contrast microscope, fluorescence inverted phase contrast microscope, and flow cytometry (FACS). The results showed that CA12 protein expression in MCF-7 and MCF-7 TaxR cells was significantly higher than that in MCF-10 cell. The growth rate of CA12-siRNA treated MCF-7 TaxR cells with paclitaxel (PTX) co-culture was markedly declined at 48 hours. Phase contrast microscope, fluorescence inverted phase contrast microscope, and FACS showed that apoptotic cells in the CA12-siRNA treated MCF-7 TaxR groups were significantly higher than that in CA12-siRNA treated MCF-7 cells. The expressions of pro-apoptotic proteins, Bax and Bid, were dramatically increased in CA12 siRNA treated MCF-7 TaxR cells. The expression quantity of the downstream effective molecules caspase-9, caspase-7, and the activated proteins of poly (ADP-ribose) polymerase (PARP), also were significantly increased. Our results indicated that the application of PTX combined silencing CA12 was able to activate the mitochondrial apoptosis pathway and promote MCF-7 TaxR apoptosis. CA12 silencing in the PTX-resistant breast cancer cell can reverse the sensitivity of PTX.

The Herbal Formula JI017 Induces ER Stress via Nox4 in Breast Cancer Cells.

Chemotherapy is a powerful anti-tumor therapeutic strategy; however, resistance to treatment remains a serious concern. To overcome chemoresistance, combination therapy with anticancer drugs is a potential strategy. We developed a novel herbal extract, JI017, with lower toxicity and lesser side effects. JI017 induced programmed cell death and excessive unfolded protein response through the release of intracellular reactive oxygen species (ROS) and calcium in breast cancer cells. JI017 treatment increased the expression of endoplasmic reticulum (ER) stress markers, including p-PERK, p-eIF2α, ATF4, and CHOP, via the activation of both exosomal GRP78 and cell lysate GRP78. The ROS inhibitors diphenyleneiodonium and N-acetyl cysteine suppressed apoptosis and excessive ER stress by inhibiting Nox4 in JI017-treated breast cancer cells. Furthermore, in paclitaxel-resistant breast cancer cell lines, MCF-7R and MDA-MB-231R, a combination of JI017 and paclitaxel overcame paclitaxel resistance by blocking epithelial-mesenchymal transition (EMT) processes, such as the downregulation of E-cadherin expression and the upregulation of HIF-1α, vimentin, Snail, and Slug expression. These findings suggested that JI017 exerts a powerful anti-cancer effect in breast cancer and a combination therapy of JI017 and paclitaxel may be a potential cancer therapy for paclitaxel resistant breast cancer.

Silencing of circHIPK3 Sensitizes Paclitaxel-Resistant Breast Cancer Cells to Chemotherapy by Regulating HK2 Through Targeting miR-1286.

BackgroundResistance development to paclitaxel (PTX) has become a major obstacle in the successful treatment of breast cancer (BC). Circular RNAs (circRNAs) have been identified as essential regulators in PTX resistance of BC. Here, we explored the precise roles of circRNA homeodomain interacting protein kinase 3 (circHIPK3, circ_0000284) in PTX resistance of BC.MethodsThe expression levels of circHIPK3, microRNA (miR)-1286, and hexokinase 2 (HK2) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Ribonuclease R (RNase R) assay was used to confirm the stability of circHIPK3. Cellular localization of circHIPK3 was assessed by subcellular localization assay. The half maximal inhibitory concentration (IC50) value for PTX was measured by Cell Counting Kit-8 (CCK-8) assay. Cell colony formation, cell cycle distribution, and apoptosis were gauged by colony formation assay and flow cytometry, respectively. Animal studies were performed to evaluate the role of circHIPK3 in vivo. The direct relationship between miR-1286 and circHIPK3 or HK2 was verified by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays.ResultsOur results showed that circHIPK3 was up-regulated in PTX-resistant BC tissues and cells compared with the sensitive counterparts. The silencing of circHIPK3 promoted PTX sensitivity of PTX-resistant BC cells in vitro and in vivo. CircHIPK3 directly targeted miR-1286, and miR-1286 acted as a downstream mediator of circHIPK3 function in vitro. HK2 was a direct target of miR-1286, and circHIPK3 modulated HK2 expression through miR-1286. The increased expression of miR-1286 sensitized PTX-resistant BC cells to PTX in vitro by down-regulating HK2.ConclusionOur findings demonstrated that the silencing of circHIPK3 sensitized PTX-resistant BC cells to PTX therapy at least in part via the regulation of the miR-1286/HK2 axis.

An in vivo genome-wide shRNA screen identifies BCL6 as a targetable biomarker of paclitaxel resistance in breast cancer.

Paclitaxel is a common breast cancer drug; however, some tumors are resistant. The identification of biomarkers for paclitaxel resistance or sensitivity would enable the development of strategies to improve treatment efficacy. A genome‐wide in vivo shRNA screen was performed on paclitaxel‐treated mice with MDA‐MB‐231 tumors to identify genes associated with paclitaxel sensitivity or resistance. Gene expression of the top screen hits was associated with tumor response (resistance or sensitivity) among patients who received neoadjuvant chemotherapy containing paclitaxel. We focused our validation on screen hit B‐cell lymphoma 6 (BCL6), which is a therapeutic target in cancer but for which no effects on drug response have been reported. Knockdown of BCL6 resulted in increased tumor regression in mice treated with paclitaxel. Similarly, inhibiting BCL6 using a small molecule inhibitor enhanced paclitaxel treatment efficacy both in vitro and in vivo in breast cancer models. Mechanism studies revealed that reduced BCL6 enhances the efficacy of paclitaxel by inducing sustained G1/S arrest, concurrent with increased apoptosis and expression of target gene cyclin‐dependent kinase inhibitor 1A. In summary, the genome‐wide shRNA knockdown screen has identified BCL6 as a potential targetable resistance biomarker of paclitaxel response in breast cancer.

Bioinformatics Analysis of TIMP1, HK2 and IGFBP7 as Potential Biomarkers and Therapeutic Targets of Paclitaxel Resistance in Breast Cancer

Background: Paclitaxel is widely used as an adjuvant therapy in the treatment of breast cancer, yet its effectiveness decreases due to resistance problems. We conducted the present study to identify the potential paclitaxel resistance biomarkers and therapeutic targets in breast cancer employing bioinformatics approach. Method: The present systematic bioinformatic study included a microarray data obtained from Gene Expression Omnibus database, which are respectively cell lines and tumor data from patients. We carried out Gene ontology, Kyoto Encyclopedia Genes, and Genome pathway enrichment analysis with The Database for Annotation, Visualization and Integrated. The protein-protein interaction network was analyzed with STRING-DB and visualized with Cytoscape. We confirmed of the reliability of the hub genes in paclitaxel sensitive and resistant breast cancer cells utilizing ONCOMINE. The prognostic value of the hub genes was evaluated using Kaplan-Meier survival curves. Results: Gene ontology analysis revealed that differential expressed genes take part in cell adhesion, located in cellular component and paly a negative role in the regulation of reactive oxygen species. The protein-protein interaction network analysis, confirmed with ONCOMINE and Kaplan Meier survival, revealed three hub genes (TIMP1, HK2, and IGFBP7). Kyoto Encyclopedia Genes and Genome pathway enrichment analysis revealed the regulation of HIF-1 signaling pathway. Kaplan Meier survival plot showed that patients with high mRNA of TIMP1, HK2, and IGFBP7 had significantly worse overall survival than those in the low expression level group. Conclusion: TIMP1, HK2, and IGFBP7 are not only biomarkers, but also potential targets to circumvent paclitaxel resistance in breast cancer.

Positive regulation of PFKFB3 by PIM2 promotes glycolysis and paclitaxel resistance in breast cancer.

BackgroundBreast cancer (BC) is one of the most common female malignancies in the world. Chemotherapeutic resistance is the major cause of BC therapy failure, leading to tumor recurrence and metastasis. Studies have illustrated the close relationship between glycolysis and BC progression and drug resistance. The key glycolysis regulator, PFKFB3 makes a difference during BC progression and drug resistance. However, the mechanism remains to be unknown.MethodsMass spectrometry analyses were used to found that PIM2 was a potential new binding protein of PFKFB3. Co‐immunoprecipitated and western blot were used to verify the interaction between PIM2 and PFKFB3 in BC and the molecular mechanism by which PIM2 phosphorylates PFKFB3 in regulating the protein function. PFKFB3 mutant forms were used to demonstrate the need for PFKFB3 in BC drug resistance.ResultsWe identified that PIM2 is a new binding protein of PFKFB3. We used biochemical methods to determine that PIM2 can directly bind and change the phosphorylation of PFKFB3 at Ser478 to enhance PFKFB3 protein stability through the ubiquitin‐proteasome pathway. Importantly, phosphorylation of PFKFB3 at Ser478 promoted glycolysis, BC cell growth, and paclitaxel resistance together with PIM2 in vitro and in vivo.ConclusionOur study demonstrates that PIM2 mediates PFKFB3 phosphorylation thus regulates glycolysis and paclitaxel resistance to promote tumor progression in BC and provides preclinical evidence for targeting PFKFB3 as a new strategy in BC treatment to battle paclitaxel resistance.

MicroRNA-155-5p promotes tumor progression and contributes to paclitaxel resistance via TP53INP1 in human breast cancer

MicroRNAs (miRNAs/miRs) are small, non-coding RNAs that are reported to serve numerous important regulatory functions; however, the role of miRNAs in regulating breast cancer cell biology remains poorly understood. Accumulating evidence has demonstrated that miRNAs orchestrate multiple cellular functions and serve crucial roles in cell differentiation and cancer development, either by acting as tumor suppressors or oncogenes. In particular, miR-155-5p expression levels have been found to be upregulated and serve as a prognostic marker in numerous types of solid cancer, including human breast cancer. More than half of patients with breast cancer benefit from treatment with adjuvant paclitaxel chemotherapy following the early postoperative period. Despite the initial response to intensive combination chemotherapy, the majority of most patients will eventually acquire resistance to the drug and succumb to their disease. Therefore, further investigations into the association between miRNAs and the mechanism of paclitaxel resistance are required. The results of the present study revealed a strong positive association between miR-155-5p expression levels and the paclitaxel resistance, as the expression levels of miR-155-5p were upregulated in resistant cells. MiR‐155-5p was further validated to regulate paclitaxel resistance using gain- and loss‐of‐function experiments. TP53INP1 was identified as a direct target gene of miR-155-5p by combining the results from the prediction algorithm based on free energy minimization and reverse transcription-quantitative PCR (qRT-PCR) analysis. Also, miR-155-5p was suggested to be a key regulator of paclitaxel resistance in tumor cells, as it increased cell viability and motility, and promoted resistance to paclitaxel‐induced apoptosis. The transfection with miR-155-5p inhibitors re-sensitized the paclitaxel-resistant breast cancer cells, while the overexpression of miR-155-5p led to an increase in the resistance to paclitaxel. Furthermore, the overexpression of the target gene, TP53INP1, contributed to the re-sensitivity of drug-resistant cells to paclitaxel. The subsequent combination of the knockdown of miR-155-5p and the overexpression of TP53INP1 conferred paclitaxel sensitivity in resistant cells. These results may enhance the understanding of the molecular mechanisms underlying breast cancer progression and resistance to chemotherapy, and suggested that miR-155-5p or TP53INP1 may serve as novel therapeutic approaches to combat resistance to therapy, as well as the proliferation and evasion of apoptosis in breast cancer.

Abstract PS16-10: Tao kinase 3 augment the resistance to microtubule-disrupting agents in breast cancer cells

Abstract Breast cancer is the leading type of cancer in women worldwide. It is a heterogeneous disease that contains several subtypes with substantial differences in pathology and clinical outcome. With the advancements in imaging and targeted therapy, many patients with early stages of this disease have a great opportunity to be cured. However, recurrence or metastasis of tumor still occurs in patients even after successful primary treatment. Chemotherapy remains one of the most important treatment for advanced breast cancer. Anti-microtubule agents including taxanes, eribulin and vinca-alkaloids constitutes the major anti-breast cancer chemotherapeutic category, while chemoresistance remains a thorny issue for advanced disease. We aimed to discover novel candidate regulators for chemoresistance in breast cancer. A lentiviral-based, high-throughput shRNA platform was developed for screening the variation of global kinome to find new therapeutic targets in paclitaxel-resistant breast cancer cells. The underlying molecular mechanisms was further explored by global phosphoprotein array and expression microarray. The serine/threonine kinase, TAO Kinase 3 (TAOK3), was identified from 724 kinase genes. Knockdown of TAOK3 exhibited the most profound reduction of IC50 values in response to paclitaxel treatment in breast cancer cells. High expression of TAOK3 was related to poor prognosis in breast cancer patients after adjuvant chemotherapy. Furthermore, the expression of TAOK3 also decreased the sensitivity to other anti-microtubule drugs, including eribulin and vinorelbine. Expression microarray analysis revealed that NF-κB signaling plays the major regulation roles in TAOK3-associated resistance. Taken together, our results showed that TAOK3 increases resistance to anti-microtubule drug through upregulating the NF-κB signaling in breast cancer. Inhibitors that disrupt TAOK3 and NF-κB signaling pathway may overcome the drug resistance for patients treated with anti-microtubule agents. Citation Format: Chih-Yeu Fang, Tsung-Ching Lai, Michael Hsiao. Tao kinase 3 augment the resistance to microtubule-disrupting agents in breast cancer cells [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-10.

Nuclear Her2 contributes to paclitaxel resistance in breast cancer cells.

Translocation of full-length Her2 receptor into nucleus was reported by some studies. Here, we tested whether nuclear Her2 contributes to paclitaxel resistance in Her2-overexpressing breast cancer cells. Breast cancer cell was transfected with plasmids containing cDNA of wild-type Her2 or mutant-type Her2 lacking the nuclear localization signal (NLS) sequence which is required for Her2 nuclear transport. Cell resistance to paclitaxel was analyzed. Paclitaxel-resistant breast cancer cell was also developed and nuclear Her2 expression was tested. Then, correlation between nuclear Her2 and resistance to paclitaxel were analyzed. Expression of importin β1 was decreased to downregulate nuclear Her2 level and cell resistance to paclitaxel was tested. We found that Her2 overexpression increases Her2 nuclear expression and cells resistance to paclitaxel in MCF-7 cells. In the paclitaxel resistant cell (SK-BR-3/R), nuclear Her2 expression is upregulated compared with parental SK-BR-3 cells. Increased expression of nuclear Her2 after short-time (48 h) treatment of paclitaxel was also observed in SK-BR-3 cells. Further downregulation of Her2 nuclear expression through blocking expression of importin β1 sensitizes the cells to paclitaxel. The analysis showed that the Her2 nuclear expression increases the survivin expression which leads to resistance to paclitaxel. Her2 nuclear expression decreases paclitaxel-induced apoptosis. However, co-immunoprecipitation was applied, and the physical interaction of nuclear Her2 and survivin was not detected. We show for the first time that nuclear Her2 contributes to paclitaxel resistance in breast cancer cells which suggests that nuclear Her2 as a potential target to sensitize breast cancers to paclitaxel treatment.

Ursolic Acid Enhances Cytotoxicity of Doxorubicin-Resistant Triple-Negative Breast Cancer Cells via ZEB1-AS1/miR-186-5p/ABCC1 Axis.

Background: Triple-negative breast cancer (TNBC) is the most serious subtype of breast cancer (BC) and has been a great health threat to females. Although chemotherapeutic agent contributes a lot to TNBC treatment, drug resistance has been a great obstacle for chemotherapies. Ursolic acid (UA), a pentacyclic triterpenoid compound, was reported to reverse paclitaxel resistance in BC. However, whether UA could affect the resistance of TNBC cells to other drugs such as doxorubicin (DOX) remains to be discovered. Materials and Methods: MTT assay, EdU assay, colony formation assay, and flow cytometry analysis were implemented to detect the viability, proliferation, and apoptosis of DOX-resistant MDA-MB-468 and MDA-MB-436 cells with or without UA treatment. Mechanism assays including RIP, RNA pull-down, and luciferase reporter assays verified the interaction between RNAs. Results: UA treatment hindered the growth and mitigated the DOX resistance of DOX-resistant MDA-MB-468 and MDA-MB-436 cells. Mechanistically, multidrug resistance-associated protein 1 (ABCC1) expression was downregulated by UA treatment. MiR-186-5p was verified to target ABCC1. Further, UA-inhibited ZEB1-AS1 (zinc finger E-box binding homeobox 1 antisense RNA 1) was verified as a competitive endogenous RNA (ceRNA) to upregulate ABCC1 through sponging miR-186-5p. Importantly, UA treatment impaired the malignant phenotypes of DOX-resistant MDA-MB-468 and MDA-MB-436 cells through ZEB1-AS1/ABCC1 axis. Conclusion: UA promotes TNBC cell sensitivity to DOX through inactivating ZEB1-AS1/miR-186-5p/ABCC1 signaling.

Kinase shRNA screening reveals that TAOK3 enhances microtubule-targeted drug resistance of breast cancer cells via the NF-\u03baB signaling pathway

BackgroundChemotherapy is currently one of the most effective treatments for advanced breast cancer. Anti-microtubule agents, including taxanes, eribulin and vinca-alkaloids are one of the primary major anti-breast cancer chemotherapies; however, chemoresistance remains a problem that is difficult to solve. We aimed to discover novel candidate protein targets to combat chemoresistance in breast cancer.MethodsA lentiviral shRNA-based high-throughput screening platform was designed and developed to screen the global kinome to find new therapeutic targets in paclitaxel-resistant breast cancer cells. The phenotypes were confirmed with alternative expression in vitro and in vivo. Molecular mechanisms were investigated using global phosphoprotein arrays and expression microarrays. Global microarray analysis was performed to determine TAOK3 and genes that induced paclitaxel resistance.ResultsA serine/threonine kinase gene, TAOK3, was identified from 724 screened kinase genes. TAOK3 shRNA exhibited the most significant reduction in IC50 values in response to paclitaxel treatment. Ectopic downregulation of TAOK3 resulted in paclitaxel-resistant breast cancer cells sensitize to paclitaxel treatment in vitro and in vivo. The expression of TAOK3 also was correlated to sensitivity to two other anti-microtubule drugs, eribulin and vinorelbine. Our TAOK3-modulated microarray analysis indicated that NF-κB signaling played a major upstream regulation role. TAOK3 inhibitor, CP43, and shRNA of NF-κB both reduced the paclitaxel resistance in TAOK3 overexpressed cells. In clinical microarray databases, high TAOK3 expressed breast cancer patients had poorer prognoses after adjuvant chemotherapy.ConclusionsHere we identified TAOK3 overexpression increased anti-microtubule drug resistance through upregulation of NF-κB signaling, which reduced cell death in breast cancer. Therefore, inhibition of the interaction between TAOK3 and NF-κB signaling may have therapeutic implications for breast cancer patients treated with anti-microtubule drugs.7Jqasva6SWS6RazCnN3hPhVideo abstract

EDIL3 promotes epithelial\u2013mesenchymal transition and paclitaxel resistance through its interaction with integrin \u03b1V\u03b23 in cancer cells

Epithelial–mesenchymal transition (EMT) has recently been associated with tumor progression, metastasis, and chemotherapy resistance in several tumor types. We performed a differential gene expression analysis comparing paclitaxel-resistant vs. paclitaxel-sensitive breast cancer cells that showed the upregulation of EDIL3 (EGF Like Repeats and Discoidin I Like Domains Protein 3). This gene codifies an extracellular matrix protein that has been identified as a novel regulator of EMT, so we studied its role in tumor progression and paclitaxel response. Our results demonstrated that EDIL3 expression levels were increased in paclitaxel-resistant breast and prostate cancer cells, and in subsets of high-grade breast and prostate tumors. Moreover, we observed that EDIL3 modulated the expression of EMT markers and this was impaired by cilengitide, which blocks the EDIL3–integrin αVβ3 interaction. EDIL3 knockdown reverted EMT and sensitized cells to paclitaxel. In contrast, EDIL3 overexpression or the culture of cells in the presence of EDIL3-enriched medium induced EMT and paclitaxel resistance. Adding cilengitide resensitized these cells to paclitaxel treatment. In summary, EDIL3 may contribute to EMT and paclitaxel resistance through autocrine or paracrine signaling in cancer cells. Blockade of EDIL3–integrin αVβ3 interaction by cilengitide restores sensitivity to paclitaxel and reverts EMT in paclitaxel-resistant cancer cells. Combinations of cilengitide and taxanes could be beneficial in the treatment of subsets of breast and prostate cancers.

Abstract 2775: Her2 expression level is positively correlated with paclitaxel response in human breast cancer xenograft mouse model

Abstract Introduction: The overall goal of this study is to quantify amount and heterogeneity of Her2 expression and determine if its expression affects its response to chemotherapy in breast cancer. Paclitaxel (PTX) is one of the most commonly used first-line drugs in the treatment of breast cancer. However, it is reported that overall PTX response rate is 30%-60%, as first-line treatment of metastatic breast cancer, and the main treatment failure is PTX resistance. Thus, novel discoveries of molecular mechanisms that account for ineffective response or resistance to chemotherapy is critical in breast cancer treatment. This study evaluates whether Her2 expression level is correlated with PTX treatment efficacy in a controlled pre-clinical mouse model. Experimental Design: BT474 (1 × 107) or MDA-MB-231 (2 × 106) cells were subcutaneously injected into athymic nude mice (n = 10). Mice were enrolled in the study once they achieved tumor volume of 200 mm3. Tumor size were measured with caliper weekly or every 3 days after experiment started. In vivo Her2 expression level was determined by [89Zr]-Pertuzumab (Her2 antibody) positron emission tomography/computed tomography (PET/CT) imaging at one week prior to initiation of treatment. PTX (15 mg/kg) was administrated via i.v. at day 0 and day 3. In vivo tumor metabolism was quantified by 2-deoxy-2-[fluorine-18]fluoro- D-glucose ([18F]-FDG) PET/CT imaging at day 0, day 3 and day 6. Mean, standard deviation, and frequency histogram of standard uptake value (SUV) was quantified. Tumors were harvested at day 6 for histological analysis. Pearson's correlation test was used as statistical analysis. Results: BT474 (Her2+) tumors had significantly increased [89Zr]-Pertuzumab uptake compared to MDA-MB-231 (Her2-) tumors, which indicated the specificity and accuracy of [89Zr]-Pertuzumab PET/CT imaging. [89Zr]-Pertuzumab SUV varies within BT474 tumors, and tumor growth rate was positively correlated with mean Her2 expression (R2=0.9011, p=0.001). During PTX treatment, tumor volumes did not reveal significant changes. However, [18F]-FDG SUV concentration, normalized to percent changes from day 0 to day 3, was negatively correlated with initial [89Zr]-pertuzumab SUV concentration (R2=0.3654, p=0.05). Conclusion: Preliminary results show Her2 expression level is positively correlated with chemotherapy response in mouse models of human breast cancer. This discovery will help studies of PTX resistance in breast cancer and potentially guide clinical administration of combined therapies. [89Zr]-Pertuzumab PET/CT imaging quantitively measured Her2 expression level in vivo, and [18F]-FDG PET/CT imaging revealed the early signals of drug treatment efficacy. Citation Format: Yun Lu, Adriana I. Massicano, Suzanne E. Lapi, Meng Li, Patrick N. Song, Anna G. Sorace. Her2 expression level is positively correlated with paclitaxel response in human breast cancer xenograft mouse model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2775.

Long-chain non-coding RNA MALAT1 regulates paclitaxel resistance of breast cancer cells by targeting miR-485-3p

To investigate the role of long-chain non-coding RNA MALAT1 in modulating paclitaxel resistance in breast cancer cells. Breast cancer SK-BR-3 cells were treated with gradient concentrations of paclitaxel to induce paclitaxel resistance of the cells. The resistant cells were transfected with si-NC, si-MALAT1, pcDNA, pcDNA-MALAT1, miRNC, miR-485-3p mimics, si-MALAT1+anti-miR-NC, or si-MALAT1+anti-miR-485-3p via liposomes. Following the transfections, the cells were examined for changes in IC50 of paclitaxel using MTT assay; the protein expression of P-gp, Bcl-2 and Bax were detected with Western blotting, and a dual luciferase reporter assay was used to detect the binding of MALAT1 to miR-485-3p. Compared with paclitaxel-sensitive SK-BR-3 cells, paclitaxel-resistant SK-BR-3 cells showed significantly increased the IC50 of paclitaxel with up-regulated MALAT1 expression and down-regulated miR-485-3p expression (P < 0.05). Silencing MALAT1 or overexpressing miR-485-3p obviously lowered the IC50 of paclitaxel and the expression of P-gp and Bcl-2 and increased the expression of Bax in SK-BR-3/PR cells (P < 0.05). miR-485-3p was identified as the target of MALAT1, and inhibiting miR-485-3p significantly reverse the effect of MALAT1 silencing on IC50 of paclitaxel and the expressions of P-gp, Bcl-2 and Bax in SK-BR-3/PR cells (P < 0.05). MALAT1 can modulate paclitaxel resistance in breast cancer cells possibly by targeting miR-485-3p to down-regulate P-gp and Bcl-2 and up-regulate Bax.

Circ-RNF111 contributes to paclitaxel resistance in breast cancer by elevating E2F3 expression via miR-140-5p.

BackgroundCircular RNAs (circRNAs) have been demonstrated to act as key regulators in the chemoresistance of human cancers, including breast cancer (BC). Here, we aimed to explore the role of circ‐RNF111 in paclitaxel (PTX) resistance of BC.MethodsQuantitative real‐time polymerase chain reaction (qRT‐PCR) was employed to determine the expression of circ‐RNF111, microRNA‐140‐5p (miR‐140‐5p) and E2F transcription factor 3 (E2F3) mRNA. The half maximal inhibitory concentration (IC50) of PTX, cell viability, colony formation and cell invasion were assessed by cell counting kit‐8 (CCK‐8) assay, colony formation assay and transwell assay, respectively. Glucose consumption and lactate production were determined by specific kits. A murine xenograft model was established to investigate the role of circ‐RNF111 in PTX resistance of BC in vivo. Dual‐luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to verify the relationship between miR‐140‐5p and circ‐RNF111 or E2F3. Western blot assay was conducted to examine the protein level of E2F3.ResultsCirc‐RNF111 was upregulated in PTX‐resistant BC tissues and cells. Circ‐RNF111 knockdown restrained IC50 of PTX, cell viability, colony numbers, cell invasion and glycolysis in PTX‐resistant BC cells in vitro and enhanced PTX sensitivity in vivo. MiR‐140‐5p was a target of circ‐RNF111 and miR‐140‐5p expression was negatively correlated with circ‐RNF111 expression in BC tissues. The effect of circ‐RNF111 knockdown on PTX resistance was rescued by miR‐140‐5p deletion. Additionally, miR‐140‐5p could interact with E2F3 and negatively regulate E2F3 expression. Moreover, miR‐140‐5p suppressed IC50 of PTX, cell viability, colony numbers, cell invasion and glycolysis by targeting E2F3.ConclusionsCirc‐RNF111 improved PTX resistance of BC by upregulating E2F3 via sponging miR‐140‐5p.