Paclitaxel-resistant Cell Line Research Articles

Elemene Injection Overcomes Paclitaxel Resistance in Breast Cancer through AR/RUNX1 Signal: Network Pharmacology and Experimental Validation.

Paclitaxel (PTX) is a cornerstone chemotherapy for Breast Cancer (BC), yet its impact is limited by emerging resistance. Elemene Injection (EI) has shown potential in overcoming chemotherapy resistance. However, the efficacy by which EI restores PTX sensitivity in BC and the implicated molecular mechanism remain uncharted. Network pharmacology and bioinformatic analysis were conducted to investigate the targets and mechanisms of EI in overcoming PTX resistance. A paclitaxel-resistant MCF-7 cell line (MCF-7PR) was established. The efficacy of EI and/or PTX in inhibiting cell viability was evaluated using sulforhodamine B assay, while cell proliferation was assessed using EdU staining. Furthermore, protein and gene expression analysis was performed through Western blotting and qPCR. The EI containing three active components exhibited a multifaceted impact by targeting an extensive repertoire of 122 potential molecular targets. By intersecting with 761 differentially expressed genes, we successfully identified 9 genes that displayed a direct association with resistance to PTX in BC, presenting promising potential as therapeutic targets for the EI to effectively counteract PTX resistance. Enrichment analysis indicated a significant correlation between these identified targets and critical biological processes, particularly DNA damage response and cell cycle regulation. This correlation was further substantiated through meticulous analysis of single-cell datasets. Molecular docking analysis revealed robust binding affinities between the active components of the EI and the identified molecular targets. Subsequently, in vitro experiments unequivocally demonstrated the dose- and time-dependent inhibitory effects of the EI on both PTX-resistant and sensitive BC cell lines, effectively mitigating the resistance phenotype associated with PTX administration. Furthermore, our findings have indicated EI to effectively suppress the protein expression levels of AR and RUNX1 in MCF-7 and MCF-7PR cells under PTX treatment, as well as downregulate the mRNA expression levels of stem-like properties' markers, KLF4 and OCT4, in these cell lines. Elemene Injection (EI) application has exhibited a significant capability to mitigate PTX resistance in BC, which has been achieved through targeted suppression of the AR/RUNX1 axis, revealing a key strategy to overcome chemotherapeutic resistance.

Exosome-Mediated Transfer of ALDH2 in Nasopharyngeal Carcinoma Cells Confers Increased Resistance to Paclitaxel Treatment.

Nasopharyngeal carcinoma (NPC) is an aggressive and highly metastatic malignant tumor. Despite recent therapeutic advances, resistance to Taxol (the generic name of paclitaxel) therapy remains a major challenge in clinical management. Therefore, it is imperative to explore the potential mechanisms of paclitaxel resistance in NPC. This study aimed to investigate the expression of aldehyde dehydrogenase 2 (ALDH2) in NPC cells and its critical role in paclitaxel resistance. Paclitaxel-resistant cell line CNE1/Taxol (CNE1-TR), a drug-resistant cell line, was established by exposing the CNE1 nasopharyngeal carcinoma cell line to progressively increasing concentrations of paclitaxel. Furthermore, we investigated the role of ALDH2 in paclitaxel resistance and the function of exosomes using cell culture, Western blotting, reverse transcription-polymerase chain reaction (RT-PCR), Cell Counting Kit-8 (CCK-8), and nanoparticle tracking analysis. The results showed that in the presence of paclitaxel, the CNE1-TR cells manifested higher survival rate and half-maximal inhibitory concentration (IC50) value compared to the parental cell line, indicating strong resistance to paclitaxel. CNE1-TR cells had significantly upregulated mRNA and protein levels of ALDH2. In addition, exosome analysis showed that CNE1-TR cells were able to deliver ALDH2 via exosomes, increasing paclitaxel resistance in the recipient cells. We observed that the ALDH2 expression levels and paclitaxel resistance in CNE1-TR cells were effectively reduced by blocking the release of exosomes. ALDH2 is not only a key molecular marker indicative of therapeutic efficacy, but also a potential therapeutic target for developing novel anticancer strategies. By blocking the exosomal transport of ALDH2 or directly inhibiting its activity, it may be possible to overcome paclitaxel resistance, thus improving the success rate of clinical treatment.

Abstract LB261: Establishment and characterization of a paclitaxel-resistant human triple-negative breast cancer cell line

Abstract Triple-negative breast cancer (TNBC) is a lethal female cancer, of which paclitaxel resistance is a major factor limiting treatment outcomes, and continued unmet needs for more effective therapeutic strategies are arduous. The aim of this study is to establish a paclitaxel-resistant TNBC cell line and to characterize the underlying mechanism for potential strategies to overcome the resistance. The human TNBC cell line MDA-MB-231 was exposed to stepwise escalating paclitaxel concentration from 0.5 to 50 nM and a resistant cell line T50R was selected. The IC50 of paclitaxel was 2 and >100 nM in MDA-MB-231 and T50R cells respectively. However, T50R cells required 50 nM Taxol for proliferation. When cultured in the absence of paclitaxel, T50R cells arrested at mitotic stage with high levels of spindle abnormalities. Since induction of microtubule (MT) stabilization plays critical roles in paclitaxel’s potency, we thus measure cell MT polymerization and kinetochore-MT stability. The results showed that, compared to parental MDA-MB-231 cells, the interphase MTs were less polymerized and the kinetochore-MTs were less stable in T50R cells, indicating that the MT dynamic instability was significantly increased in T50R cells. Thus, the increased dynamic unstable MTs in T50R cells might contribute to drug requirement for cell growth and counteract with paclitaxel effects, and consequently lead to drug-resistant phenotype. Identification and interference of endogenous regulators involved in the increased MT instability in T50R cells might provide leads to overcome paclitaxel resistance. Citation Format: Ling-Huei Yih, Hsiao-Hui Kuo, Chieh-Ting Fang. Establishment and characterization of a paclitaxel-resistant human triple-negative breast cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB261.

Abstract 2012: Extrachromosomal DNA promotes drug resistance in pancreatic ductal adenocarcinoma cells

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in Western Countries and has been predicted to become the second leading cause of cancer-related deaths by 2030. Difficulties in early detection as well as strong chemoresistance of the disease prevent the successful treatment of PDAC patients. Although we have previously identified CYP3A5 as a mediator of paclitaxel resistance, further targets remain elusive. Thus, we set out to identify these CYP3A5-independent mechanisms which could lead to the identification of novel biomarkers and drug targets to improve PDAC treatment. For this project, pancreatic cancer cell lines were derived from treatment-naive PDAC samples and exposed to increasing paclitaxel concentrations to induce resistance. The cells were characterized on a genomic and transcriptomic level using short-read sequencing as well as optical mapping to characterize potential candidate genes in vitro. In our results, multi-drug resistance protein 1 (ABCB1) was highly upregulated in our differential gene expression analysis and confirmed as a CYP3A5-independent mediator of acquired paclitaxel resistance. Its overexpression was mediated by extrachromosomal DNA (ecDNA) amplifications upon drug treatment. These circular DNAs were characterized by short-read sequencing as well as long-read optical mapping, which revealed the structures of the most abundant molecules. Furthermore, we investigated the biogenesis of the ecDNA. Current models propose different events such as chromothripsis or breakage-fusion-bridge cycles to initiate ecDNA formation, however, knowledge about the underlying mechanisms remains limited. To study the ecDNA dynamics, we generated ecDNA-negative cell clones from a paclitaxel resistant PDAC cell line. Interestingly, our clonal cell population appears to show the capacity for ecDNA de novo generation and quickly adapts to changing selective pressure such as therapy. This finding might unveil new insights into the triggers of ecDNA formation and its dynamic regulation. In conclusion, our results suggest the clinical relevance of ABCB1 as a mediator of paclitaxel resistance in PDAC and we currently validate our findings in patient samples. However, ABCB1 inhibition is associated with severe side effects. Instead, the regulatory pathways associated with enhanced ABCB1 expression and the formation of ecDNAs might provide therapeutic potential. Additionally, the characterization of ecDNA de novo generation might unveil new insights into how cancer cells adapt to changing selective pressures. Citation Format: Tim Vorberg, Manuel Reitberger, Bernardo Rodriguez-Martin, Maja Starostecka, Ornella Kossi, Vanessa Vogel, Jan Korbel, Andreas Trumpp, Martin Sprick. Extrachromosomal DNA promotes drug resistance in pancreatic ductal adenocarcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2012.

Abstract 4754: Targeted inhibition of the HNF1A/SHH axis by triptolide overcomes paclitaxel resistance in non-small cell lung cancer

Abstract Paclitaxel resistance is commonly associated with a poor prognosis in non-small cell lung cancer (NSCLC) patients, and there are no promising pharmacological treatments for paclitaxel resistance. Here, we constructed human NSCLC-derived paclitaxel-resistant cell lines as an effective tool to explore the mechanisms of chemoresistance. We discovered that triptolide, a monomer compound extracted from Tripterygium wilfordii Hook F, was able to reverse paclitaxel resistance by reducing ABCB1 expression in vivo and in vitro. Through high-throughput sequencing, we found that the SHH-initiated Hedgehog signaling pathway played an important role in this process. More importantly, triptolide could directly target HNF1A, a transcription factor of SHH, and inhibited HNF1A/SHH expression, ensuing in attenuation of Hedgehog signaling. Clinically, we found a positive correlation between HNF1A and SHH expression in both NSCLC tumor tissue microarrays and cancer network databases. These findings illuminated a novel molecular mechanism through which triptolide effectively targets and inhibits HNF1A, thereby impeding the activation of the Hedgehog signaling pathway and reducing the expression of ABCB1. This finding indicated the potential clinical application of triptolide and provided promising prospects in targeting the HNF1A/SHH pathway as a therapeutic strategy for NSCLC patients with paclitaxel resistance. Citation Format: Peichao Li, ngbing Li, Lingxiao Yang, Lei Liu, Xiaogang Zhao. Targeted inhibition of the HNF1A/SHH axis by triptolide overcomes paclitaxel resistance in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4754.

YAP/TEAD involvement in resistance to paclitaxel chemotherapy in lung cancer.

The Yes-associated protein (YAP) oncoprotein has been linked to both metastases and resistance to targeted therapy of lung cancer cells. We aimed to investigate the effect of YAP pharmacological inhibition, using YAP/TEA domain (TEAD) transcription factor interaction inhibitors in chemo-resistant lung cancer cells. YAP subcellular localization, as a readout for YAP activation, cell migration, and TEAD transcription factor functional transcriptional activity were investigated in cancer cell lines with up-regulated YAP, with and without YAP/TEAD interaction inhibitors. Parental (A549) and paclitaxel-resistant (A549R) cell transcriptomes were analyzed. The half-maximal inhibitory concentration (IC50) of paclitaxel or trametinib, which are Mitogen-Activated protein kinase and Erk Kinase (MEK) inhibitors, combined with a YAP/TEAD inhibitor (IV#6), was determined. A three-dimensional (3D) microfluidic culture device enabled us to study the effect of IV#6/paclitaxel combination on cancer cells isolated from fresh resected lung cancer samples. YAP activity was significantly higher in paclitaxel-resistant cell lines. The YAP/TEAD inhibitor induced a decreased YAP activity in A549, PC9, and H2052 cells, with reduced YAP nuclear staining. Wound healing assays upon YAP inhibition revealed impaired cell motility of lung cancer A549 and mesothelioma H2052 cells. Combining YAP pharmacological inhibition with trametinib in K-Ras mutated A549 cells recapitulated synthetic lethality, thereby sensitizing these cells to MEK inhibition. The YAP/TEAD inhibitor lowered the IC50 of paclitaxel in A549R cells. Differential transcriptomic analysis of parental and A549R cells revealed an increased YAP/TEAD transcriptomic signature in resistant cells, downregulated upon YAP inhibition. The YAP/TEAD inhibitor restored paclitaxel sensitivity of A549R cells cultured in a 3D microfluidic system, with lung cancer cells from a fresh tumor efficiently killed by YAP/TEAD inhibitor/paclitaxel doublet. Evidence of the YAP/TEAD transcriptional program's role in chemotherapy resistance paves the way for YAP therapeutic targeting.

Targeted inhibition of the HNF1A/SHH axis by triptolide overcomes paclitaxel resistance in non-small cell lung cancer

Paclitaxel resistance is associated with a poor prognosis in non-small cell lung cancer (NSCLC) patients, and currently, there is no promising drug for paclitaxel resistance. In this study, we investigated the molecular mechanisms underlying the chemoresistance in human NSCLC-derived cell lines. We constructed paclitaxel-resistant NSCLC cell lines (A549/PR and H460/PR) by long-term exposure to paclitaxel. We found that triptolide, a diterpenoid epoxide isolated from the Chinese medicinal herb Tripterygium wilfordii Hook F, effectively enhanced the sensitivity of paclitaxel-resistant cells to paclitaxel by reducing ABCB1 expression in vivo and in vitro. Through high-throughput sequencing, we identified the SHH-initiated Hedgehog signaling pathway playing an important role in this process. We demonstrated that triptolide directly bound to HNF1A, one of the transcription factors of SHH, and inhibited HNF1A/SHH expression, ensuing in attenuation of Hedgehog signaling. In NSCLC tumor tissue microarrays and cancer network databases, we found a positive correlation between HNF1A and SHH expression. Our results illuminate a novel molecular mechanism through which triptolide targets and inhibits HNF1A, thereby impeding the activation of the Hedgehog signaling pathway and reducing the expression of ABCB1. This study suggests the potential clinical application of triptolide and provides promising prospects in targeting the HNF1A/SHH pathway as a therapeutic strategy for NSCLC patients with paclitaxel resistance.Schematic diagram showing that triptolide overcomes paclitaxel resistance by mediating inhibition of the HNF1A/SHH/ABCB1 axis.

CTEN-induced TGF-β1 expression facilitates EMT and enhances paclitaxel resistance in bladder cancer cells.

To investigate the role of C-terminal tensin-like (CTEN) in mediating chemotherapy resistance via epithelial-mesenchymal transition (EMT) in bladder cancer (BC) cells, through the regulation of transforming growth factor-β1 (TGF-β1) expression. Lentiviral vectors were used to create CTEN overexpression and knockdown constructs, which were then introduced into paclitaxel-resistant BC cell lines. The effects of CTEN manipulation on cell proliferation and drug sensitivity was assessed using the CCK-8 assay, and apoptosis was evaluated by flow cytometry. The expression levels of CTEN, TGF-β1, and EMT markers were quantified by RT-qPCR and Western blot analysis. The interaction between CTEN and TGF-β1 and its effect on TGF-β1 methylation were studied using bisulfite sequencing PCR and co-immunoprecipitation. Overexpression of CTEN in BC cells was associated with decreased paclitaxel efficacy, reduced apoptosis, and elevated levels of TGF-β1 and EMT-related proteins. CTEN was found to bind TGF-β1, inhibiting its methylation and thereby promoting TGF-β1 upregulation. This increase in TGF-β1 expression facilitated the EMT process and enhanced drug resistance in BC cells. The induction of TGF-β1 expression by CTEN promotes EMT and increases chemotherapy resistance in BC cells. Targeting CTEN or the EMT pathway could improve chemosensitivity in treatment-resistant BC, suggesting a novel therapeutic strategy to enhance chemotherapy effectiveness.

Resensitizing Paclitaxel-Resistant Ovarian Cancer via Targeting Lipid Metabolism Key Enzymes CPT1A, SCD and FASN.

Epithelial ovarian cancer (EOC) is a lethal gynecological cancer, of which paclitaxel resistance is the major factor limiting treatment outcomes, and identification of paclitaxel resistance-related genes is arduous. We obtained transcriptomic data from seven paclitaxel-resistant ovarian cancer cell lines and corresponding sensitive cell lines. Define genes significantly up-regulated in at least three resistant cell lines, meanwhile they did not down-regulate in the other resistant cell lines as candidate genes. Candidate genes were then ranked according to the frequencies of significant up-regulation in resistant cell lines, defining genes with the highest rankings as paclitaxel resistance-related genes (PRGs). Patients were grouped based on the median expression of PRGs. The lipid metabolism-related gene set and the oncological gene set were established and took intersections with genes co-upregulated with PRGs, obtaining 229 co-upregulated genes associated with lipid metabolism and tumorigenesis. The PPI network obtained 19 highly confidential synergistic targets (interaction score > 0.7) that directly associated with CPT1A. Finally, FASN and SCD were up-stream substrate provider and competitor of CPT1A, respectively. Western blot and qRT-PCR results confirmed the over-expression of CPT1A, SCD and FASN in the A2780/PTX cell line. The inhibition of CPT1A, SCD and FASN down-regulated cell viability and migration, pharmacological blockade of CPT1A and SCD increased apoptosis rate and paclitaxel sensitivity of A2780/PTX. In summary, our novel bioinformatic methods can overcome difficulties in drug resistance evaluation, providing promising therapeutical strategies for paclitaxel-resistant EOC via taregting lipid metabolism-related enzymes.

Mechanisms of cytotoxic activity of pyrrole-carboxamides against multidrug-resistant tumor cell sublines

Introduction. Mitotic poisoning agents (MPAs) affecting the dynamic state of the microtubules, are the well-known and effective chemotherapeutic agents. Mitotic poisoning agents are binding to the microtubules, and thereby interfere with tubulin polymerization or depolymerization dynamic state, resulting in the cell cycle arrest in M-phase (mitotic catastrophe) and subsequent apoptotic cell death. We reported previously about potent cytotoxic activities against the pyrrole-carboxamides (PCs) (PC-61 and PC-84) against broad spectrum of cancer cell lines, including triple negative breast cancer, lung and prostate cancer.Aim. To examine the cytotoxic activities of PC-61 and PC-84 against multidrug-resistant cancer cell lines indicated above.Materials and methods. Studу was performed on the triple-negative paclitaxel-resistant breast cancer cell line HCC1806 Tx-R and doxorubicin-resistant osteosarcoma SaOS-2 Dox-R cell line.Results. The cytotoxic activity of PCs was due to the inhibition of tubulin polymerization. Immunofluorescence staining data revealed PC’s ability to interfere with tubulin’s assembly in multidrug-resistant cancer cell lines. As an outcome of inhibition of tubulin polymerization, PCs induced cell cycle arrest in M-phase, and further led to apoptotic cell death of cancer cells.Conclusion. Collectively, we demonstrated potent cytotoxic activity of PCs against cancer cell lines with multidrug-resistant phenotype, which arising the possibilities to develop novel and effective anti-tumor agents that belongs to mitotic poisoning agents

Combined PI3K Inhibitor and Eribulin Enhances Anti-Tumor Activity in Preclinical Models of Paclitaxel-Resistant, PIK3CA-Mutated Endometrial Cancer.

Endometrial cancer stands as the predominant gynecological malignancy in developed nations. For advanced or recurrent disease, paclitaxel-based chemotherapy is the standard front-line therapy. However, paclitaxel resistance eternally develops. Based on the high prevalence of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutation, reaching 50%, in endometrial cancer, we preclinically investigated the effectiveness of a combination of a phosphatidylinositol 3-kinase (PI3K) inhibitor with eribulin, a post-paclitaxel therapy for breast cancer, in treating paclitaxel-resistant, PIK3CA-mutated endometrial cancer. We generated paclitaxel-resistant cell lines from PIK3CA-mutated endometrial cancer cell lines by gradually increasing the concentration of paclitaxel in cell cultures. We observed that the PI3K/AKT and epithelial-mesenchymal transition (EMT) pathways in paclitaxel-resistant cells were significantly upregulated compared with those in parental cells. Then, we demonstrated that the combination of alpelisib (a PI3K inhibitor) and eribulin more effectively suppressed the cellular growth of paclitaxel-resistant cells in in vitro and in vivo xenograft models. Mechanistically, we demonstrated that the effect of the combination could be enhanced by inhibiting both the PI3K/AKT and EMT pathways. Therefore, we suggest that paclitaxel resistance is associated with the activation of the PIK3/AKT pathway in PIK3CA-mutated endometrial cancer, and the combination of a PI3K inhibitor and eribulin merits further clinical investigation.

Activity of Lysosomal ABCC3, ABCC5 and ABCC10 is Responsible for Lysosomal Sequestration of Doxorubicin and Paclitaxel-Oregongreen488 in Paclitaxel-Resistant Cancer Cell Lines.

Cancer cell multidrug resistance induced by paclitaxel contributes to the high failure rates of chemotherapy and relapse of the disease. Several mechanisms have been described that underlie the observed resistance, including the overexpression of ABCB1 (P-glycoprotein), which represents an ATP-binding cassette (ABC) transmembrane protein, and its functional occurrence in lysosomal membranes is linked to drug accumulation in these organelles. Using clinically-relevant models of paclitaxel-resistant triple-negative breast cancer and non-small cell lung cancer cell lines, we provide evidence for the role of ABCC subfamily members in the lysosomal sequestration of drugs in multidrug resistant phenotypes. Proteins expression level and its cellular localisation was measured using Western Blot and confocal microscopy. Drug accumulation was analysed by confocal microscopy and flow cytometry. Drug cytotoxicity was tested using resasurin assay and anexin V propidium iodide staining. Regardless of the alteration in gene expression, paclitaxel induced the intracellular redistribution of ABCC3, ABCC5 and ABCC10 and their enrichment in lysosomes. The use of ABCC inhibitors and transient silencing of these three genes limited the accumulation of doxorubicin and paclitaxel-OregonGreen488 in lysosomes, while having little impact on the total drug level inside cells. The cancer cells were also sensitized to various structurally unrelated chemotherapeutics of differing acidity. The results suggest that lysosome membranes anchored ABCC proteins which remained functionally active and were capable to load chemotherapeutics into lysosomes in paclitaxel-resistant cancer cells. Therefore, targeting of lysosomal ABCC transporters may help to overcome paclitaxel-induced resistance by reducing the accumulation of drugs in lysosomes.

CRISPR/Cas9-Mediated CtBP1 Gene Editing Enhances Chemosensitivity and Inhibits Metastatic Potential in Esophageal Squamous Cell Carcinoma Cells.

Innovative therapeutic strategies for esophageal squamous cell carcinoma (ESCC) are urgently required due to the limited effectiveness of standard chemotherapies. C-Terminal Binding Protein 1 (CtBP1) has been implicated in various cancers, including ESCC. However, the precise expression patterns and functional roles of CtBP1 in ESCC remain inadequately characterized. In this study, we aimed to investigate CtBP1 expression and its role in the resistance of ESCC to paclitaxel, an effective chemotherapeutic agent. Western blotting and immunofluorescence were applied to assess CtBP1 expression in the TE-1 and KYSE-50 cell lines. We observed the marked expression of CtBP1, which was associated with enhanced proliferation, invasion, and metastasis in these cell lines. Further, we successfully generated paclitaxel resistant ESCC cell lines and conducted cell viability assays. We employed the CRISPR/Cas9 genome editing system to disable the CtBP1 gene in ESCC cell lines. Through the analysis of the drug dose-response curve, we assessed the sensitivity of these cell lines in different treatment groups. Remarkably, CtBP1-disabled cell lines displayed not only improved sensitivity but also a remarkable inhibition of proliferation, invasion, and metastasis. This demonstrates that CtBP1 may promote ESCC cell malignancy and confer paclitaxel resistance. In summary, our study opens a promising avenue for targeted therapies, revealing the potential of CtBP1 inhibition to enhance the effectiveness of paclitaxel treatment for the personalized management of ESCC.

Computational drug repositioning for the identification of new agents to sensitize drug-resistant breast tumors across treatments and receptor subtypes.

Drug resistance is a major obstacle in cancer treatment and can involve a variety of different factors. Identifying effective therapies for drug resistant tumors is integral for improving patient outcomes. In this study, we applied a computational drug repositioning approach to identify potential agents to sensitize primary drug resistant breast cancers. We extracted drug resistance profiles from the I-SPY 2 TRIAL, a neoadjuvant trial for early stage breast cancer, by comparing gene expression profiles of responder and non-responder patients stratified into treatments within HR/HER2 receptor subtypes, yielding 17 treatment-subtype pairs. We then used a rank-based pattern-matching strategy to identify compounds in the Connectivity Map, a database of cell line derived drug perturbation profiles, that can reverse these signatures in a breast cancer cell line. We hypothesize that reversing these drug resistance signatures will sensitize tumors to treatment and prolong survival. We found that few individual genes are shared among the drug resistance profiles of different agents. At the pathway level, however, we found enrichment of immune pathways in the responders in 8 treatments within the HR+HER2+, HR+HER2-, and HR-HER2- receptor subtypes. We also found enrichment of estrogen response pathways in the non-responders in 10 treatments primarily within the hormone receptor positive subtypes. Although most of our drug predictions are unique to treatment arms and receptor subtypes, our drug repositioning pipeline identified the estrogen receptor antagonist fulvestrant as a compound that can potentially reverse resistance across 13/17 of the treatments and receptor subtypes including HR+ and triple negative. While fulvestrant showed limited efficacy when tested in a panel of 5 paclitaxel resistant breast cancer cell lines, it did increase drug response in combination with paclitaxel in HCC-1937, a triple negative breast cancer cell line. We applied a computational drug repurposing approach to identify potential agents to sensitize drug resistant breast cancers in the I-SPY 2 TRIAL. We identified fulvestrant as a potential drug hit and showed that it increased response in a paclitaxel-resistant triple negative breast cancer cell line, HCC-1937, when treated in combination with paclitaxel.

The CCL2-CCR2 Axis Contributes to Migration of Cabazitaxel-resistant Prostate Cancer Cells.

Developing resistance to cabazitaxel is a major challenge in patients with docetaxel- and castration-resistant prostate cancer (CRPC) since it is frequently administered as a last resort. We have previously reported that CCL2 induces resistance to the antiproliferative effect of cabazitaxel in DU145-TxR/CxR prostate cancer cell lines. However, how CCL2 induces resistance to the antimigration effect of cabazitaxel remains unclear. We established a cabazitaxel-resistant cell line, DU145-TxR/CxR, from a previously established paclitaxel-resistant cell line, DU145-TxR, which was confirmed to show docetaxel resistance. We performed migration assay and analyzed the expression of epithelial-mesenchymal transition markers using DU145-TxR/CxR with or without CCL2 silencing with small interfering RNA (siRNA) transfection. Cabazitaxel inhibited the migration of DU145 cells through the inactivation of STAT3. A CCR2 (a specific receptor of CCL2) antagonist suppressed the migration of DU145-TxR and DU145-TxR/CxR cells under cabazitaxel treatment. Western blotting revealed that the CCR2 antagonist inhibited STAT3 phosphorylation in DU145-TxR and DU145-TxR/CxR cells under cabazitaxel treatment. CCL2 silencing with siRNA in DU145-TxR and DU145-TxR/CxR cells decreased migration through STAT3 and p38 inactivation. Furthermore, CCL2 activated AKT, and CCR2 antagonist inhibited AKT phosphorylation in DU145-TxR and DU145-TxR/CxR cells with recovery of sensitivity to cabazitaxel under cabazitaxel treatment. The CCL2-CCR2 axis is a key contributor to resistance to the antimigration effect of cabazitaxel in prostate cancer cells. CCL2-CCR2 axis inhibition may be a potential therapeutic target against chemoresistant CRPC in combination with cabazitaxel.

Abstract 2846: The mitochondrial dynamics of breast cancer regulate P-glycoprotein expression

Abstract Multidrug efflux transporter ABCB1 (MDR1) encodes P-glycoprotein (P-gp), a well-known transporter that is believed to contribute to the development of multidrug resistance (MDR). Furthermore, P-gp plays an important role in drug disposition, pharmacokinetics, and drug development. Thus, P-gp transporter expression, function, and modulation of MDR in human cancers have been extensively studied. In spite of significant progress in understanding P-gp, ABCB1 regulation and its impact on clinical trials remain unclear. Through sequential exposure of SUM159PT to paclitaxel, we created a paclitaxel resistant triple negative breast cancer cell line SUM159PT-PAC200. We observed a significant increase in the phosphorylation of the mitochondrial fission protein dynamin related protein kinase 1 (Drp1) at Ser616 and its upstream kinase ERK1/2a of Drp1. Through the use of CRISPR-Cas9 technology, we knocked out the gene encoding Drp1 in PAC200. When Drp1 is genetically abated (PAC200 Drp1-/-), paclitaxel resistance is significantly reversed. It was also observed that clonogenicity under paclitaxel pressure was significantly decreased in both the PAC200/Drp1-/- line and the partial KO Drp1-/+ line, compared to the PAC200 line. Parallelly, a significant increase in the expression of P-gp transporters was also observed in the PAC200 cell line. As a surprise, genetically targeting Drp1 in PAC200 cells (i.e., PAC200 Drp1/KO) reduced P-gp expression to virtually undetectable levels. Until now, there has been no description of how Drp1 and P-gp are related. Furthermore, PAC200 Drp1/KO cells showed reduced proliferation, migration, and invasion potential, while their soft agar clonogenic capacities were almost nonexistent, suggesting that Drp1 plays a key role in breast cancer proliferation and dissemination. It appears that modulating or targeting Drp1 might be a useful strategy for modulating P-gp in the development and maintenance of the cancer's MDR phenotype, proliferation, and invasion. Understanding how mitochondrial dynamics determine the transactivation and coordination of drug-resistant P-gp phenotypes would make Drp1 an attractive molecular target for cancer treatment. Citation Format: David Terrero, Saloni Malla, Dayanidhi Raman, Jayachandra Babu, Amit Tiwari. The mitochondrial dynamics of breast cancer regulate P-glycoprotein expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2846.

Abstract 4943: SP-1-39 as a novel tubulin inhibitor overcomes taxane resistance in castration-resistant prostate cancer model

Abstract Colchicine binding site inhibitors have proven to have potent anti-cancer efficacy in many cancer types. Inhibition of tubulin polymerization, a popular mechanism of action for anti-cancer activities, will be activated once the inhibitor interacts with the colchicine binding site. We have previously developed and reported a potent tubulin inhibitor (SB-216). One of its derivatives, the compound SP-1-39, a pyrimidine dihydroquinoxalinone derivative, has been recently designed to further improve the potency and therapeutic properties. Here, we examined the impact of SP-1-39 on preclinical prostate cancer models in vitro and in vivo. EBI competition assay confirmed that SP-1-39 binds to colchicine-binding site and inhibits the formation of EBI:β-tubulin adduct. The anti-proliferation abilities of SP-1-39 was tested against melanoma, breast cancer, pancreatic cancer, prostate cancer, and paclitaxel-resistant prostate cancer cell lines by MTS assay, and defined IC50 values of 0.2-4 nmol/L. Other in vitro results showed SP-1-39 disrupted microtubule network, induced cell cycle arrest at G2/M phase, and caused apoptosis in paclitaxel-resistant human prostate cancer cells (PC-3/TxR). For in vivo study, 2.5mg/kg SP-1-39 significantly attenuated tumor growth in PC-3/TxR xenograft mouse model, inhibited angiogenesis, and overcame taxane resistance. Our findings demonstrate the preclinical therapeutic efficacy and safety of SP-1-39, and provide support for further exploration of novel tubulin inhibitors as anti-cancer agent for cancer therapy. Citation Format: Rui Wang, Kelli L. Hartman, Satyanarayana Pochampally, Duane D. Miller, Wei Li. SP-1-39 as a novel tubulin inhibitor overcomes taxane resistance in castration-resistant prostate cancer model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4943.

Abstract 1689: The role of circular RNAs in triple negative breast cancer and chemotherapy resistance

Abstract Background: Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer-related death among women worldwide. Triple negative breast cancer (TNBC) represents 15-20% of all breast cancers and is an aggressive subtype. Chemotherapy-based treatments remain the standard of care for TNBC. Unfortunately, chemotherapy resistance is common, and for these patients, outcomes are poor and alternative treatment strategies remain an unmet need. circRNAs are a newly identified class of noncoding RNA molecules with covalently closed circular structures. An increasing number of recent studies including ours have indicated that circRNAs play crucial roles in regulating tumor development and chemoresistance. However, the role of circRNAs in the process of chemotherapy resistance and TNBC progression is not clear. Materials and Methods: As a first step towards identifying circRNAs that participate in the development of chemoresistance in TNBC cells, and to determine if targeting such circRNAs is a novel and efficacious therapeutic strategy, doxorubicin-resistant (Doxo-R), paclitaxel-resistant (PTX-R), and double-resistant (DP-R) cell lines were generated from MDA-MB-231. Human circRNA microarrays were utilized to profile the expression of approximately 14,000 known circRNAs in normal breast tissue, matched patient-derived xenografts (PDX) generated prior to and following neoadjuvant chemotherapy (NAC), and TNBC chemosensitive and chemoresistant cell lines. Top hits were validated using RT-PCR. Results: circRNA microarray profiling identified 429 and 310 transcripts differentially expressed in doxorubicin and paclitaxel resistant cells, respectively, compared to parental chemosensitive cell lines (|FC| ≥ 1.5; p value < 0.05). In comparison to pre-NAC derived xenografts, 1,396 circRNAs were dysregulated among post-NAC PDX models. Further, three circRNAs (hsa_circ_001388, hsa_circ_104652, and hsa_circ_061260) were upregulated in Doxo-R, PTX-R, and post-NAC PDX samples compared to their respective controls. Among these three circRNAs, hsa_circ_001388 (also known as circNSD2) was the only transcript also predicted to be translated into a novel and uncharacterized protein given the presence of a high confidence translation initiation site and IRES sequence. Ongoing studies are aimed at determining the role of circNSD2 protein in breast cancer carcinogenesis, progression, and response to standard of care chemotherapeutics and the mechanistic process by which this protein functions. Additionally, the efficacy of specifically targeting this circRNA as a novel therapeutic approach is being explored. Conclusions: Increasing knowledge of the important functions of circRNAs underlying drug resistance will provide new opportunities for developing efficacious therapeutic strategies and prognostic/predictive biomarkers for TNBC. Citation Format: Xiyin Wang, Michael J. Emch, Xiaojia Tang, Jia Yu, Krishna R. Kalari, Liewei Wang, Matthew P. Goetz, John R. Hawse. The role of circular RNAs in triple negative breast cancer and chemotherapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1689.

GSK2801 Reverses Paclitaxel Resistance in Anaplastic Thyroid Cancer Cell Lines through MYCN Downregulation.

Anaplastic thyroid cancer (ATC) is a very rare, but extremely aggressive form of thyroid malignancy, responsible for the highest mortality rate registered for thyroid cancer. Treatment with taxanes (such as paclitaxel) is an important approach in counteracting ATC or slowing its progression in tumors without known genetic aberrations or those which are unresponsive to other treatments. Unfortunately, resistance often develops and, for this reason, new therapies that overcome taxane resistance are needed. In this study, effects of inhibition of several bromodomain proteins in paclitaxel-resistant ATC cell lines were investigated. GSK2801, a specific inhibitor of BAZ2A, BAZ2B and BRD9, was effective in resensitizing cells to paclitaxel. In fact, when used in combination with paclitaxel, it was able to reduce cell viability, block the ability to form colonies in an anchor-independent manner, and strongly decrease cell motility. After RNA-seq following treatment with GSK2801, we focused our attention on MYCN. Based on the hypothesis that MYCN was a major downstream player in the biological effects of GSK2801, we tested a specific inhibitor, VPC-70619, which showed effective biological effects when used in association with paclitaxel. This suggests that the functional deficiency of MYCN determines a partial resensitization of the cells examined and, ultimately, that a substantial part of the effect of GSK2801 results from inhibition of MYCN expression.

Abstract P2-22-02: The role of circular RNAs in triple-negative breast cancer and chemotherapy resistance

Abstract Background: Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer-related death among women worldwide. Triple-negative breast cancer (TNBC) is an aggressive subtype representing 15-20% of all breast cancers. TNBC often ends with a poor clinical outcome due to high histological grade and recurrence rates with few treatment options. Chemotherapy remains the standard of care for TNBC treatment, but unfortunately, patients frequently develop resistance, and alternative treatment strategies for the chemoresistant disease remain a major unmet need. circRNAs are newly identified non-coding RNA molecules with covalently closed circular structures. Recently, an increasing number of studies have indicated that circRNAs play crucial roles in regulating tumor development and chemoresistance. However, the role of circRNAs in the process of TNBC chemotherapy resistance is not yet fully clear. Materials and methods: To study the molecular functions of circRNAs in TNBC chemoresistance, identify alternative biomarkers for chemotherapy-resistant disease, and elucidate novel therapeutic targets, doxorubicin-resistant (Doxo-R) and paclitaxel-resistant (Taxol-R) cell lines were generated using TNBC models. Results: Doxo-R and Taxol-R cells exhibited a 3- and 12-fold decrease in sensitivity to their respective chemotherapeutic agents. We identified a large spectrum of gene expression changes that were acquired in Doxo-R and Taxol-R cells. The expression of circRNAs was profiled using circRNA microarrays, and top hits were validated using RT-PCR. We identified 429 and 310 circRNAs that were differentially expressed in Doxo-R and Taxol-R resistant cells respectively compared to the parental chemosensitive cell line (|FC| ≥ 1.5; p value < 0.05). Of these, 66 were commonly differentially expressed between the two chemotherapy resistant models. Conclusions: These results revealed that Doxo-R and Taxol-R TNBC cell lines were successfully established and serve as good models for studying the mechanisms of chemotherapy resistance and the regulatory roles of circRNAs in the development of chemoresistance in TNBC. Increasing knowledge of the important functions of circRNAs underlying drug resistance will provide new opportunities for developing efficacious therapeutic strategies and prognostic/predictive biomarkers for TNBC. Citation Format: Xiyin Wang, Michael Emch, John Hawse. The role of circular RNAs in triple-negative breast cancer and chemotherapy resistance [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 P2-22-02.