MDR1 Expression Research Articles

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

All patients with metastatic breast cancer (MBC) expressing estrogen receptor-α (ESR1) will eventually develop resistance to endocrine therapies. In up to 40% of patients, this resistance is caused by activating mutations in the ligand-binding domain (LBD) of ESR1. Accumulating clinical evidence indicate adverse outcomes for these patients, beyond that expected by resistance to endocrine therapy. Here we aimed to study the role of ESR1 mutations in conferring chemoresistance in BC cells. MCF-7 cells harboring Y537S and D538G ESR1 mutations (mut-ER) were employed to study the response to chemotherapy drugs, paclitaxel and doxorubicin, using viability and apoptotic assay in vitro, and tumor growth in vivo. JNK/c-Jun/MDR1 pathway was studied using qRT-PCR, western-blot, gene-reporter and ChIP assays. MDR1 expression was analyzed in clinical samples using IHC. Cell harboring ESR1 mutations displayed relative chemoresistance compared to WT-ER, evidenced by higher viability and reduced apoptosis as well as resistance to paclitaxel in vivo. To elucidate the underlying mechanism, MDR1 expression was examined and elevated levels were observed in mut-ER cells, and in clinical BC samples. MDR1 is regulated by the c-Jun pathway, and we showed high correlation between these two genes in BC using TCGA databases. Accordingly, we detected higher JNK/c-Jun expression and activity in ESR1-mutated cells, as well as increased occupancy of c-Jun in MDR1 promoter. Importantly, JNK inhibition decreased MDR1 expression and restored sensitivity to chemotherapy. Taken together, these data indicate that ESR1 mutations confer chemoresistance through activation of the JNK/MDR1 axis. These finding suggest a novel treatment option for BC tumors expressing ESR1 mutations.

Trichophyton indotineae Erg1Ala448Thr Strain Expressed Constitutively High Levels of Sterol 14-α Demethylase Erg11B mRNA, While Transporter MDR3 and Erg11A mRNA Expression Was Induced After Addition of Short Chain Azoles

Trichophyton indotineae is an emerging pathogen causing recalcitrant skin infections and exhibiting multiple resistances to azoles and allylamines. Squalene epoxidase erg1Ala448Thr mutants often show association with azole resistance. RT-PCR gene expression analysis helps to elucidate the connection between ergosterol biosynthesis regulation and efflux control through the activation of multidrug resistance (MDR) and major facilitator superfamily (MFS1) transporters as well as heat shock proteins (HSP). Several T. indotineae isolates demonstrated a heat-dependent increase of Erg11B transcripts combined with downregulation of Erg1, suggesting a protective role for Erg11B. They also showed persistent upregulation of MFS1. The addition of fluconazole or voriconazole induced the expression of Erg11A, MDR3 and, to a lesser extent, Erg11B and Erg1. The azole-resistant erg1Ala448Thr mutant UKJ 476/21 exhibited exceptionally high transcript levels of sterol 14-αdemethylase Erg11B, combined with the inability of HSP60 and HSP90 to respond to increasing growth temperatures. Itraconazole demonstrated similar effects in a few T. indotineae isolates, but terbinafine did not enhance Erg1 transcription at all. Overexpression of Erg11B may explain the multiple azole resistance phenotype, whereas Erg11B point mutations are not associated with resistance to azoles used for medical treatment.

Antineoplastic Activity of Sodium Caseinate in a Cytarabine-Resistant Mouse Acute Myeloid Leukemia Cell Line.

Acute myeloid leukemia (AML) is a hematological neoplasm of rapid and progressive onset, and is the most common form of leukemia in adults. Chemoresistance to conventional treatments such as cytarabine (Ara-C) and daunorubicin is a main cause of relapse, recurrence, metastasis, and high mortality in AML patients. It is known that sodium caseinate (SC), a salt derived from casein, a milk protein, inhibits growth and induces apoptosis in acute myeloid leukemia cells but not in normal hematopoietic cells. However, it is unknown whether SC retains its antileukemic effect in cytarabine-resistant AML cell lines. To evaluate the antineoplastic effect of SC in cytarabine-resistant leukemia models. The SC inhibits the growth and induces apoptosis in parental WEHI-3 AML cells. Here, we generated two cytarabine-resistant sublines, WEHI-CR25 and WEHI-CR50, which exhibit 6- and 16-fold increased resistance to cytarabine, respectively, compared to the parental WEHI-3 cells. Thus, these sublines mimic a chemoresistant model. We demonstrate that WEHI-CR25 and WEHI-CR50 cells retain sensitivity to SC, similar to parental WEHI-3 cells. This sensitivity results in inhibited cell proliferation, induced apoptosis, and increased expression of ENT1 and dCK, molecules involved in the entry and metabolism of Ara-C, while decreasing MDR1 expression. Additionally, we observed that SC prolonged the survival of WEHI-CR50 tumor-bearing mice, despite their resistance to Ara-C. This is the first evidence that SC, a milk protein, may inhibit proliferation and induce apoptosis in cytarabine-resistant cells.

Retracted] RNAi‑mediated EZH2 depletion decreases MDR1 expression and sensitizes multidrug‑resistant hepatocellular carcinoma cells to chemotherapy

Retracted] RNAi‑mediated EZH2 depletion decreases MDR1 expression and sensitizes multidrug‑resistant hepatocellular carcinoma cells to chemotherapy

LncRNA PVT1 silencing inhibits gastric cancer cells’ progression via enhancing chemosensitivity to paclitaxel

Gastric cancer (GC) is one of the leading causes of cancer-related deaths worldwide because of its high morbidity and the absence of effective therapies. Even though paclitaxel is a powerful anticancer chemotherapy drug, recent studies have indicated its ineffectiveness against GC cells. Long non-coding RNA (lncRNA) PVT1 has a high expression in GC cells and increases the progression of tumors via inducing drug resistance. In the present study, the effects of the siRNA-mediated lncRNA PVT1 gene silencing along with paclitaxel treatment on the rate of apoptosis, growth, and migration of AGS GC cells were investigated. AGS cells were cultured and then transfected with siRNA PVT1 using electroporation. The MTT test was used to examine the effect of treatments on the viability of cultured cells. Furthermore, the flow cytometry method was used to evaluate the impact of treatments on the cell cycle process and apoptosis induction in GC cells. Finally, the mRNA expression of target genes was assessed using the qRT-PCR method. The results showed that lncRNA PVT1 gene suppression, along with paclitaxel treatment, reduces the viability of cancer cells and significantly increases the apoptosis rate of cancer cells and the number of cells arrested in the G2/M phase compared to the control group. Based on the results of qRT-PCR, combined treatment significantly decreased the expression of MMP3, MMP9, MDR1, MRP1, Bcl-2, k-Ras, and c-Myc genes and increased the expression of the Bax gene compared to the control group. The results of our study showed that lncRNA PVT1 gene targeting, together with paclitaxel treatment, induces apoptosis, inhibits growth, alleviates drug resistance, and reduces the migratory capability of GC cells. Therefore, there is a need for further investigations to evaluate the feasibility and effectiveness of this approach in vivo in animal models.

Characterization of Chemoresistance in Pancreatic Cancer: A Look at MDR-1 Polymorphisms and Expression in Cancer Cells and Patients.

Pancreatic malignancy is the fourth cause of cancer-related death in Western countries and is predicted to become the second leading cause of cancer-related mortality by 2030. The standard therapies (FOLFIRINOX and gemcitabine with nab-paclitaxel) are not resolutive because this type of cancer is also characterized by a high chemoresistance, due in part to the activity of the ATP Binding Cassette (ABC) pumps accounting for the reduction in the intracellular concentration of the drugs. In this work, we analyze the occurrence of single-nucleotide polymorphisms (SNPs) in the MDR-1 gene, in different pancreatic cancer cell lines, and in tissues from pancreatic cancer patients by DNA sequencing, as well as the expression levels of MDR-1 mRNA and protein, by qRT-PCR and Western Blot analysis. We found that gemcitabine-resistant cells, in conjunction with homozygosis of analyzed SNPs, showed high MDR-1 basal levels with further increases after gemcitabine treatment. Nevertheless, we did not observe in the human PDAC samples a correlation between the level of MDR-1 mRNA and protein expression and SNPs. Preliminary, we conclude that in our small cohort, these SNPs cannot be used as molecular markers for predicting the levels of MDR-1 mRNA/protein levels and drug responses in patients with PDAC.

Abstract PO2-05-09: Beyond endocrine resistance: estrogen receptor (ESR1) activating mutations mediate chemotherapy resistance through the JNK/c-Jun MDR1 pathway in breast cancer

Abstract Purpose: All patients with metastatic breast cancer (MBC) expressing estrogen receptor-α (ESR1) will eventually develop resistance to endocrine therapies. In up to 40% of patients, this resistance is caused by activating mutations in the ligand-binding domain (LBD) of ESR1. Accumulating clinical evidence indicate adverse outcomes for these patients, beyond that expected by resistance to endocrine therapy. We hypothesized that ESR1 mutations may also confer resistance to chemotherapy. Experimental Design: MCF-7 cells harboring Y537S and D538G ESR1 mutations (mut-ER) were employed to study response to chemotherapy using viability and apoptotic assay in vitro, and tumor growth in vivo. JNK/c-Jun/MDR1 pathway was studied using qRT-PCR, western-blot, gene-reporter and ChIP assays. MDR1 expression was analyzed in clinical samples using IHC. Results: Cell harboring ESR1 mutations displayed relative chemoresistance, evidenced by higher viability and reduced apoptosis as well as resistance to paclitaxel in vivo. To elucidate the underlying mechanism, MDR1 expression was examined and elevated levels were observed in mut-ER cells, and in clinical BC samples. MDR1 is regulated by the JNK/cJun pathway, and indeed, we detected higher JNK/cJun expression and activity in mut-ER cells, as well as increased occupancy of c-Jun in MDR1 promoter. Importantly, JNK inhibition decreased MDR1 expression, particularly of D538G-cells, and reduced viability in response to chemotherapy. Conclusions: Taken together, these data indicate that ESR1 mutations confer chemoresistance in BC through activation of the JNK/MDR1 axis. Targeting this pathway may restore sensitivity to chemotherapy and serve as a novel treatment strategy for MBC patients carrying ESR1 mutations. Citation Format: Marwa Taya, Keren Merenbakh-Lamin, Asia Zubkov, Zohar Honig, Ori Mayer, Noam Shomron, Wolf Ido, Tami Rubinek. Beyond endocrine resistance: estrogen receptor (ESR1) activating mutations mediate chemotherapy resistance through the JNK/c-Jun MDR1 pathway in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-05-09.

Evaluating the Dynamics of Cyp3a11, MRP2 and MDR1 Modulation by IL-22

Background: IL-22 is a cytokine produced by a number of immune cell populations, including type 3 innate lymphoid cells. We have shown previously that IL-22 suppresses the expression Cyp3a11 in the mouse intestine, a gene that encodes a drug metabolizing. In addition, IL-22 reduces the expression of MRP2 and MDR1, the gene that encodes as Multidrug resistance protein 2 or 1 involve in the drug detoxification. Modulation of these proteins can influence the oral bioavailability of drugs. Aims: The aim of this study was to define the existence of reversible modulation of IL-22 on Cyp3a11, MRP2 and MDR1 expression and protein level or activity in the intestinal epithelium. Methods: In vitro enteroids of the mouse small intestine were cultured in 3D in the presence/absence of IL-22 for 5 days. Cyp3a11, MRP2 and MDR1 transcript expression was evaluated by qPCR followed by protein analysis to define their modulation and activity at varying timepoints after IL-22 treatment cessation. Results: IL-22 reduced the expression and activity of Cyp3a11 which normalized soon after stopping IL-22 treatment. This action of IL-22 are made without any modulation of protein level of Cyp3a11. In the same way, IL-22 reduces the expression and the protein level of MRP2 and MDR1, an effect normalizes after treatment cessation. Conclusions: IL-22 suppresses the expression and the activity of Cyp3a11 without any modulation of the protein level unlike at the level of MRP2 and MDR1 which are reduced. These data suggest that regulation of gene expression, protein or enzyme activity by IL-22 may have variable effects: Cyp3a11 is ATP-independent, whereas MRP2 and MDR1 are ATP-dependent. This direct or indirect enzymatic modulation of these proteins must be taken into account when assessing the role of the drug in the biology of the intestinal mucosa. Dr. Lloyd Sutherland Chair in IBD/GI Research/Weston Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Disrupting the interaction between a p53 gain-of-function mutant and the transcriptional co-activator PC4 reverses drug resistance in cancer cells.

PC4 is a chromatin-associated protein and transcriptional coactivator whose role in gene regulation by wild-type p53 is now well known. Little is known about the roles of PC4 in tumor cells bearing mutant p53 genes. We show that PC4 associates with one of the tumor-associated gain-of-function p53 mutants, R273H. This association drives its recruitment to two promoters, UBE2C and MDR1, known to be responsible for imparting aggressive growth and resistance to many drugs. Here, we introduced a peptide that disrupts the PC4-R273Hp53 interaction to tumor cells bearing the R273HTP53 gene, which led to a lowering of MDR1 expression and abrogation of drug resistance in a mutant-specific manner. The results suggest that the PC4-R273Hp53 interaction may be a promising target for reducing proliferation and drug resistance in tumors.

Differential synergistic effects of palbociclib and doxorubicin on doxorubicin-resistant cancer cells with diverse tumor origins

Palbociclib is a dual inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6). Palbociclib has frequently been studied in breast cancer cells and has also been linked to function of P-glycoprotein (P-gp), main protein responsible for cancer drug resistance. However, the effect of Palbociclib on cancer drug resistance and specifically doxorubicin-resistant cells overexpressing P-gp have limitedly been studied in the literature. Here, we aimed to decipher the possible synergistic effects of Palbociclib and Doxorubicin combination treatment in doxorubicin-resistant not only breast cancer, which has restrictedly been studied previously, but leukemia and cervical cancer cell lines in the presence of sensitive counterparts to totally explore the mechanistic properties of the Palbociclib in cancer drug resistance. Our results underlined that Palbociclib differentially displayed synergistic effect with doxorubicin in a cell type-specific manner and increased the efficacy of Doxorubicin in Doxorubicin-resistant cells. As a monotherapy, palbociclib has been shown to decrease the expression of MDR-1 in doxorubicin-resistant cells, and when used in combination with doxorubicin, it has been shown to increase the accumulation of doxorubicin in the cell and consequently induce apoptosis. This is the first report that proposes the Palbociclib as a candidate for combination therapy to limit the Doxorubicin resistance in different cancer origins in clinics.

Effect of Melatonin on Chemoresistance Exhibited by Spheres Derived from Canine Mammary Carcinoma Cells.

Mammary cancer is a frequent disease in female dogs, where a high proportion of cases correspond to malignant tumors that may exhibit drug resistance. Within the mammary tumor microenvironment, there is a cell subpopulation called cancer stem cells (CSCs), which are capable of forming spheres in vitro and resisting anti-tumor treatments, partly explaining the recurrence of some tumors. Previously, it has been described that spheres derived from canine mammary carcinoma cells CF41.Mg and REM 134 exhibit stemness characteristics. Melatonin has shown anti-tumor effects on mammary tumor cells; however, its effects have been poorly evaluated in canine mammary CSCs. This study aimed to analyze the effect of melatonin on the chemoresistance exhibited by stem-like neoplastic cells derived from canine mammary carcinoma to cytotoxic drugs such as doxorubicin and mitoxantrone. CF41.Mg and REM 134 cells were cultured in high-glucose DMEM supplemented with fetal bovine serum and L-glutamine. The spheres were cultured in ultra-low attachment plates in DMEM/F12 medium without fetal bovine serum and with different growth factors. The CD44+/CD24-/low phenotype was analyzed by flow cytometry. The viability of sphere-derived cells (MTS reduction) was studied in the presence of melatonin (0.1 or 1 mM), doxorubicin, mitoxantrone, and luzindole. In addition, the gene (RT-qPCR) of the multidrug resistance bombs MDR1 and ABCG2 were analyzed in the presence of melatonin. Both cell types expressed the MT1 gene, which encodes the melatonin receptor MT1. Melatonin 1 mM does not modify the CD44+/CD24-/low phenotype; however, the hormone reduced viability (p < 0.0001) only in CF41.Mg spheres, without inducing an additive effect when co-incubated with cytotoxic drugs. These effects were independent of the binding of the hormone to its receptor MT1, since, by pharmacologically inhibiting them, the effect of melatonin was not blocked. In CF41.Mg spheres, the relative gene expression of ABCG2 and MDR1 was decreased in response to the hormone (p < 0.001). These results indicate that melatonin negatively modulates the cell survival of spheres derived from CF41.Mg cells, in a way that is independent of its MT1 receptor. These effects did not counteract the resistance to doxorubicin and mitoxantrone, even though the hormone negatively regulates the gene expression of MDR1 and ABCG2.

Valproate modulates the activity of multidrug resistance efflux pumps, as a chemoresistance factor in gastric cancer cells.

Drug resistance is one of the most critical problems in gastric cancer therapy. This study was performed to investigate the valproic acid effects on the proliferation of sensitive and resistant cell lines of human gastric cancer, and to explore the mechanism of the agent on multi drug resistance and apoptosis genes. The cytotoxicity effect of valproic acid on the EPG85.257 and EPG85.257RDB cells was assessed by the MTT assay, and the IC50 concentration was evaluated. Apoptosis, genotoxicity, and drug resistance pump activity were evaluated using comet assay, Real-time PCR, and flow cytometry, respectively. Cell proliferation was assayed using a scratch test. Dose-dependent toxicity was recorded after treatment of cells with valproic acid. Valproic acid represented a significant growth inhibition on EPG85.257 cells with IC50 values of 5.84 µM and 4.78 µM after 48 h and 72 h treatment, respectively. In contrast, the drug-resistant counterpart represented 8.7 µM and 7.02 µM IC50 values after the same treatment time. Valproic acid induced PTEN, Bcl2, P53, Bax, P21, and caspase3 expression in EPG85.257 cells, whereas p21, p53, PTEN, and ABCB1 were overexpressed in EPG5.257RDB. Valproic acid hindered cell migration in both cell lines (P < 0.01). Valproate genotoxicity was significantly higher in the parent cells than in their resistant EPG85.257RDB counterparts. Valproate led to a 62% reduction in the daunorubicin efflux of the MDR1 pump activity. Valproate can affect drug resistance in gastric cancer via a unique mechanism independent of MDR1 expression.

Up-regulation of CDR1 and MDR1 efflux pump genes and fluconazole resistance are involved in recurrence in Candida albicans-induced vulvovaginal candidiasis

Recurrent vulvovaginal candidiasis (RVVC) due to fluconazole resistance in Candida albicans isolates causes a wide range of complications. A number of 63 Candida albicans isolates obtained from vulvovaginal candidiasis (VVC) were identified by Internal Transcribed Spacer-Restriction Fragment Length Polymorphism (ITS-RFLP). Antifungal susceptibility testing was performed by broth microdilution method according to the CLSI protocol. The role of CDR1 and MDR1 genes in progress of VVC to RVVC was examined and the activity of virulence-related enzymes was assessed. Candida albicans was diagnosed in 62.4 % cases, of which 22.2 % were confirmed as RVVC. Voriconazole was the most active drug among five tested antifungals. The mean expression level of CDR1 and MDR1 was higher in RVVC isolates compared to multidrug azole-resistant VVC isolates. Our results demonstrated that the expression of CDR1 and MDR1 and the level of phospholipase and proteinase activities could be quite important to induce fluconazole resistance in C. albicans and to progress of VVC to become RVVC in involved patients.

Pan-azole-resistant Meyerozyma guilliermondii clonal isolates harbouring a double F126L and L505F mutation in Erg11.

Meyerozyma guilliermondii is a yeast species responsible for invasive fungal infections. It has high minimum inhibitory concentrations (MICs) to echinocandins, the first-line treatment of candidemia. In this context, azole antifungal agents are frequently used. However, in recent years, a number of azole-resistant strains have been described. Their mechanisms of resistance are currently poorly studied. The aim of this study was consequently to understand the mechanisms of azole resistance in several clinical isolates of M. guilliermondii. Ten isolates of M. guilliermondii and the ATCC 6260 reference strain were studied. MICs of azoles were determined first. Whole genome sequencing of the isolates was then carried out and the mutations identified in ERG11 were expressed in a CTG clade yeast model (C. lusitaniae). RNA expression of ERG11, MDR1 and CDR1 was evaluated by quantitative PCR. A phylogenic analysis was developed and performed on M. guilliermondii isolates. Lastly, invitro experiments on fitness cost and virulence were carried out. Of the ten isolates tested, three showed pan-azole resistance. A combination of F126L and L505F mutations in Erg11 was highlighted in these three isolates. Interestingly, a combination of these two mutations was necessary to confer azole resistance. An overexpression of the Cdr1 efflux pump was also evidenced in one strain. Moreover, the three pan-azole-resistant isolates were shown to be genetically related and not associated with a fitness cost or a lower virulence, suggesting a possible clonal transmission. In conclusion, this study identified an original combination of ERG11 mutations responsible for pan-azole-resistance in M. guilliermondii. Moreover, we proposed a new MLST analysis for M. guilliermondii that identified possible clonal transmission of pan-azole-resistant strains. Future studies are needed to investigate the distribution of this clone in hospital environment and should lead to the reconsideration of the treatment for this species.

EGR1 mediates MDR1 transcriptional activity regulating gemcitabine resistance in pancreatic cancer

BackgroundGemcitabine is a cornerstone drug for the treatment of all stages of pancreatic cancer and can prolong the survival of patients with pancreatic cancer, but resistance to gemcitabine in pancreatic cancer patients hinders its efficacy. The overexpression of Early growth response 1(EGR1) in pancreatic ductal adenocarcinoma as a mechanism of gemcitabine chemoresistance in pancreatic cancer has not been explored. The major mechanisms of gemcitabine chemoresistance are related to drug uptake, metabolism, and action. One of the common causes of tumor multidrug resistance (MDR) to chemotherapy in cancer cells is that transporter proteins increase intracellular drug efflux and decrease drug concentrations by inducing anti-apoptotic mechanisms. It has been reported that gemcitabine binds to MDR1 with high affinity. The purpose of this research was to investigate the potential mechanisms by which EGR1 associates with MDR1 to regulate gemcitabine resistance in pancreatic cancer cells.MethodsThe following in vitro and in vivo techniques were used in this research to explore the potential mechanisms by which EGR1 binds to MDR1 to regulate gemcitabine resistance in pancreatic cancer cells. Cell culture; in vitro and in vivo study of EGR1 function by loss of function analysis. Binding of EGR1 to the MDR1 promoter was detected using the ChIP assay. qRT-PCR, Western blot assays to detect protein and mRNA expression; use of Annexin V apoptosis detection assay to test apoptosis; CCK8, Edu assay to test cell proliferation viability. The animal model of pancreatic cancer subcutaneous allograft was constructed and the tumours were stained with hematoxylin eosin and Ki-67 expression was detected using immunohistochemistry.FindingsWe revealed that EGR1 expression was increased in different pancreatic cancer cell lines compared to normal pancreatic ductal epithelial cells. Moreover, gemcitabine treatment induced upregulation of EGR1 expression in a dose- and time-dependent manner. EGR1 is significantly enriched in the MDR1 promoter sequence.Upon knockdown of EGR1, cell proliferation was impaired in CFPAC-1 and PANC-1 cell lines, apoptosis was enhanced and MDR1 expression was decreased, thereby partially reversing gemcitabine chemoresistance. In animal experiments, knockdown of EGR1 enhanced the inhibitory effect of gemcitabine on tumor growth compared with the sh-NC group.ConclusionsOur study suggests that EGR1 may be involved in the regulation of MDR1 to enhance gemcitabine resistance in pancreatic cancer cells. EGR1 could be a novel therapeutic target to overcome gemcitabine resistance in pancreatic cancer.

Inhibition of the NMDA Currents by Probenecid in Amygdaloid Kindling Epilepsy Model.

Epilepsy is characterized by a sustained depolarization and repeated discharge of neurons, attributed to overstimulation of N-methyl-D-aspartate receptors (NMDAr). Herein, we propose that probenecid (PROB), an inhibitor of the activity of some ATP binding-cassette transporters (ABC-transporters) can modify NMDAr activity and expression in amygdaloid kindled model. Some studies have suggested that NMDAr expression could be regulated by inhibiting the activity of P-glycoprotein (MDR1) and drug resistance protein-1 (MRP1). Besides, PROB was found to interact with other proteins with proven activity in the kindling model, such as TRPV2 channels, OAT1, and Panx1. Administering PROB at two doses (100 and 300mg/kg/d) for 5 d decreased after-discharge duration and Racine behavioral scores. It also reduced the expression of NR2B and the activity of total NOS and the expression of nNOS with respect to the kindling group. In a second protocol, voltage-clamp measurements of NMDA-evoked currents were performed in CA1 hippocampal cells dissociated from control and kindled rats. PROB produced a dose-dependent reduction in NMDA-evoked currents. In neurons from kindled rats, a residual NMDA-evoked current was registered with respect to control animals, while a reduction in NMDA-evoked currents was observed in the presence of 20mM PROB. Finally, we evaluated the expression of MRP1 and MDR1 in order to establish a relationship between the reduction of kindling parameters, the inhibition of NMDA-type currents, and the expression of these transporters. Based on our results, we conclude that at the concentrations used, PROB inhibits currents evoked by NMDA in dissociated neurons of control and kindled rats. In the kindling model, at the tested doses, PROB decreases the after-discharge duration and Racine behavioral score in the kindling model. We propose a mechanism that could be dependent on the expression of ABC-type transporters.

Gastric cancer mesenchymal stem cells promote tumor glycolysis and chemoresistance by regulating B7H3 in gastric cancer cells.

Despite surgical treatment combined with multidrug therapy having made some progress, chemotherapy resistance is the main cause of recurrence and death of gastric cancer (GC).Gastric cancer mesenchymal stem cells (GCMSCs)have been reported to be correlated with the limited efficacy of chemotherapy in GC, but the mechanism of GCMSCs regulating GC resistance needs to be further studied. The gene set enrichment analysis (GSEA)was performed to explore the glycolysis-related pathways heterogeneity across different cell subpopulations. Glucose uptake and lactate production assays were used to evaluate the importance of B7H3 expression in GCMSCs-treated GCcells. The therapeutic efficacy of oxaliplatin (OXA)and paclitaxel (PTX)was determined using CCK-8and colony formation assays. Signaling pathways altered by GCMSCs-CMwere revealed by immunoblotting. The expression of TNF-αin GCMSCs and bone marrow mesenchymal stem cells (BMMSCs)was detected by western blot analysis and qPCR. Our results showed that the OXA and PTX resistance of GC cells were significantly enhanced in the GCMSCs-CMtreated GC cells. Acquired OXA and PTX resistance was characterized by increased cell viability for OXA and PTX, the formation of cell colonies, and decreased levels of cell apoptosis, which were accompanied by reduced levels of cleaved caspase-3 and Bax expression, and increased levels of Bcl-2, HK2, MDR1, and B7H3 expression. Blocking TNF-αin GCMSCs-CM,B7H3 knockdown or the use of 2-DG,a key enzyme inhibitor of glycolysis in GC cells suppressed the OXA and PTX resistance of GC cells that had been treated with GCMSCs-CM. This study shows that GCMSCs-CMderived TNF-α could upregulate the expression of B7H3 of GC cells to promote tumor chemoresistance. Our results provide a new basis for the treatment of GC.

Timosaponin AⅢ induces drug-metabolizing enzymes by activating constitutive androstane receptor (CAR) via dephosphorylation of the EGFR signaling pathway.

The current study aimed to assess the effect of timosaponin AⅢ (T-AⅢ) on drug-metabolizing enzymes during anticancer therapy. The in vivo experiments were conducted on nude and ICR mice. Following a 24-day administration of T-AⅢ, the nude mice exhibited an induction of CYP2B10, MDR1, and CYP3A11 expression in the liver tissues. In the ICR mice, the expression levels of CYP2B10 and MDR1 increased after a three-day T-AⅢ administration. The in vitro assessments with HepG2 cells revealed that T-AⅢ induced the expression of CYP2B6, MDR1, and CYP3A4, along with constitutive androstane receptor (CAR) activation. Treatment with CAR siRNA reversed the T-AⅢ-induced increases in CYP2B6 and CYP3A4 expression. Furthermore, other CAR target genes also showed a significant increase in the expression. The up-regulation of murine CAR was observed in the liver tissues of both nude and ICR mice. Subsequent findings demonstrated that T-AⅢ activated CAR by inhibiting ERK1/2 phosphorylation, with this effect being partially reversed by the ERK activator t-BHQ. Inhibition of the ERK1/2 signaling pathway was also observed in vivo. Additionally, T-AⅢinhibited the phosphorylation of EGFR at Tyr1173 and Tyr845, and suppressed EGF-induced phosphorylation of EGFR, ERK, and CAR. In the nude mice, T-AⅢ also inhibited EGFR phosphorylation. These results collectively indicate that T-AⅢ is a novel CAR activator through inhibition of the EGFR pathway.

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

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

PYGO2-MDR1 Axis in Multiple Myeloma Patients with 1q21 Amplification As Promising Target to Overcome Carfilzomib Resistance

Multiple myeloma (MM) is a hematological malignancy characterized by an accumulation of clonal plasma cells (PCs) in the bone marrow (BM). Gain and/or amplification of 1q21 (1q21+) is one of the most frequent secondary cytogenetic abnormalities present in MM patients and in smoldering MM (SMM). The incidence and copy number alteration (CNA) of the 1q21 region increase during the progression and relapse of the disease; in fact, additional copies of 1q21 can be detected in around 40% of newly diagnosed MM (NDMM) and 70% of relapse-refractory MM (RRMM). Recently, 1q21+ has been identified as an adverse prognostic factor in MM patients and its presence has been correlated with a shorter duration of the response in MM patients treated with carfilzomib (CFZ)-based regimens. Thus, the identification of possible target genes in 1q21 region is an emerging unmet medical need in MM patients. PYGOPUS2 (PYGO2) is a gene located in the 1q21 chromosomal region and it is a downstream target of β-catenin, and it has been shown to promote the transcription of gene target of the Wnt-signaling, forming a nuclear complex with β-catenin/BCL9. Different studies have reported that upregulation of PYGO2 is involved in tumorigenesis in breast and lung cancers, showing that overexpression of PYGO2 leads to drug resistance by promoting the activation of the multidrug resistance 1 polypeptide ( MDR1). The expression profile of PYGO2 in1q21+ MM patients and its possible role in CFZ resistance is still unknown and have been investigated in this study. Firstly, we purified CD138 + PCs from BM samples from 18 NDMM and from 11 SMM patients. Fluorescent in situ hybridization (FISH) analysis was performed on purified CD138 + PCs for all patients to access CNA in the region 1q21. A score indicating the number of 1q21 copies was calculated based on the FISH hybridization pattern of each patient. 14/29 (48%) patients presented CNA in the 1q21 region. The expression profile of all 29 samples was generated using GeneChip ClariomD Arrays (Affymetrix Inc., Santa Clara, CA, USA) . The sam package was used to identify differentially expressed genes between 1q21+ and control samples. Our analysis identified PYGO2 to be significantly upregulated in patients with 1q21+ when compared with controls (p= 0.0008). Additionally, we found a positive correlation between gene expression and the 1q21 copy number (p= &amp;lt;0.0001, r= 0.6738). Secondly, we evaluated PYGO2 mRNA expression levels in several human myeloma cell lines (HMCLs) with 1q21 CNA. Our results showed that PYGO2 is overexpressed in HMCLs with 1q21 CNA. Previous studies in solid tumor have shown that PYGO2 is upregulated in drug resistant cancer cells, and this upregulation results in overexpression of MDR1; indeed, we evaluated the mRNA expression of PYGO2 in CFZ-resistant HMCLs. Interestingly our results showed that both PYGO2 and MDR1 expression are upregulated in CFZ-R cells. We next examined the relationship between PYGO2 and MDR1. We generated a PYGO2-knockdown in JJN3 MM cell line using short hairpin RNA (shRNA) lentivectors. Knockdown of PYGO2 resulted in a significant decrease in expression PYGO2, a decrease in cell viability, and a reduction in MDR1 expression. In conclusion, our results show that PYGO2 expression is significantly upregulated in MM patients carrying 1q21+ and that its expression is correlated with 1q21 copy number. Moreover, we found that the PYGO2-MDR1 axis is involved in the CFZ resistance in HMCLs MM cells suggesting that PYGO2 could be a possible future druggable target in 1q21+ MM patients.