Induced Paclitaxel Resistance Research Articles

IMPA2 blocks cervical cancer cell apoptosis and induces paclitaxel resistance through p53-mediated AIFM2 regulation.

Cervical cancer continues to be a concern, and the prognosis of locally advanced cervical cancer remains poor. IMPA2 was previously identified as a potential oncogene and regulator of tumor apoptosis. In this study, we aim to further elucidate the underlying mechanisms of IMPA2 gene in the regulation of cervical cancer apoptosis. First, we identify AIFM2 as an upregulated gene in IMPA2-silenced cervical cancer cells, and inhibition of AIFM2 reverses IMPA2 knockdown-induced apoptosis. Further study reveals that AIFM2 regulates cell apoptosis in a mitochondrial-dependent manner with a redistribution of mitochondrial membrane potential and intracellular Ca 2+ levels. However, the analysis of the STRING database and our experimental results show that AIFM2 has little effect on cervical cancer progression and survival. Further mechanistic study demonstrates that IMPA2 and AIFM2 silencing inhibits apoptosis by activating p53. Meanwhile, the knockdown of IMPA2 enhances the chemosensitivity of cervical cancer cells by strengthening paclitaxel-induced apoptosis. Based on the above results, the IMPA2/AIFM2/p53 pathway may be a new molecular mechanism for paclitaxel treatment of cervical cancer and an effective strategy to enhance the sensitivity of cervical cancer cells to paclitaxel. Our findings display a novel function of IMPA2 in regulating cell apoptosis and paclitaxel resistance mediated by a disturbance of AIFM2 and p53 expression, potentially making it a novel therapeutic target for cervical cancer treatment.

Exosomal lncRNA NEAT1 induces paclitaxel resistance in breast cancer cells and promotes cell migration by targeting miR-133b

The lncRNA nuclear paraspeckle assembly transcript 1 (lncRNA NEAT1) has been associated with the development, metastasis and drug resistance of breast cancer (BC). However, the mechanisms underlying NEAT1-induced paclitaxel resistance in the microenvironment of BC remain unclear. In this study, NEAT1 expression was found to be high in paclitaxel-resistant BC cells (SKBR3/PR cells) and exosomes derived from these cells. NEAT1 promoted the migration of BC cells and their resistance to paclitaxel, whereas its downregulation reduced the drug resistance. In addition, downregulation of NEAT1 decreased the migration and proliferation of BC cells by inhibiting the expression of CXCL12 by reducing the adsorption of miR-133b. Furthermore, inhibition of miR-133b reversed the interference of NEAT1 and CXCL12 in paclitaxel resistance, migration and proliferation of BC cells. Knockdown of NEAT1 in a xenograft-bearing mouse model remarkably inhibited cancer progression and improved the response to paclitaxel. Altogether, this study revealed that SKBR3/PR cell-derived exosomal lncRNA NEAT1 can induce paclitaxel resistance and cell migration and growth in the tumour microenvironment of BC and may serve as a new target for the clinical treatment of BC.

Hypoxia-induced paclitaxel resistance in cervical cancer modulated by miR-100 targeting of USP15

ObjectiveHypoxia, which occurs during the development of cervical cancer, confers chemotherapy resistance. MicroRNA expression is regulated by hypoxia and is associated with the onset and progression of certain types of cancer. MicroRNA-100 (miR-100) is a microRNA, associated with nasopharyngeal and oral squamous cell carcinomas, whose expression is decreased in cervical cancer. This study aims to ascertain the effect of hypoxia on expression levels of both miR-100 and its target genes, as well as exploring the sensitivity to paclitaxel under hypoxic conditions. MethodsWe investigated the effect of hypoxia on miR-100 expression. We also explored the regulators of paclitaxel response under hypoxic conditions of cervical cancer. ResultsUsing RT-qPCR, we found that expression of miR-100 in cervical cancer cell lines SiHa and HeLa is significantly higher under hypoxic conditions (1% O2). We also confirmed that human ubiquitin-specific protease 15 (USP15) is the one of the target proteins of miR-100. Hypoxia and overexpression of miR-100 both reduced the activity of the luciferase reporter containing the 3′-untranslated region of USP15, which contains the miR-100 binding site. Furthermore, a western blot analysis showed that hypoxia suppresses the expression of the USP15 protein, while RT-qPCR showed hypoxia-induced downregulation of USP15 mRNA levels. We also discovered that overexpression of miR-100 induces paclitaxel resistance, thereby reducing the drug’s therapeutic effect on cell death. ConclusionsOur results are consistent with the hypothesis that cervical cancer cells overexpress miR-100 in response to hypoxia and that miR-100 is a facilitator of USP15 downregulation and inactivation.

Nogo-B receptor increases glycolysis and the paclitaxel resistance of estrogen receptor-positive breast cancer via the HIF-1α-dependent pathway.

Chemotherapy can improve the prognosis and overall survival of breast cancer patients, but chemoresistance continues a major problem in clinical. Most breast cancer is estrogen receptor (ER) positive but responds less to neoadjuvant or adjuvant chemotherapy than ER-negative breast cancer. The Nogo-B receptor (NgBR) increases the chemoresistance of ER-positive breast cancer by facilitating oncogene signaling pathways. Here, we further investigated the potential role of NgBR as a novel target to overcome glycolysis-dependent paclitaxel resistance in ER-positive breast cancer. NgBR knockdown inhibited glycolysis and promoted paclitaxel-induced apoptosis by attenuating HIF-1α expression in ER-positive breast cancer cells via NgBR-mediated estrogen receptor alpha (ERα)/hypoxia-inducible factor-1 alpha (HIF-1α) and nuclear factor-kappa B subunit (NF-κB)/HIF-1α signaling pathways. A ChIP assay further confirmed that NgBR overexpression not only facilitates ERα binding to HIF-1α and GLUT1 genes but also promotes HIF-1α binding to GLUT1, HK2, and LDHA genes, which further promotes glycolysis and induces paclitaxel resistance. In conclusion, our study suggests that NgBR expression is essential for maintaining the metabolism and paclitaxel resistance of ER-positive breast cancer, and the NgBR can be a new therapeutic target for improving chemoresistance in ER-positive breast cancer.

Generation of Two Paclitaxel-Resistant High-Grade Serous Carcinoma Cell Lines With Increased Expression of P-Glycoprotein.

Debulking surgery followed by chemotherapy are the standard of care for high-grade serous carcinoma. After an initial good response to treatment, the majority of patients relapse with a chemoresistant profile, leading to a poor overall survival. Chemotherapy regimens used in high-grade serous carcinomas are based in a combination of classical chemotherapeutic drugs, namely, Carboplatin and Paclitaxel. The mechanisms underlying drug resistance and new drug discovery are crucial to improve patients’ survival. To uncover the molecular mechanisms of chemoresistance and test drugs capable of overcoming this resistant profile, it is fundamental to use good cellular models capable of mimicking the chemoresistant disease. Herein, we established two high-grade serous carcinoma cell lines with intrinsic resistance to Carboplatin and induced Paclitaxel resistance (OVCAR8 PTX R C and OVCAR8 PTX R P) derived from the OVCAR8 cell line. These two chemoresistant cell line variants acquired an enhanced resistance to Paclitaxel-induced cell death by increasing the drug efflux capacity, and this resistance was stable in long-term culture and following freeze/thaw cycles. The mechanism underlying Paclitaxel resistance resides in a significant increase in P-glycoprotein expression and, when this drug efflux pump was blocked with Verapamil, cells re-acquired Paclitaxel sensitivity. We generated two high-grade serous carcinoma cell lines, with a double-chemoresistant (Carboplatin and Paclitaxel) phenotype that mimics the majority of tumor recurrences in ovarian cancer context. This robust tool is suitable for preliminary drug testing towards the development of therapeutic strategies to overcome chemoresistance.

Paclitaxel exposure downregulates miR-522 expression and its downregulation induces paclitaxel resistance in ovarian cancer cells

Paclitaxel resistance is a critical challenge in ovarian cancer treatment. This study aimed to identify microRNAs (miRNAs) that modulate paclitaxel resistance for use as potential therapeutic targets in such settings. Paclitaxel-resistant cell lines were established using two ovarian cancer cell lines: SKOV3ip1 and HeyA8. The evaluation of miRNA polymerase chain reaction (PCR) arrays indicated that the expression of miR-522-3p was downregulated in paclitaxel-resistant cells. The restoration of miR-522-3p sensitized the resistant cells to paclitaxel, and its downregulation desensitized the parental cells. Using PCR arrays, we focused on E2F2, with the luciferase reporter assay revealing that it was a direct target for miR-522-3p. The paclitaxel-resistant cells showed stronger E2F2 expression than the parental cells, while E2F2 inhibition sensitized the resistant cells to paclitaxel. Forced E2F2 expression in the parental cells led to the acquisition of paclitaxel resistance, while miR-522-3p inhibited E2F2 expression and was associated with retinoblastoma protein phosphorylation attenuation, which resulted in G0/G1 arrest. The effects of miR-522-3p and E2F2 in ovarian cancer were examined using public databases, revealing that low miR-522-3p expression and high E2F2 expression were associated with significantly poorer overall survival. In conclusion, miR-522-3p attenuated the degree of paclitaxel resistance in vitro through the downregulation of E2F2; miR-522-3p supplementation may be a therapeutic target for paclitaxel-resistant ovarian cancer.

Altered Mitochondrial Dynamics, Biogenesis, and Functions in the Paclitaxel-Resistant Lung Adenocarcinoma Cell Line A549/Taxol

BackgroundChemoresistance is a primary hindrance for current cancer treatments. The influence of abnormal mitochondria in chemotherapy resistance is not well known. To explore the correlation between mitochondria and acquired chemoresistance, this work studied alterations in mitochondrial dynamics, biogenesis, and functions for paclitaxel-resistant cancer cell line A549/Taxol and its parental line A549.Material/MethodsMitochondrial morphology was observed by transmission electron microscopy and confocal microscopy. We measured the mitochondrial mass and mitochondrial membrane potential using fluorescent dyes. The glucose metabolic profile and ATP (adenosine triphosphate) content were determined by bioluminescent cell assays. Seahorse bio-energy analyzer XF24 was used to detect the mitochondrial respiratory function. The expressions of mitochondrial dynamics and biogenesis related genes were quantified using real-time polymerase chain reaction.ResultsWe observed fusion morphology of the mitochondrial network in A549/Taxol cells, with upregulation of fusion genes (Mfn1 and Mfn2) and downregulation of fission gene Fis1. In A549/Taxol cells, mitochondrial mass showed a significant decrease, while the mitochondrial biogenesis pathway was strongly activated. Despite the decreased mitochondrial membrane potential, the capability for mitochondrial respiration was not impaired in A549/Taxol cells.ConclusionsOur study revealed a series changes of mitochondrial characteristics in paclitaxel-resistant cells. Mfn1 and Mfn2 and PGC-1α increased, while Fis1 expression and mitochondrial oxidative phosphorylation decreased in A549/Taxol cell lines. These changes to mitochondrial fusion, fission, and biological function contributed to the occurrence of paclitaxel resistance in tumor cells which induced paclitaxel resistance.

EPHB6 mutation induces cell adhesion-mediated paclitaxel resistance via EPHA2 and CDH11 expression

Mutations affect gene functions related to cancer behavior, including cell growth, metastasis, and drug responses. Genome-wide profiling of cancer mutations and drug responses has identified actionable targets that can be utilized for the management of cancer patients. Here, the recapitulation of pharmacogenomic data revealed that the mutation of EPHB6 is associated with paclitaxel resistance in cancer cells. Experimental data confirmed that the EPHB6 mutation induces paclitaxel resistance in various cancer types, including lung, skin, and liver cancers. EPHB6 mutation-induced paclitaxel resistance was mediated by an interaction with EPHA2, which promotes c-Jun N-terminal kinase (JNK)-mediated cadherin 11 (CDH11) expression. We demonstrated that EPHB6-mutated cells acquire cell adhesion-mediated drug resistance (CAM-DR) in association with CDH11 expression and RhoA/focal adhesion kinase (FAK) activation. Targeted inhibition of EPHA2 or CDH11 reversed the acquired paclitaxel resistance, suggesting its potential clinical utility. The present results suggest that the EPHB6 mutation and its downstream EPHA2/JNK/CDH11/RhoA/FAK signaling axis are novel diagnostic and therapeutic targets for overcoming paclitaxel resistance in cancer patients.

Downregulation of miR-194-5p induces paclitaxel resistance in ovarian cancer cells by altering MDM2 expression.

Paclitaxel is a first-line drug for treating epithelial ovarian cancer (EOC). However, prognosis for patients with advanced stage cancer remains poor due to primary or acquired drug resistance. Therefore, overcoming chemoresistance is one of the greatest challenges in treating EOC. In this study, we identified microRNAs (miRNA) that regulate paclitaxel resistance and tested their potential utility as therapeutic targets. Paclitaxel-resistant cell lines were established using two EOC cell lines: SKVO3ip1 and HeyA8. miRNA PCR arrays showed that miR-194-5p was downregulated in paclitaxel-resistant cells. Forced expression of miR-194-5p resensitized resistant cells to paclitaxel. Conversely, miR-194-5p inhibition induced paclitaxel resistance in parental cells. In silico analysis and luciferase reporter assay revealed that MDM2 is a direct target of miR-194-5p. MDM2 was upregulated in paclitaxel resistant cells compared with parental cells. MDM2 inhibition also resensitized resistant cells to paclitaxel and forced MDM2 induced paclitaxel resistance in parental cells. miR-194-5p induced p21 upregulation and G1 phase arrest in resistant cells by downregulating MDM2. Furthermore, a public database showed that high MDM2 expression was associated with a shorter progression-free survival in EOC patients treated with paclitaxel. Collectively, our results show that restoring miR-194-5p expression resensitizes EOCs to paclitaxel, and this may be exploited as a therapeutic option.

Isobavachalcone sensitizes cells to E2-induced paclitaxel resistance by down-regulating CD44 expression in ER+ breast cancer cells.

Oestrogen receptor (ER) is expressed in approximately 60%‐70% of human breast cancer. Clinical trials and retrospective analyses have shown that ER‐positive (ER+) tumours are more tolerant to chemotherapeutic drug resistance than ER‐negative (ER−) tumours. In addition, isobavachalcone (IBC) is known as a kind of phytoestrogen with antitumour effect. However, the underlying mechanism of IBC in ER+ breast cancer needs to be elucidated further. Our in vitro experiments showed that IBC could attenuate 17β‐estradiol (E2)‐induced paclitaxel resistance and that E2 could stimulate CD44 expression in ER+ breast cancer cells but not in ER− cells. Meanwhile, E2 could promote ERα expression to render ER+ breast cancer cells resistant to paclitaxel. Furthermore, we established paclitaxel‐resistant breast cancer cell lines and determined the function of ERα in the enhancement of paclitaxel resistance via the regulation of CD44 transcription. IBC down‐regulated ERα and CD44 expression and thus inhibited tumour growth in paclitaxel‐resistant xenograft models. Overall, our data demonstrated for the first time that IBC could decrease CD44 expression level via the ERα pathway and make ER+ breast cancer cells sensitive to paclitaxel treatment.

Interleukin-22 promotes triple negative breast cancer cells migration and paclitaxel resistance through JAK-STAT3/MAPKs/AKT signaling pathways

PurposeOwing to the absence of any targeted therapies, triple negative breast cancer (TNBC), which accounts for approximately 15% of all breast cancers, typically have a poorer outcome. In an attempt to find out the TNBCs' microenvironment, we ran into the endogenous expression of a newly discovered cytokine known as Interleukin (IL)-22. MethodsThirty TNBC patients were recruited and the levels of IL-22 producing (Th22) cells in tumor, paratumor and normal breast tissues were measured. Then we studied the role and mechanism of IL-22 in migration and paclitaxel resistance in TNBC cells MDA-MB 468 and MDA-MB 231. ResultsThe prevalence of Th22 cells were gradually increased in normal, paratumor and tumor tissues. In vitro, IL-22 promotes TNBC cells migration and induces paclitaxel resistance. Importantly, IL-22 exposure of TNBC cells resulted in JAK-STAT3/MAPKs/AKT signaling pathways activated. ConclusionIL-22 may play an accelerating role in the development of TNBC and may open a new therapeutic approach for TNBC.

Mesenchymal stem cells drive paclitaxel resistance in ErbB2/ErbB3-coexpressing breast cancer cells via paracrine of neuregulin 1

We had previously demonstrated that increased expression of ErbB3 is required for ErbB2-mediated paclitaxel resistance in breast cancer cells. In the present study, we have explored the possible role of mesenchymal stem cells (MSCs) in regulating the paclitaxel-sensitivity of ErbB2/ErbB3-coexpressing breast cancer cells. We show that human umbilical cord-derived MSCs express significantly higher level of neuregulin-1 as compared with ErbB2/ErbB3-coexpressing breast cancer cells themselves. Coculture or treatment with conditioned medium of MSCs not only decreases the anti-proliferation effect of paclitaxel on ErbB2/ErbB3-coexpressing breast cancer cells, but also significantly inhibits paclitaxel-induced apoptosis. We further demonstrate that this MSCs-drived paclitaxel resistance in ErbB2/ErbB3-coexpressing breast cancer cells could be attributed to upregulation of Survivin via paracrine effect of NRG-1/ErbB3/PI-3K/Akt signaling, as either specific knockdown expression of ErbB3, or blocking of downstream PI-3K/Akt signaling, or specific inhibition of Survivin can completely reverse this effect. Moreover, targeted knockdown of NRG-1 expression in MSCs abrogates theirs effect on paclitaxel sensitivity of ErbB2/ErbB3-coexpressing breast cancer cells. Taken together, our study indicate that paracrine of NRG-1 by MSCs induces paclitaxel resistance in ErbB2/ErbB3-coexpressing breast cancer cells through PI-3K/Akt signaling-dependent upregulation of Survivin. Our findings suggest that simultaneously targeting mesenchymal stem cells in tumor microenvironment may be a novel strategy to overcome paclitaxel resistance in patients with ErbB2/ErbB3-coexpressing breast cancer.

Single-cell Transcriptome Analyses Reveal Molecular Signals to Intrinsic and Acquired Paclitaxel Resistance in Esophageal Squamous Cancer Cells

Paclitaxel is widely used in the combination chemotherapy for many cancers including esophageal squamous cell carcinoma (ESCC). However, the paclitaxel resistance occurs frequently in treating ESCC and the mechanism is not fully understood yet. The heterogeneity of gene expression within the drug-resistant cancer cells may be one of the major factors contributing to its resistance. In the present study, we successfully induced paclitaxel resistance in ESCC cell line KYSE-30 through low dose and long-term treatment of paclitaxel. Gene expression profiles were measured utilizing population RNA-seq and single-cell RNA-seq (scRNA-seq). 37 single cells from KYSE-30 cells and 73 single cells from paclitaxel resistant KYSE-30 cells (Taxol-R) were subjected to scRNA-seq. Weighted gene co-expression network analysis (WGCNA) of scRNA-seq data revealed two major subpopulations in both KYSE-30 and Taxol-R cancer cells. Two subpopulations based on the KRT19 expression levels in KYSE-30 cells exhibited different paclitaxel sensitivity, suggesting the existence of an intrinsic paclitaxel resistance in KYSE-30 cells. In addition, the Taxol-R cells that acquired the resistance to paclitaxel through induction were characterized with higher expressions of proteasomes but a lower expression of HIF-1 signaling genes. Furthermore, we showed that carfilzomib (CFZ), a proteasome inhibitor, could attenuate the paclitaxel resistance in Taxol-R cancer cells through activating the HIF-1 signaling. Our new finding may pave a way leading to an improvement in the treatment on cancers including ESCC by combining CFZ with paclitaxel as a novel approach for cancer therapy.

Abstract 3436: MiR-194 modulates paclitaxel resistance in ovarian cancer cells through the regulation of MDM-2 expression

Abstract Purpose: Ovarian cancer is the most lethal gynecologic malignancy. Platinum-paclitaxel combination chemotherapy is the current standard postoperative care for patients with advanced ovarian cancer. Despite developments in current chemotherapy regimens in the last decades, overall 5-year survival rate for patients with advanced disease remains approximately 30%, mainly due to primary or acquired drug resistance. Therefore, overcoming chemoresistance remains the great challenge in ovarian cancer management. However, mechanisms underlying chemoresistance to paclitaxel have not been fully understood. The aim of this study is to identify key miRNAs which regulate paclitaxel resistance in ovarian cancer and to pursue those potential as therapeutic targets. Methods: Using two serous ovarian cancer cell lines, SKVO3ip1 and HeyA8, paclitaxel resistant ovarian cancer cell lines were established by a continuous exposure of paclitaxel. MiRNA PCR arrays were performed and miR-194 was found to be one of down-regulated miRNAs in paclitaxel-resistant cell lines. The effect of miR-194 on paclitaxel resistance was assessed by transducing the precursor miRNA into ovarian cancer cells. In silico analysis revealed MDM-2 is one possible putative target of miR-194 and it was assessed using luciferase reporter assay. Since MDM-2 is one of key regulator of P53 ubiquitination, the effect of miR-194 on cell cycle in ovarian cancer cell was assessed. To analyze the impact of miR-194 and MDM-2 expression on patient survival, Kaplan-Meier and log-rank methods were used using The Cancer Genome Atlas (TCGA) dataset. Results: While IC50 values of SKOV3ip1 and HeyA8 were 5.0 nM and 3.5 nM respectively, those of paclitaxel resistant cell lines, named as SKOV3ip1-TR and HeyA8-TR, were both over 300 nM. In both resistant cell lines, miR-194 was found to be down-regulated compared with their parental cell lines. Up-regulation of miR-194 sensitized resistant cells to paclitaxel. Conversely, its downregulation induced paclitaxel resistance in parental cells. Luciferase reporter assay revealed that miR-194 directly suppressed MDM-2 transcriptional activity. In resistant cell lines, MDM-2 was up-regulated compared with their parental cell lines. MiR-194 induced P21 upregulation and G1 phase arrest in resistant cell lines through the downregulation of MDM-2. In TCGA dataset, high MDM-2 expression was associated with shorter progression-free survival of ovarian cancer patients (concrete data, p=0.046). Conclusion: miR-194 acts as a tumor suppressor miRNA through the sensitization to paclitaxel and can be considered as a therapeutic option to overcome paclitaxel-resistant in ovarian cancer patients. Citation Format: Koji Nakamura, Kenjiro Sawada, Akihiko Yoshimura, Erica Nakatsuka, Yasuto Kinose, Seiji Mabuchi, Tadashi Kimura. MiR-194 modulates paclitaxel resistance in ovarian cancer cells through the regulation of MDM-2 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3436. doi:10.1158/1538-7445.AM2017-3436

Abstract 5894: Stromal fibroblasts from metastatic breast cancer promote proliferation and migration of breast cancer cell and regulate its stemness

Abstract Breast cancer is one of the most common causes of cancer-related death in women in the world. Although most research is focused on the tumor cells, it is important to elucidate the molecular nature of the tumor-stromal relationship to truly understand the biology of breast tumor growth. Cancer-associated fibroblasts (CAFs) are the major cellular constituents in the tumor microenvironment. CAFs are in direct contact with the adjacent cancer cells and crosstalk with them through soluble cytokines, growth factors and exosomes during carcinogenesis in human breast cancer. To determine for the first time the function of CAFs in metastatic sites of breast cancer, we collected CAFs from human patients of metastatic sites (m-CAFs) and compared them with CAFs from primary tumor (p-CAFs) and normal fibroblasts (NFs). We found that m-CAFs expressed higher levels of α-smooth muscle actin (SMA) compared with p-CAFs and NFs. The proliferation of MDA-MB-436 breast cancer cells was significantly increased by treatment with conditioned medium (CM) from m-CAFs compared with p-CAFs in vitro. Also, CM by m-CAFs induced paclitaxel resistance in MDA-MB-436 cells, suggesting that soluble factors produced by m-CAFs promote chemo-resistance, to a greater extent than CM from p-CAFs. The migration and invasion ability of MDA-MB-436 cells co-cultured with m-CAFs CM was significantly greater than that of the p-CAFs CM. m-CAFs outperformed the p-CAFs and NFs in terms of their ability to promote sphere-forming activity in a 3D in vitro culture model. Furthermore, m-CAFs protected MDA-MB-436 cells from the cytotoxic effects of doxorubicin more than p-CAFs in this spheroid 3-D culture. When MDA-MB-436 cells were sorted from these spheroids and grew in the respective CMs, they showed higher expression of the stemness markers of CD44+CD24- compared with cells obtained from p-CAFs/MDA-MB-436 spheroids. Also these cells from the co-culture with m-CAFs experienced with the cadherin switch. Nanog has been demonstrated to promote chemoresistant and epithelial-mesenchymal transition (EMT). The results displayed that MDA-MB-436 cells incubated with the CM from the m-CAFs had a higher expression of Nanog compared with that of p-CAFs. RNA-seq results showed that IGF2 is one of the most different genes between m-CAFs and p-CAFs, its expression was more significant in m-CAFs co-cultured with MDA-MB-436 cells than p-CAFs. The signaling pathway in MDA-MB-436 cells were comparable between co-cultured with m-CAFs CM and the IGF2 stimulation at 2 hours. All these data illustrates that fibroblasts isolated from metastatic site differed in terms of their ability to promote breast cancer cells progression compared to p-CAFs. Our data suggests that m-CAFs are more potent than p-CAFs in inducing the proliferation, migration, drug resistance and cancer stemness of breast cancer cells in an in vitro model. Citation Format: Yirui Gui, Adriana Aguilar-Mahecha, Marguerite Buchanan, Mark Basik. Stromal fibroblasts from metastatic breast cancer promote proliferation and migration of breast cancer cell and regulate its stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5894. doi:10.1158/1538-7445.AM2017-5894

Modulation of HAT activity by the BRCA2 N372H variation is a novel mechanism of paclitaxel resistance in breast cancer cell lines

Paclitaxel stabilizes microtubule polymerization, enhances microtubule assembly, and G2/M arrests, leading to cell death. Paclitaxel resistance has been attributed to a variety of mechanisms. In the present study, we define a new resistance mechanism to paclitaxel based on BRCA2 variation. Chemo-sensitivity to paclitaxel based on the variations was compared. Restoration of paclitaxel sensitivity was induced indirectly with combined treatment of paclitaxel and HDAC inhibitor. Variant and wild type of BRCA2 clones were obtained from wild and variant cells, respectively. Chemo-sensitivity, P/CAF and BubR1 expression and acetylation, BRCA2-P/CAF and BRCA2-BubR1 interactions, and HAT activities of the clones with BRCA2 variation were compared. We identified an association between chemo-sensitivity and BRCA2 N372H variation. The IC50 of paclitaxel in heterozygous variation was higher than that of wild type. There were no differences in basic expression levels of BRCA2 among variant types. However, P/CAF expression, of BRCA2-P/CAF interaction, and HAT activity were significantly lower in heterozygous variants than in the wild type. After HDAC inhibitor treatment, HAT activity and paclitaxel sensitivity were restored in variant cells. Cell lines transformed from wild to variant or from variant to wild showed reciprocal changes in P/CAF expression, BRCA2-P/CAF interaction, HAT activity, and paclitaxel sensitivity. Forced expression of the BRCA2 heterozygous variant induced paclitaxel resistance due to altered HAT activity (p<0.001). This was reversed by the TSA combination. Restoration of wild BRCA2 from variant type improved paclitaxel sensitivity (p<0.001). Modulation of HAT activity by BRCA2 N372H variation is a new mechanism of paclitaxel resistance in breast cancer.

Targeting Smoothened Sensitizes Gastric Cancer to Chemotherapy in Experimental Models

BackgroundThe Hedgehog pathway receptor smoothened (SMO) has critical roles in tumor progression. However, whether SMO is a key factor regulating gastric cancer chemotherapy resistance is unknown.Material/MethodsWe investigated the potential functions of SMO in inducing gastric cancer paclitaxel resistance in clinical samples, gastric cancer cell lines (424GC and AGS), and subcutaneous syngeneic mouse models.ResultsWe found high SMO expression in paclitaxel-resistant gastric cancer clinical samples. Paclitaxel gastric cancer cells had higher SMO expression than in drug-sensitive cells. Upregulating SMO expression induced paclitaxel resistance in gastric cells lines via enhancing cell proliferation and inhibiting apoptosis. The combination of IPI-926, an inhibitor of SMO, with paclitaxel decreased cell viability of paclitaxel-resistant gastric cancer cells in vitro and controlled tumor growth in animal models.ConclusionsThe Hedgehog pathway receptor SMO is an important regulator of gastric cancer paclitaxel resistance and could be a target for sensitizing paclitaxel-resistant tumors.

DUSP1 induces paclitaxel resistance through the regulation of p-glycoprotein expression in human ovarian cancer cells

The heterogeneity and genetic instability of ovarian cancer cells often lead to the development of drug resistance, closely related with the increased cancer-related mortality. In this study, we investigated the role of dual-specificity phosphatase 1 (DUSP1) in the development of the resistance in human ovarian cancer cells against paclitaxel. Overexpression of DUSP1 in HeyA8 human ovarian cancer cells (HeyA8-DUSP1) up-regulated the expression of the drug efflux pump, p-glycoprotein. Consequently, HeyA8-DUSP1 cells are highly resistant to paclitaxel, with the resistance comparable to that of a multi-drug resistance cell line (HeyA8-MDR). Moreover, over expression of DUSP1 significantly increased the activation of p38 MAPK, leaving the activation of ERK1/2 and JNK1/2 unaffected. Pharmacological suppression of p38 MAPK activity prevents the up-regulation of p-glycoprotein expression and the consequent resistance against paclitaxel in HeyA8-DUSP1 cells. By contrast, HeyA8-MDR cells expressed a significantly higher level of DUSP1, but treatment with small interference RNA against DUSP1 significantly suppressed the expression of p-glycoprotein and the resistance against paclitaxel in HeyA8-MDR cells. Ectopic expression of MKK3, an upstream activator of p38 MAPK, significantly up-regulated the expression of p-glycoprotein and increased the consequent resistance against paclitaxel in HeyA8 cells. Collectively, these data indicated that DUSP1 may induce the resistance against paclitaxel through the p38 MAPK-mediated overexpression of p-glycoprotein in human ovarian cancer cells.

Non-adherent culture induces paclitaxel resistance in H460 lung cancer cells via ERK-mediated up-regulation of βIVa-tubulin

Circulating tumor cells (CTCs) are metastasizing epithelial cancer cells that adapt to survive when floating in bloodstream during metastasis. This condition can be mimicked in vitro by using non-adherent cell culture. The chemosensitivity of CTCs appears to correlate with the response of metastatic cancer patients to therapy, but chemoresistance is also frequently observed in advanced stage cancer patients, who have never previously received chemotherapy. We hypothesize that adaptation of epithelial cancer cells to become floating CTCs could lead to development of chemoresistance. Here, we explore whether chemoresistance is induced in epithelial cancer cells when cultured under non-adherent conditions. Increased paclitaxel-specific resistance was observed in floating cells compared to attached cells in H460, MCF-7, and HepG2 human cancer cell lines, by 15.6-, 3.9-, and 2.6-fold increases in IC50 values, respectively. qRT-PCR analysis showed that a paclitaxel-resistant β-tubulin isotype, βIVa-tubulin, was the most up-regulated gene compared with other β-tubulin isotypes in H460 floating cells, concomitant with elevated ERK activation. ERK inhibitor treatment could attenuate the up-regulation of βIVa-tubulin, and decreased the paclitaxel resistance of H460 floating cells, even though other β-tubulin isotypes were up-regulated when the ERK activation was blocked. In conclusion, we show induction of paclitaxel resistance in epithelial cancer cells, when floating in non-adherent culture, and this might occur with CTCs of cancer patients.

Abstract 4378: miR-100 mediates resistance to paclitaxel in cervical cancer cells

Abstract Objectives: Hypoxia occurs during development of cervical cancer and is considered to correlate with its resistance to chemotherapy. We found that hypoxia induced miR-100 whose expression was low in cervical cancer. We sought to determine the regulators of the paclitaxel response during hypoxia in cervical cancer. Methods and Results: We found that the expression of miR-100 was significantly higher under hypoxic condition (1% O2) by realtime RT-PCR. According to the results of in silico analysis, miR-100 targeted human ubiquitin-specific protease (USP) 15. Hypoxia and overexpression of miR-100 respectively decreased the activity of luciferase-reporter containing the 3′-untranslated region (UTR) of USP15 with the predicted miR-100-binding site in cervical cancer cell lines, SiHa and HeLa. By western blot analysis, hypoxia enhanced the endogenous USP15 protein expression. USP15 mRNA level was also up-regulated by hypoxia using realtime RT-PCR. Overexpression of miR-100 induced paclitaxel resistance reducing apoptosis using MTT assays and annexin V staining. Conclusions: Our results provide evidence that regulation of USP15 expression by miR-100 represents a mechanism for paclitaxel resistance in cervical cancer. Our results suggest that miR-100 and USP15 could be a therapeutic target in paclitaxel-resistant cervical cancer. Citation Format: Rina Kato, Hirotaka Nishi, Yuzo Nagamitsu, Toru Sasaki, Keiichi Isaka. miR-100 mediates resistance to paclitaxel in cervical cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4378. doi:10.1158/1538-7445.AM2014-4378