Docetaxel-resistant Cells Research Articles

Abstract 4396: Targeting survivin to overcome docetaxel resistance in prostate cancer

Abstract Despite therapeutic advancements, castration-resistant prostate cancer (CRPC) remains the second most common cause of cancer-related mortality in men. Docetaxel is the first cytotoxic agent to show modest improvements in overall survival rate in patients with CRPC. Unfortunately, over half of these patients do not respond to treatment and ultimately all develop resistance. The mechanism mediating docetaxel resistance remains unknown. However, survivin, an inhibitor of apoptosis (IAP) family member, and known mediator of chemo-resistance has been previously associated with docetaxel resistance, as inhibition of survivin expression sensitized prostate cancer cells to docetaxel in vitro, and a small molecule inhibitor targeting survivin expression led to a significant regression in prostate cancer xenograft tumors when given in combination with docetaxel. However, how survivin may mediate docetaxel resistance in prostate cancer remains unknown. In this study, we tested the hypothesis that overexpression of nuclear survivin contributes to docetaxel resistance in prostate cancer cells. First, utilizing western blot to assess protein level and methylene blue assays to determine ability to proliferate under cytotoxic conditions, we found that survivin expression and docetaxel IC50 correlates strongly in five prostate cancer cell lines. Furthermore, using paired parental drug sensitive and stepwise selected docetaxel resistant cell lines, we determined that resistant cells overexpress survivin as compared to parental cells, and knockdown of survivin decreases resistance to docetaxel. Our studies suggest that survivin is likely implicated in CRPC and treatment with a direct survivin inhibitor may sensitize resistant cells to docetaxel. We are in the process of determining how this IAP may mechanistically mediate docetaxel resistance. Citation Format: Robert C. Peery. Targeting survivin to overcome docetaxel resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4396.

Cdc20/p55 mediates the resistance to docetaxel in castration-resistant prostate cancer in a Bim-dependent manner.

At least to date, no effective treatment for advanced castration-resistant prostate cancer (CRPC) has been established. Recent studies indicated that cell division cycle 20 homolog (Cdc20) overexpression is associated with poor prognosis in patients with castration-resistant prostate cancer. However, the mechanism of Cdc20 in the development of docetaxel resistance in CRPC remains elusive. In this study, the transcription of Cdc20 was confirmed in three independent CRPC cell lines derived from different tissues, including LNCaP, PC3, and DU145. Docetaxel resistant (DR) cell lines were generated within the background of DU145 and PC3. The protein levels of Cdc20 and the biological phenotype were detected in both wild-type and DR cell lines. To further explore the mechanism of Cdc20 overexpression, stable cell lines with Cdc20 or Bcl-2 interacting mediator of cell death (Bim) deprivation were generated and examined for biological parameters. In addition, a specific Cdc20 inhibitor was used in DR cell lines to explore the potential solution for docetaxel resistant CRPC. Here, we identified Cdc20 is overexpressed in docetaxel resistant CRPC cell lines, including LNCaP, PC3, and DU145. We also reported that DR cell lines, which mimic the recurrent prostate cancer cells after docetaxel treatment, have higher levels of Cdc20 protein compared with the CRPC cell lines. Interestingly, the protein levels of Bim, an E3 ligase substrate of Cdc20, were decreased in DR cell lines compared with the wild-type, while the mRNA levels were similar. More importantly, in DR cell lines, the biological phenotype induced by Cdc20 deletion could be significantly reversed by the additional knockdown of Bim. As a result, docetaxel resistant prostate cancer cells treated with the pharmacological Cdc20 inhibitor became sensitive to docetaxel treatment. In conclusion, our data collectively demonstrated that Cdc20 overexpression facilitates the docetaxel resistant of the CRPC cell lines in a Bim-dependent manner. Furthermore, additionally targeting Cdc20 might be a promising solution for the treatment of the CRPC with docetaxel resistance.

Abstract A144: Sensitization of docetaxel-resistant breast cancer cells to docetaxel by the VRAC modulator SCO-101

Abstract Introduction: Breast cancer is the most frequent cancer among women worldwide, and the aggressive subtype triple-negative breast cancer (TNBC) comprises 15% of breast cancer cases. Taxanes are a key component of the (neo)adjuvant treatment for TNBC; however, taxane resistance develops in one in five neoadjuvant or adjuvant taxane-treated patients. Resistance to anticancer drugs represents the main cause of cancer-related deaths. Thus, resensitization of taxane-resistant cancer cells constitutes a highly unmet medical need. Materials and Methods: SCO-101 is a modulator of the Volume Regulated Anion Channel (VRAC) complex. It is an oral drug and has previously passed 4 phase I clinical trials where it demonstrated excellent PK (T1/2=15 hours) and only limited toxicity. In this study we used an isogenic pair of parental (sensitive) and docetaxel (taxane)-resistant MDA-MB-231 human TNBC cells (Hansen et al.,Tumor Biol 2015:36:4327-38). Cell viability in vitro during 72h or 120h exposure to SCO-101 and/or docetaxel was addressed by MTT and Presto blue assays and cell counting. Potential targets for SCO-101 were investigated by transient knockdown of LRRC8a (obligatory component of VRAC) followed by cell viability assay, and by studying Pgp-mediated substrate efflux. Results: In parental MDA-MB-231 cells, SCO-101 and docetaxel each had a dose-dependent effect on cell viability and no combinatorial effects were observed between the drugs. When exposing docetaxel-resistant cells to the combination of SCO-101 and docetaxel, a significant inhibitory effect on cell viability compared to either drug alone was observed, suggesting an additive or synergistic effect between these two drugs with SCO-101 resensitizing the resistant cells to docetaxel. By knockdown of LRRC8a no differences in the response to SCO-101 and/or docetaxel were observed, suggesting that the anticancer effects of SCO-101 are not dependent on this obligatory VRAC component. Alternatively, preliminary experimental interrogation of the Pgp drug efflux pump suggests that SCO-101 affects this well-known docetaxel resistance mechanism. Conclusion and Future Perspectives: These preclinical studies demonstrate that SCO-101 sensitizes docetaxel-resistant breast cancer cells to docetaxel through a mechanism not dependent on LRRC8a expression. Currently, xenotransplant models of mice are being established to examine the in vivo inhibitory effects of SCO-101 on tumor growth. Additionally, we are preparing a clinical phase II study enrolling metastatic TNBC patients who have failed prior docetaxel treatment. These patients will be reexposed to the combination of docetaxel and SCO-101. End-point will be objective response rate according to RECIST 1.1. Citation Format: Sofie O. Bagger, Jørgen Drejer, Nils Brünner, Signe L. Nielsen, Palle Christoffersen, Jan Stenvang. Sensitization of docetaxel-resistant breast cancer cells to docetaxel by the VRAC modulator SCO-101 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A144.

MiR-361-5p suppresses chemoresistance of gastric cancer cells by targeting FOXM1 via the PI3K/Akt/mTOR pathway.

Gastric cancer is a prevalent cancer and chemotherapy is a main treatment for patients. Docetaxel is commonly used as a chemotherapeutic drug for gastric cancer patients. With the increasing emergence of docetaxel resistance, exploring the mechanism of chemoresistance may improve prognosis of patients. In this study, we found that overexpressed miR-361-5p suppressed chemoresistance to docetaxel of gastric cancer cells (SGC-7901, MKN-28) by decreasing IC50 values of docetaxel while increasing cell apoptosis rate, especially in docetaxel resistant SGC-7901 cells. Further researches revealed that overexpressed miR-361-5p inhibited chemoresistance through inhibiting autophagy with a characteristic of declined number of LC3+ puncta, decreased expression of Beclin-1 and the ratio of LC3 II/I and increased expression of p62. Bioinformatics study and Luciferase reporter assay indicated that FOXM1 was a target of miR-361-5p and FOXM1 was negatively regulated by miR-361-5p in gastric cancer. Simultaneously, overexpression of FOXM1 counteracted the inhibitory effects of miR-361-5p on chemoresistance of gastric cancer cells through activating autophagy, further certifying the targeting relationship between the two. Moreover, overexpressed miR-361-5p activated the PI3K/Akt/mTOR pathway. The adding of PI3K inhibitor LY294002 played an opposite role to miR-361-5p mimic by inducing autophagy and chemoresistance to docetaxel of gastric cancer cells compared with docetaxel + miR-361-5p mimic group, indicating that miR-361-5p suppressed autophagy-induced chemoresistance via the PI3K/Akt/mTOR pathway in gastric cancer cells. In conclusion, we found that miR-361-5p suppressed autophagy-induced chemoresistance of gastric cancer cells through targeting FOXM1 via the PI3K/Akt/mTOR pathway, providing a foundation for the mechanism research and treatment of gastric cancer.

Resensitization of Akt Induced Docetaxel Resistance in Breast Cancer by \u2018Iturin A\u2019 a Lipopeptide Molecule from Marine Bacteria Bacillus megaterium

Development of the resistance is the major problem in cancer therapy. Docetaxel is a taxol alkaloid that is frequently used in metastatic breast cancer. However, resistance often limits the usefulness of this drug in many breast cancer patients. Manipulation of resistant cells to re-sensitize to the therapeutic effect of docetaxel is current strategy to overcome this problem. Here, we have introduced ‘Iturin A’ as a potent chemosensitizer in docetaxel resistant breast cancer cells. Combination of Iturin A and docetaxel treatment significantly hampered the proliferation of docetaxel resistant MDA-MB-231 and MDA-MB-468 breast cancer cells. Cell cycle analysis also showed massive amount of apoptotic population (Sub G0/G1) in combination therapy. A number of apoptotic and anti-apoptotic proteins were significantly altered in dual drug treated groups. Caspase 3 dependent cell death was observed in dual treatment. Molecular mechanism study showed that over-expression of Akt and its downstream signaling pathway was associated with docetaxel resistance. Iturin A significantly reduced Akt signaling pathway in resistant cells. This mechanistic action might be the reason behind the chemo-sensitization effect of Iturin A in docetaxel resistant breast cancer cells. In conclusion, Iturin A resensitized the resistant breast cancer cells to docetaxel therapy by inhibiting Akt activity.

ABCB1 Mediates Cabazitaxel-Docetaxel Cross-Resistance in Advanced Prostate Cancer.

Advancements in research have added several new therapies for castration-resistant prostate cancer (CRPC), greatly augmenting our ability to treat patients. However, CRPC remains an incurable disease due to the development of therapeutic resistance and the existence of cross-resistance between available therapies. Understanding the interplay between different treatments will lead to improved sequencing and the creation of combinations that overcome resistance and prolong survival. Whether there exists cross-resistance between docetaxel and the next-generation taxane cabazitaxel is poorly understood. In this study, we use C4-2B and DU145 derived docetaxel-resistant cell lines to test response to cabazitaxel. Our results demonstrate that docetaxel resistance confers cross-resistance to cabazitaxel. We show that increased ABCB1 expression is responsible for cross-resistance to cabazitaxel and that inhibition of ABCB1 function through the small-molecule inhibitor elacridar resensitizes taxane-resistant cells to treatment. In addition, the antiandrogens bicalutamide and enzalutamide, previously demonstrated to be able to resensitize taxane-resistant cells to docetaxel through inhibition of ABCB1 ATPase activity, are also able to resensitize resistant cells to cabazitaxel treatment. Finally, we show that resensitization using an antiandrogen is far more effective in combination with cabazitaxel than docetaxel. Collectively, these results address key concerns in the field, including that of cross-resistance between taxanes and highlighting a mechanism of cabazitaxel resistance involving ABCB1. Furthermore, these preclinical studies suggest the potential in using combinations of antiandrogens with cabazitaxel for increased effect in treating advanced CRPC. Mol Cancer Ther; 16(10); 2257-66. ©2017 AACR.

KIF11 is required for proliferation and self-renewal of docetaxel resistant triple negative breast cancer cells.

Development of chemoresistance remains a major hurdle for triple negative breast cancer treatment. Previous studies suggest that CD44+/CD24- cells, subpopulation of cancer stem cells with self-renewing and tumor-initiating capacities, are partly responsible for chemoresistance and therapeutic failure of triple negative breast cancer. Therefore, novel agents that target cancer stem cells (CSCs) may improve the clinical outcome. KIF11 (kinesin family member 11), overexpressed in many cancer cells, is a molecular motor protein that plays essential role in mitosis. In this study, we assess its role in docetaxel resistant triple negative breast cancer (TNBC). We found that the expression of KIF11 was significantly increased in CD44+/CD24- subpopulation of docetaxel resistant TNBC cells. Knockdown of KIF11 resulted in a significant decrease in the percentage of CSCs and mammosphere formation. KIF11 knockdown also inhibits cell growth and induces cell cycle G2/M arrest followed by cell mitosis and apoptosis. Further docetaxel resistant TNBC xenograft models demonstrated that KIF11 inhibitor exerts growth inhibitory effect in vivo. Of note, we also found that KIF11 was highly expressed in TNBC and its expression was correlated with shorter disease free survival time. All these data indicate that KIF11 is critical for proliferation and self-renewal in TNBC tumor cells in vitro and in vivo, suggesting that KIF11 may be a promising therapeutic target for treating chemoresistant TNBC.

Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a common mechanism of disease progression and a prognostic-determinant feature of malignant tumors. In prostate cancer (PC), resistance to MTAs such as the taxane Docetaxel dictates treatment failure as well as progression towards lethal stages of disease that are defined by a poor prognosis and high mortality rates. Though studied for decades, the array of mechanisms contributing to acquired resistance are not completely understood, and thus pose a significant limitation to the development of new therapeutic strategies that could benefit patients in these advanced stages of disease. In this protocol, we describe the generation of Docetaxel-resistant prostate cancer cell lines that mimic lethal features of late-stage prostate cancer, and therefore can be used to study the mechanisms by which acquired chemoresistance arises. Despite potential limitations intrinsic to a cell based model, such as the loss of resistance properties over time, the Docetaxel-resistant cell lines produced by this method have been successfully used in recent studies and offer the opportunity to advance our molecular understanding of acquired chemoresistance in lethal prostate cancer.

Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel.

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a common mechanism of disease progression and a prognostic-determinant feature of malignant tumors. In prostate cancer (PC), resistance to MTAs such as the taxane Docetaxel dictates treatment failure as well as progression towards lethal stages of disease that are defined by a poor prognosis and high mortality rates. Though studied for decades, the array of mechanisms contributing to acquired resistance are not completely understood, and thus pose a significant limitation to the development of new therapeutic strategies that could benefit patients in these advanced stages of disease. In this protocol, we describe the generation of Docetaxel-resistant prostate cancer cell lines that mimic lethal features of late-stage prostate cancer, and therefore can be used to study the mechanisms by which acquired chemoresistance arises. Despite potential limitations intrinsic to a cell based model, such as the loss of resistance properties over time, the Docetaxel-resistant cell lines produced by this method have been successfully used in recent studies and offer the opportunity to advance our molecular understanding of acquired chemoresistance in lethal prostate cancer.

Ginger Phytochemicals Inhibit Cell Growth and Modulate Drug Resistance Factors in Docetaxel Resistant Prostate Cancer Cell.

Ginger has many bioactive compounds with pharmacological activities. However, few studies are known about these bioactive compounds activity in chemoresistant cells. The aim of the present study was to investigate the anticancer properties of ginger phytochemicals in docetaxel-resistant human prostate cancer cells in vitro. In this study, we isolated 6-gingerol, 10-gingerol, 4-shogaol, 6-shogaol, 10-shogaol, and 6-dehydrogingerdione from ginger. Further, the antiproliferation activity of these compounds was examined in docetaxel-resistant (PC3R) and sensitive (PC3) human prostate cancer cell lines. 6-gingerol, 10-gingerol, 6-shogaol, and 10-shogaol at the concentration of 100 μM significantly inhibited the proliferation in PC3R but 6-gingerol, 6-shogaol, and 10-shogaol displayed similar activity in PC3. The protein expression of multidrug resistance associated protein 1 (MRP1) and glutathione-S-transferase (GSTπ) is higher in PC3R than in PC3. In summary, we isolated the bioactive compounds from ginger. Our results showed that 6-gingerol, 10-gingerol, 6-shogaol, and 10-shogaol inhibit the proliferation of PC3R cells through the downregulation of MRP1 and GSTπ protein expression.

RBPJ contributes to acquired docetaxel resistance in prostate cancer cells

Our previous work has shown that depletion of recombination signal-binding protein J (RBPJ) results in reduced cell growth in prostate cancer cells. In this study, we aimed to investigate the function of RBPJ in the chemoresistance of prostate cancer. The expression of RBPJ was quantified in docetaxel-resistant and parental prostate cancer cells. Loss- and gain-of-function experiments were conducted to explore the regulatory role of RBPJ in prostate cancer sensitivity to docetaxel. The pro-apoptotic effect of RBPJ silencing was checked in docetaxel-resistant prostate cancer cells. We found that docetaxel-resistant PC3-DR and DU145-DR cells expressed 3–5-fold high levels of RBPJ than parental PC3 and DU145 cells. Short hairpin RNA-mediated knockdown of RBPJ inhibited cell proliferation and colony formation and reversed docetaxel resistance in docetaxel-resistant prostate cancer cells. In contrast, overexpression of RBPJ promoted cell growth, colony formation, and docetaxel resistance in parental prostate cancer cells. Downregulation of RBPJ induced apoptosis in docetaxel-resistant cells, which was accompanied by enhanced cleavage of caspase-3. In addition, RBPJ silencing or overexpression markedly modulated the expression of the Bcl-2 family members including Bcl-2, Bcl-xL, Mcl-1, Bax, and Bak. Altogether, RBPJ contributes to acquisition of docetaxel resistance in prostate cancer cells and may thus represent a potential target for overcoming chemotherapeutic resistance in this malignancy.

Elacridar, a third-generation ABCB1 inhibitor, overcomes resistance to docetaxel in non-small cell lung cancer.

Docetaxel is a third-generation chemotherapeutic drug that is widely used in the treatment of patients with non-small cell lung cancer (NSCLC). However, the majority of patients with NSCLC eventually acquire resistance to the treatment. In the present study, the mechanism of acquired resistance to docetaxel treatment in lung cancer cells was investigated. The three NSCLC cell lines, H1299 with wild-type epidermal growth factor receptor (EGFR), EGFR-mutant HCC4006 and HCC827, and experimentally established docetaxel-resistant (DR) cells, H1299-DR, HCC827-DR, and HCC4006-DR were used with stepwise increases in concentrations of docetaxel. It was demonstrated that the established cell lines showed resistance to docetaxel and EGFR-tyrosine kinase inhibitors (TKIs). Molecular analysis revealed that all of the resistant cell lines highly expressed ATP binding cassette subfamily B member 1 (ABCB1), which is also known as P-glycoprotein or MDR1. Furthermore, HCC827-DR and HCC4006-DR cells exhibited a cancer stem cell-like marker and epithelial-to-mesenchymal transition features, respectively. Elacridar (GF120918), a third-generation inhibitor of ABCB1, was able to overcome resistance to docetaxel. Additionally, knockdown of ABCB1 using small interfering RNA (si)-ABCB1 recovered sensitivity to docetaxel. However, elacridar and si-ABCB1 could not recover sensitivity to EGFR-TKIs in established resistant cells. The results of the present study revealed that docetaxel-resistant NSCLC cells also acquired cross-resistance to EGFR-TKI therapy through mechanisms other than ABCB1, that ABCB1 serves an important role in acquired resistance to docetaxel in lung cancer, and that combination therapy with elacridar can overcome ABCB1-mediated docetaxel resistance.

Prostate Tumor Overexpressed-1 (PTOV1) promotes docetaxel-resistance and survival of castration resistant prostate cancer cells.

Metastatic prostate cancer is presently incurable. The oncogenic protein PTOV1, first described in prostate cancer, was reported as overexpressed and significantly correlated with poor survival in numerous tumors. Here, we investigated the role of PTOV1 in prostate cancer survival to docetaxel and self-renewal ability. Transduction of PTOV1 in docetaxel-sensitive Du145 and PC3 cells significantly increased cell survival after docetaxel exposure and induced docetaxel-resistance genes expression (ABCB1, CCNG2 and TUBB2B). In addition, PTOV1 induced prostatospheres formation and self-renewal genes expression (ALDH1A1, LIN28A, MYC and NANOG). In contrast, Du145 and PC3 cells knockdown for PTOV1 significantly accumulated in the G2/M phase, presented a concomitant increased subG1 peak, and cell death by apoptosis. These effects were enhanced in docetaxel-resistant cells. Analyses of tumor datasets show that PTOV1 expression significantly correlated with prostate tumor grade, drug resistance (CCNG2) and self-renewal (ALDH1A1, MYC) markers. These genes are concurrently overexpressed in most metastatic lesions. Metastases also show PTOV1 genomic amplification in significant co-occurrence with docetaxel-resistance and self-renewal genes. Our findings identify PTOV1 as a promoter of docetaxel-resistance and self-renewal characteristics for castration resistant prostate cancer. The concomitant increased expression of PTOV1, ALDH1A1 and CCNG2 in primary tumors, may predict metastasis and bad prognosis.

Abstract 3891: Chemoresistant cancer stem cells undergo gene changes that drive tumor recurrence

Abstract Despite the efficacy of chemotherapy, resistant cells are thought to contribute to tumor recurrence. In vitro models of recurrence must mimic the complexity and heterogeneity of in vivo tumors and provide the longevity needed to capture tumor dormancy following chemotherapy. We have previously described a 3D in vitro model that simulates in vivo tumors containing cancer stem cell niches. Encapsulation of murine renal adenocarcinoma (RENCA) cells in a double layer of agarose that form spherical macrobeads allows for the development of 3D tumor colonies comprised of tumor-initiating and tumor-amplifying cells within the inner agarose matrix. In the current study, we used the RENCA macrobead in combination with chemotherapy to evaluate genetic regulation that allows tumor dormancy and drives recurrence. RENCA macrobeads treated with docetaxel (5 μg/ml) undergo a significant loss of cell mass and a period of latency followed by tumor recurrence. RNA isolated from docetaxel-resistant cells (Wk.6 post-treatment), recurrent tumors (Wk.20 post-treatment) and paired vehicle controls were characterized using whole-transcriptome profiling (GeneChip® WT Pico Kit with Transciptome Analysis Console; Affymetrix). At Wk.6 post-treatment, genes associated with a stem cell phenotype (Car12, Arrb1, Nos2, Aldh1l2) and members of the ABC1 family associated with multidrug resistance were enriched in docetaxel-resistant cells. Genes regulating the canonical Wnt signaling pathway, specifically LRP family members, were significantly up regulated. Pathway analysis revealed dysregulated focal adhesion-PI3K-Akt-mTor-signaling, including genes modulating integrin adhesion (Itga5, Itga7), ECM receptor interaction (Lama5, Lamb2, Lamc1) and actin cytoskeletal remodeling (Vcl). Supporting the quiescent nature of the docetaxel-resistant population, genes implicated in DNA replication (Dbf4, Cdc6, Mcm5), transcriptional initiation (Taf13, Gtf2h1, Polr2i) and tumor progression (Mmp3, Mmp13) were significantly down regulated. At Wk.20 post-treatment, up regulation of matrix metalloproteinases (Mmp13) and partial retention of stem cell (Nos2, Car12) and drug resistant (Abcb1) genes supports the evolution of regenerated tumors to a metastatic phenotype. Genes involved in fatty acid β-oxidation (Acsl5, Lipf), oxidative phosphorylation (ND3, ND6) and the electron transport chain (COX2) were down regulated in concert with up regulation of Me2 suggesting reduced flux through the TCA cycle and use of aerobic glycolysis in rapidly growing cells. This study further supports the RENCA macrobead as a model system to evaluate the biology of chemotherapy-resistant cells and tumor recurrence in a 3D microenvironment. These data are in line with the notion that chemotherapy-resistant cells exhibit a period of quiescence with a stem cell-like gene profile and experience an intrinsic transformation towards an invasive phenotype that drives tumor recurrence. Citation Format: Prithy C. Martis, Atira Dudley, Melissa A. Laramore, Hunter L. Gazda, Michael P. Markey, Barry H. Smith, Lawrence S. Gazda. Chemoresistant cancer stem cells undergo gene changes that drive tumor recurrence [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 3891. doi:10.1158/1538-7445.AM2017-3891

Abstract 4590: Abciximab manifests striking anti-tumor effects in sensitive and chemoresistant ovarian cancer cells

Abstract Introduction: We have recently reported myeloperoxidase (MPO) to be expressed in epithelial ovarian cancer (EOC) cells and tissues. This finding was surprising, as MPO is known to be expressed only in cells of myeloid origin. We have also found that targeting β2 integrin (CD11b/CD18), a known ligand for MPO, resulted in significant anti-tumor effects. The objective of this study was to determine if targeting CD11b with a clinically approved drug that cross-reacts with CD11b will have anti-tumor efficacy for the treatment of sensitive and chemoresistant ovarian cancer. Methods: Human EOC cell lines MDAH-2774, SKOV-3, and A2780 and their chemoresistant counterparts were utilized for this study. Cells were treated for 24 hrs with increasing doses of docetaxel (0.0075, 0.01, and 0.025 μM) or cisplatin (0.1, 0.5 and 1.0 μM) with our without abciximab (5 or 10 μg/ml), an existing clinically approved anticoagulant drug. Abciximab is a human-murine chimeric antibody Fab fragment against platelet integrin glycoprotein IIb/IIIa (GP IIb/IIIa). Cytotoxicity was determined by the TACS MTT Cell Proliferation Assay. Data was analyzed using SPSS and significant cytotoxic effects were determined by one-way analysis of variance within groups (sensitive, resistant to docetaxel or cisplatin) followed by Tukey’s post hoc tests, and independent t-tests for comparison between groups. For the synergistic effect of abciximab and chemotherapy, the automated calculation of combination index values was conducted by CompuSyn software. A combination index method provides qualitative information on the nature of compound interaction (antagonistic, additive or synergistic effect) and was used to analyze the results. Statistical significance of p<0.05 was considered significant for all analyses. Results: Abciximab had significant cytotoxic effects in sensitive (40.8 ± 10.3 and 56.7 ± 5.3%), docetaxel resistant (30.0 ± 11.6 and 46.6 ± 10.0%), and cisplatin resistant (27.3 ± 7.9 and 41.9 ±7.0%) EOC cells at the 5 and 10 μg/ml dose, respectively, as compared to untreated controls (p<0.01). Abciximab is significantly more cytotoxic to sensitive as compared to cisplatin resistant cells (p<0.01), with no significant difference in killing as compared to docetaxel resistant cells (p=0.057). Treatment with chemotherapy combined with abciximab resulted in a synergist cytotoxic effect in all sensitive EOC cell lines, and in chemoresistant MDAH-2774 and SKOV-3 cell lines. For the cisplatin resistant A2780 cell line, synergist effects were only observed when combining abciximab with 1.0 μM cisplatin. Conclusions: The observed intriguing anti-tumor effects of abcixiamb in ovarian cancer cells indicates the potential for abciximab to be repurposed as a novel therapy for ovarian cancer. Citation Format: Ghassan M. Saed, Nicole M. Fletcher, Ira Memaj. Abciximab manifests striking anti-tumor effects in sensitive and chemoresistant ovarian cancer cells [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 4590. doi:10.1158/1538-7445.AM2017-4590

Abstract 3186: Taxane resistance in prostate cancer: A role for miRNA 181a

Abstract Introduction: Docetaxel (DTX) is one of the primary drugs used for treating castration resistant prostate cancer (CRPC). Unfortunately, over time patients invariably develop resistance to DTX therapy and their disease will continue to progress. The mechanisms by which resistance develops are still incompletely understood. This study seeks to determine the involvement of miRNAs, specifically miR-181a, in DTX resistance in CRPC. Methods: Total RNA from parental C4-2B prostate cancer cells and DTX resistant C4-2B cells (C4-2B TaxR) was submitted for small RNA deep sequencing. Data was analyzed to ascertain which miRNAs expressions were most altered in C4-2B TaxR cells compared to parental cells. Having identified an increase in miR-181a in resistant cells, its expression was modulated in C4-2B and C4-2B TaxR cells by transfecting them with miR-181a mimics or antisense, respectively. Following transfection, cell number was determined after 48 h with or without DTX. Cross resistance to cabazitaxel induced by miR-181a was also determined. Western blots were used to determine ABCB1 protein expression and rhodamine assays used to assess activity. Phospho-p53 expression was assessed by western blot and apoptosis was measured by ELISA in C4-2B TaxR cells with inhibited miR-181a expression with or without DTX. Results: miR-181a is significantly upregulated in C4-2B TaxR cells compared to parental C4-2B cells as analyzed by small RNA sequencing. Overexpression of miR-181a in C4-2B cells confers DTX and cabazitaxel resistance. Knockdown of miR-181a in C4-2B TaxR cells re-sensitizes them to treatment with both DTX and cabazitaxel. miR-181a knockdown alone induced apoptosis in C4-2B TaxR cells which is further enhanced by DTX. We next assessed if miR-181a altered expression or activity of ABCB1, which is overexpressed/active in C4-2B TaxR cells and promotes resistance to DTX by pumping the drug out of cells. We found that miR-181a does not impact ABCB1 expression or activity. Since we previously demonstrated that phospho-p53 can modulate DTX sensitivity, we determined if miR-181a can alter p53 expression in C4-2B TaxR cells. Knockdown of miR-181a in C4-2B TaxR cells induced phospho-p53 expression, suggesting that miR-181a induced resistance to DTX is mediated by inhibition of phospho-p53 expression. Conclusions: Overexpression of miR-181a in C4-2B TaxR cells contributes to their resistance to DTX and inhibition of miR-181a expression can restore treatment response. This is due, in part, to modulation of p53 phosphorylation and induction of apoptosis. Note: This abstract was not presented at the meeting. Citation Format: Cameron M. Armstrong, Chengfei Liu, Wei Lou, Allen C. Gao. Taxane resistance in prostate cancer: A role for miRNA 181a [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 3186. doi:10.1158/1538-7445.AM2017-3186

Abstract 1130: Targeting the tumor-associated autoantigen alpha-enolase in prostate cancer

Abstract Prostate cancer (PCa) is the most commonly diagnosed cancer and second leading cause of cancer-related deaths in American men. African American (AA) men are more likely to be diagnosed with aggressive PCa at a younger age and twice as likely to die from the disease as European American (EA) men. In order to reduce PCa mortality and its associated racial disparities, there is a critical need to identify and target pathways responsible for PCa aggressiveness. An unexplored target is the plasminogen system, which is key to cancer cell migration and tissue invasion during metastasis. During inflammation, activation of the plasminogen pathway degrades extracellular fibrin networks; however, in the context of cancer, this activation promotes cancer tissue invasion and metastasis. Mounting evidence shows that the glycolytic enzyme alpha-enolase (ENO1) plays a vital role in both increased energy metabolism and plasminogen activation during cancer progression and metastasis. Using immunoseroproteomic profiling of anti-tumor autoantibody responses in AA and EA men with PCa, we identified several tumor-associated autoantigens with functions in glycolysis and plasminogen signaling, including ENO1, annexin A2, fructose bisphosphate aldolase, glucose-regulated protein 78, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. In a cohort of PCa (N=157) and non-PCa sera (N=183), we found a significantly higher frequency of autoantibodies to ENO1 in patients with PCa (p<0.05). Surprisingly, when we probed a panel of non-PCa and PCa cell lines with anti-ENO1 positive sera from AA and EA patients with PCa, we observed that AA patients showed increased immunoreactivity to this protein in metastatic PCa cells, with decreased reactivity in docetaxel-resistant cell lines. By contrast, the anti-ENO1 EA-PCa sera and a monoclonal mouse anti-ENO1 showed relatively uniform immunoreactivity across the same panel cell lines. This suggested that the AA-PCa patients are producing antibodies to a different ENO1 variant than EA-PCa patients. Proteomic analysis of post-translational modifications (PTM) in ENO1 showed differences in ENO1 PTM profiles between the parental PC3 bone metastatic cell line and the PC3 docetaxel-resistant cell line, which could explain the differential autoantibody reactivity observed in AA men. Given that ENO1 is up-regulated in PCa tissue, plays a role in glucose metabolism and plasminogen activation during cancer progression, and that the immune system of a cohort of AA-PCa patients recognizes an alternately modified variant of ENO1 that is upregulated in metastatic PCa, we elected to target this protein in metastatic PCa cell lines. ENO1 depletion via siRNA knockdown in PC3 cells led to increased cell death, with concomitant decrease in PCa proliferation. Taken together, these results highlighted the value of immunoproteomics as a tool to identify novel therapeutic targets, with ENO1 as a promising target in metastatic PCa. Citation Format: Tino Wilson Sanchez, Guangyu Zhang, Jitian Li, Liping Dai, Saied Mirshahidi, Nathan R. Wall, Clayton Yates, Colwick Wilson, Susanne Montgomery, Jian-Ying Zhang, Carlos A. Casiano. Targeting the tumor-associated autoantigen alpha-enolase in prostate cancer [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 1130. doi:10.1158/1538-7445.AM2017-1130

MicroRNA-181a promotes docetaxel resistance in prostate cancer cells.

Docetaxel is one of the primary drugs used for treating castration resistant prostate cancer (CRPC). Unfortunately, over time patients invariably develop resistance to docetaxel therapy and their disease will continue to progress. The mechanisms by which resistance develops are still incompletely understood. This study seeks to determine the involvement of miRNAs, specifically miR-181a, in docetaxel resistance in CRPC. Real-time PCR was used to measure miR-181a expression in parental and docetaxel resistant C4-2B and DU145 cells (TaxR and DU145-DTXR). miR-181a expression was modulated in parental or docetaxel resistant cells by transfecting them with miR-181a mimics or antisense, respectively. Following transfection, cell number was determined after 48 h with or without docetaxel. Cross resistance to cabazitaxel induced by miR-181a was also determined. Western blots were used to determine ABCB1 protein expression and rhodamine assays used to assess activity. Phospho-p53 expression was assessed by Western blot and apoptosis was measured by ELISA in C4-2B TaxR and PC3 cells with inhibited or overexpressed miR-181a expression with or without docetaxel. miR-181a is significantly overexpressed in TaxR and DU145-DTXR cells compared to parental cells. Overexpression of miR-181a in parental cells confers docetaxel and cabazitaxel resistance and knockdown of miR-181a in TaxR cells re-sensitizes them to treatment with both docetaxel and cabazitaxel. miR-181a was not observed to impact ABCB1 expression or activity, a protein which was previously demonstrated to be highly involved in docetaxel resistance. Knockdown of miR-181a in TaxR cells induced phospho-p53 expression. Furthermore, miR-181a knockdown alone induced apoptosis in TaxR cells which could be further enhanced by the addition of DTX. Overexpression of mir-181a in prostate cancer cells contributes to their resistance to docetaxel and cabazitaxel and inhibition of mir-181a expression can restore treatment response. This is due, in part, to modulation of p53 phosphorylation and apoptosis.

Calpain and AR-V7: Two potential therapeutic targets to overcome acquired docetaxel resistance in castration-resistant prostate cancer cells.

Docetaxel-based chemotherapy has been widely used as the first-line treatment for castration-resistant prostate cancer (CRPC) patients. However, the mechanisms of docetaxel-resistance remain unclear. In the present study with the establishment of 2 invitro models of docetaxel-resistant CRPC cell sublines, we firstly reported that activation of calpain may play a promotional role in the resistance of docetaxel in prostate cancer, meanwhile using the calpain inhibitor combined with docetaxel improved the efficiency of docetaxel in docetaxel-resistant cell sublines. Moreover, we also found that the expression of androgen-independent constitutively and transcriptionally active androgen receptor splice variant-7(AR-V7) remained high in the docetaxel-resistant CRPC cell subline Rv1-DR, and that it may be involved in acquired docetaxel-resistance of CRPC. However, a novel importin-β inhibitor (importazole) was only capable of slightly decreasing the transcriptional activity of the AR signaling pathway via blocking nuclear import of AR-FL and various non-specific AR-Vs, instead of AR-V7. These findings suggest that calpain and AR-V7 may serve as important biomarkers in the treatment of CRPC, and targeting calpain and AR-V7 may provide a new approach in overcoming docetaxel-resistance.

Microtubule-associated protein tau is associated with the resistance to docetaxel in prostate cancer cell lines.

Tau, a microtubule-associated protein, has been investigated primarily in neurons. Recently, tau has been explored to be associated with increased drug resistance in various kinds of cancers. We found that the tau was expressed in prostate cancer cell lines DU145 and PC-3. We also reported that recurrent prostate cancer cells after docetaxel treatment have higher levels of microtubule-associated protein tau. In vitro, inactivation of tau by gene knockdown suppressed cell proliferation and sensitized docetaxel cytotoxicity. Also, our results demonstrated that the PI3K/Akt/mTOR pathway was upregulated in DU145 docetaxel-resistant cells compared with the DU145-naïve cells. Thus, targeting tau protein and PI3K/Akt/mTOR pathway are promising strategies to enhance docetaxel response for the treatment of prostate cancer.