Increased Paclitaxel Sensitivity Research Articles

Abstract P6-10-15: Targeting MARCKS in inflammatory breast cancer increased paclitaxel sensitivity

Abstract Background. Because of its high metastatic potential, inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer. We previously demonstrated that Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) protein overexpression was associated with shorter metastasis-free survival (MFS) in IBC patients, but not in non-IBC (nIBC) patients. However, the mechanism of action of MARCKS and its particular association to poorer outcome in IBC compared to nIBC are poorly understood. Methods. We evaluated in vitro the inhibitory effect of MPS (MARCKS phosphorylation site domain), a peptide targeting MARCKS phosphorylation site domain (PSD) in single and in combination with paclitaxel treatment, on cell proliferation and cell motility in two cell lines of different phenotype (SUM149 for IBC and MDA-MB-231 for nIBC), as well as its distinct molecular mechanisms of action. We also assessed the clinical relevance of the protein expression of MARCKS and phosphatase and tensin homolog (PTEN) in a large series of IBC vs. nIBC patients. Results. In vitro, the treatment with MPS peptide impaired cell proliferation, migration, and invasion in SUM149 compared to MDA-MB-231 cells. More important, MARCKS inhibition increased paclitaxel sensitivity when using combination therapy in SUM149 cells compared to MDA-MB-231 cells. Interestingly, we could partially explain this specific inhibitory effect in IBC cells using western blot: MARCKS inhibition in single and in combination induced up and downregulation of the PTEN/AKT signaling pathway respectively in IBC compared to nIBC cells. Importantly, a negative correlation of MARCKS and PTEN was only found in the clinical IBC samples (180 patients) compared to nIBC samples (355 patients). More importantly, the group of patients with negative MARCKS and positive PTEN protein expression was associated to better 5-year MFS only in IBC patients. Conclusion. These results indicate two major findings: first, the important prognostic value of the negative correlation of MARCKS and PTEN expression in IBC patients, and second the specific role of MARCKS in regulating different downstream pathways and increasing the paclitaxel response in combination treatment in IBC compared to non-IBC. They suggest a potential IBC-specific targetable biomarker, the inhibition of which might impair disease aggressiveness and perhaps enhance patients’ survival. Citation Format: Maroua Manai, Ines Bini, Pascal Finetti, Haifa Bichiou, Carolina Reduzzi, Naziha Ben Hamida, Marc Lopez, Khaled Rahal, Karima Mrad, Mohamed Manai, Massimo Cristofanilli, Hamouda Boussen, Raoudha Doghri, Maher Kharrat, François Bertucci. Targeting MARCKS in inflammatory breast cancer increased paclitaxel sensitivity [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-15.

CHFR and Paclitaxel Sensitivity of Ovarian Cancer.

Simple SummaryStudies in tissue culture cell lines have indicated that silencing of the mitotic regulator CHFR is associated with increased paclitaxel sensitivity. More recently, it has been suggested that a UBC13-DNMT1-CHFR pathway also modulates sensitivity of ovarian cancer to paclitaxel in the clinic. Here we have credentialed an anti-CHFR monoclonal antibody for immunohistochemistry and directly examined the association of CHFR expression with outcome of paclitaxel-containing ovarian cancer therapy. While CHFR levels were higher in high grade, high stage ovarian cancer, after correction for stage and debulking status there was no association between CHFR levels and progression-free survival in high grade serous ovarian cancer treated with surgery followed by platinum/taxane therapy. Moreover, there was no association between CHFR expression and response of patient-derived ovarian cancer xenografts treated with paclitaxel monotherapy. These studies indicate that CHFR varies among ovarian cancers but is unlikely to be an independent biomarker for poor response to taxanes.The poly(ADP-ribose) binding protein CHFR regulates cellular responses to mitotic stress. The deubiquitinase UBC13, which regulates CHFR levels, has been associated with better overall survival in paclitaxel-treated ovarian cancer. Despite the extensive use of taxanes in the treatment of ovarian cancer, little is known about expression of CHFR itself in this disease. In the present study, tissue microarrays containing ovarian carcinoma samples from 417 women who underwent initial surgical debulking were stained with anti-CHFR antibody and scored in a blinded fashion. CHFR levels, expressed as a modified H-score, were examined for association with histology, grade, time to progression (TTP) and overall survival (OS). In addition, patient-derived xenografts from 69 ovarian carcinoma patients were examined for CHFR expression and sensitivity to paclitaxel monotherapy. In clinical ovarian cancer specimens, CHFR expression was positively associated with serous histology (p = 0.0048), higher grade (p = 0.000014) and higher stage (p = 0.016). After correction for stage and debulking, there was no significant association between CHFR staining and overall survival (p = 0.62) or time to progression (p = 0.91) in patients with high grade serous cancers treated with platinum/taxane chemotherapy (N = 249). Likewise, no association between CHFR expression and paclitaxel sensitivity was observed in ovarian cancer PDXs treated with paclitaxel monotherapy. Accordingly, differences in CHFR expression are unlikely to play a major role in paclitaxel sensitivity of high grade serous ovarian cancer.

Effect of stable overexpression of XAF1 gene on biological characteristics of ovarian cancer A2780 cells

To construct an ovarian cancer cell line stably overexpressing XAF1 gene and observe the effects of XAF1 gene overexpression on proliferation, apoptosis, cell cycle and sensitivity to paclitaxel of the cells. Ovarian cancer A2780 cells were transfected with the plasmids pcDNA3.1(+) or pcDNA3.1(+)-XAF1, and the cells stably Over expressing XAF1 (A2780/XAF1 cells) were screened using G418. Cell clone formation assay and CCK8 assay were used to evaluate the changes in proliferation and paclitaxel sensitivity of the transfected cells, and cell cycle and apoptosis of the cells were analyzed using flow cytometry. We successfully obtained A2780/XAF1 cells stably overexpressing XAF1, which exhibited no significant changes in cell morphology. Compared with the negative control cells (A2780/NC), A2780/XAF1 cells had lowered clone formation ability (P=0.0016) and attenuated proliferative activity on the first (P=0.009) and third (P=0.0035) days after cell adherence with also a significantly increased percentage of cells in G2-M phase (P < 0.001). A2780/XAF1 cells showed significantly higher apoptosis rates than A2780/NC cells in the absence of apoptotic stimulation, in serum-free culture or following paclitaxel induction (P < 0.001). The proliferative activity of A2780/XAF1 cells was significantly lower than that of A2780/NC cells after exposure to different paclitaxel concentrations (P < 0.001). The half inhibitory concentration of paclitaxel was significantly lower in A2780/XAF1 than in A2780/NC cells. Overexpression of XAF1 significantly inhibits the proliferation, induces cell cycle arrest, promotes apoptosis, and increases paclitaxel sensitivity in ovarian cancer cells.

Knockout of vasohibin-2 reduces tubulin carboxypeptidase activity and increases paclitaxel sensitivity in ovarian cancer.

Vasohibin‐1 (VASH1) is a VEGF‐inducible endothelium‐derived angiogenesis inhibitor, and vasohibin‐2 (VASH2), its homolog, exhibits proangiogenic activity. VASH2 is expressed by various cancer cells and accelerates tumor angiogenesis and progression. VASH2 was recently shown to exhibit tubulin carboxypeptidase (TCP) activity related to microtubule functions. Paclitaxel (PTX), an effective chemotherapeutic agent that is widely used to treat ovarian cancer, inhibits microtubule depolymerization and may interact with VASH2. We herein established several VASH2 knockout ovarian cancer cell lines using the CRISPR/Cas9 genome editing system to examine the intracellular tubulin detyrosination status and PTX chemosensitivity. The knockout of VASH2 did not affect the proliferation or sphere‐forming activity of ovarian cancer cells in vitro. A Western blot analysis of VASH2 knockout cells revealed the weak expression of detyrosinated tubulin and upregulated expression of cyclin B1. The knockout of VASH2 significantly increased chemosensitivity to PTX, but not to cisplatin in ovarian cancer cell lines. The knockout of VASH2 reduced TCP activity and increased cyclin B1 expression, resulting in increased PTX chemosensitivity in ovarian cancer cells. The inhibition of angiogenesis and regulation of microtubule activity may be achieved in ovarian cancer treatment strategies targeting VASH2.

A Non-canonical Function of BMAL1 Metabolically Limits Obesity-Promoted Triple-Negative Breast Cancer.

SummaryThe epidemiological association between disrupted circadian rhythms and metabolic diseases is implicated in increased risk of human breast cancer and poor therapeutic outcomes. To define a metabolic phenotype and the underlying molecular mechanism, we applied chronic insulin treatment (CIT) to an in vitro model of triple-negative breast cancer to directly address how BMAL1, a key circadian transcription factor, regulates cancer cell respiration and governs tumor progression. At the cellular level, BMAL1 suppresses the flexibility of mitochondrial substrate usage and the pyruvate-dependent mitochondrial respiration induced by CIT. We established an animal model of diet-induced obesity/hyperinsulinemia and observed that BMAL1 functions as a tumor suppressor in obese, but not lean, mice. Downregulation of BMAL1 is associated with higher risk of metastasis in human breast tumors. In summary, loss of BMAL1 in tumors confers advantages to cancer cells in both intrinsic mitochondrial metabolism and extrinsic inflammatory tumor microenvironment during pre-diabetic obesity/hyperinsulinemia.

EPAS1 targeting by miR-152-3p in Paclitaxel-resistant Breast Cancer.

Background: Paclitaxel plays a pivotal role in the chemotherapy of breast cancer, but resistance to this drug is an important obstacle in the treatment. It is reported that microRNA-152-3p (miR-152-3p) is involved in tamoxifen resistance in breast cancer, but whether it is involved in paclitaxel resistance in breast cancer remains unknown.Materials and methods: We examined the expression of miR-152-3p in breast cancer tissues and cells by qRT-PCR. After transfecting paclitaxel-resistant MCF-7/TAX cells with miR-152-3p mimics, we analyzed the function of miR-152-3p in these cells by MTT assay and flow cytometry. We screened the target gene, endothelial PAS domain-containing protein 1 (EPAS1), using bioinformatics analysis and verified it with the dual luciferase reporter gene experiment. The relationship between EPAS1 and miR-152-3p and their roles in paclitaxel resistance of breast cancer were further investigated using RNA interference and transfection techniques.Results: The expression of miR-152-3p in normal breast tissues and cells was markedly higher than that in breast cancer. Overexpression of miR-152-3p decreased the survival rate and increased the apoptosis rate and sensitivity of MCF-7/TAX cells to paclitaxel. We confirmed that EPAS1 is the target of miR-152-3p and is negatively regulated by this miRNA. Moreover, transfection with EPAS1 siRNA enhanced the susceptibility and apoptosis rate of MCF-7/TAX cells to paclitaxel. Co-transfection of miR-152-3p mimics and EPAS1 increased paclitaxel sensitivity and apoptosis induced by the drug.Conclusion: miR-152-3p inhibits the survival of MCF-7/TAX cells and promotes their apoptosis by targeting the expression of EPAS1, thereby, enhancing the sensitivity of these breast cancer cells to paclitaxel.

Protein Phosphatase 1H, Cyclin-Dependent Kinase Inhibitor p27, and Cyclin-Dependent Kinase 2 in Paclitaxel Resistance for Triple Negative Breast Cancers.

PurposePaclitaxel is a cytotoxic chemotherapy commonly used in patients with triple negative breast cancer (TNBC); however, the resistance to paclitaxel is a cause of poor response in the patients. The aim of this study was to examine the role of protein phosphatase 1H (PPM1H) in paclitaxel resistance in breast cancer patients.MethodsTo investigate the function of PPM1H in paclitaxel treatment, we conducted in vitro assays and molecular experiments using a stable cell line (MDA-MB-231) in which PPM1H is overexpressed. We also performed molecular analyses on patient tissue samples. Molecular expression related to PPM1H in breast cancer patients was analyzed using TCGA data.ResultsWe investigated whether PPM1H was associated with paclitaxel resistance in breast cancer. PPM1H expression was upregulated in breast cancer cells treated with paclitaxel. We also observed that overexpression of PPM1H in breast cancer cells resulted in increased sensitivity to paclitaxel in vitro. Additionally, paclitaxel treatment induced dephosphorylation of cyclin-dependent kinase (CDK) inhibitor p27 (p27), which was more evident in PPM1H-overexpressing cells. To understand how upregulation of PPM1H increases paclitaxel sensitivity, we determined the levels of p27, phospho-p27, and CDK2, since CDK2 exerts antagonistic effects against PPM1H on p27 phosphorylation. The patient-derived xenograft (PDX) tumors that did not respond to paclitaxel showed increased levels of CDK2 and phospho-p27 and decreased levels of total p27 compared to the other breast tumor tissues. The use of dinaciclib, a selective CDK inhibitor, significantly inhibited tumor growth in the PDX model.ConclusionCDK2 kinase activity was significantly upregulated in basal breast cancer tumors and was negatively correlated with p27 protein levels in the TCGA breast cancer dataset, suggesting that targeting CDK2 may be an effective treatment strategy for TNBC patients.

Circular RNA circ-PVT1 contributes to paclitaxel resistance of gastric cancer cells through the regulation of ZEB1 expression by sponging miR-124-3p.

Gastric cancer (GC) is the fifth most commonly diagnosed malignancy. Paclitaxel (PTX) is an effective first-line chemotherapy drug in GC treatment, but the resistance of PTX attenuates the therapeutic effect. Circular RNA circ-PVT1 can exert the oncogenic effect in GC. But the function of circ-PVT1 involved in PTX resistance of GC is still unknown. In the present study, the expression levels of circ-PVT1, miR-124-3p and ZEB1 in PTX-resistant GC tissues and cells were detected by quantitative real-time polymerase chain reaction (RT-qPCR). PTX resistance in PTX-resistant cells was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The protein levels of Zinc finger E-box binding homeobox 1 (ZEB1), P-glycoprotein (P-gp) and glutathione S-transferase (GST-π) were detected by Western blot assay. Cell apoptosis and invasion were measured in PTX-resistant cells by flow cytometry and transwell invasion assays, severally. The interaction between miR-124-3p and circ-PVT1 or ZEB1 was predicted by starBase software, and then verified by the dual-luciferase reporter assay. The role of circ-PVT1 in PTX resistance of GC in vivo was measured by xenograft tumor model. Our results showed that circ-PVT1 expression was up-regulated in PTX-resistant GC tissues and cells. Circ-PVT1 down-regulation enhanced PTX sensitivity in PTX-resistant GC cells by negatively regulating miR-124-3p. ZEB1 served as a direct target of miR-124-3p. Circ-PVT1 enhanced ZEB1 expression by sponging miR-124-3p. Circ-PVT1 knockdown increased PTX sensitivity of GC in vivo. Taken together, our studies disclosed that circ-PVT1 facilitated PTX resistance by up-regulating ZEB1 mediated via miR-124-3p, suggesting an underlying therapeutic strategy for GC.

DANCR sponges miR-135a to regulate paclitaxel sensitivity in prostate cancer.

Paclitaxel is one of the most common drugs for cancer treatment. LncRNA DANCR is a regulator of up-regulation in tumors. Our experiment aims to clarify the role of DANCR in paclitaxel sensitivity of prostate cancer. We found that the expression of DANCR in prostate cancer tissues and cells was significantly higher than that in normal groups. DANCR knockdown could reduce cell proliferation and induce cell apoptosis in cells. Moreover, DANCR silence enhanced the effect of paclitaxel on cell proliferation and apoptosis in prostate cancer cells. DANCR targeted and negatively regulated the expression of miR-135a. miR-135a overexpression inhibited cell proliferation and promoted cell apoptosis and paclitaxel sensitivity in prostate cancer cells. miR-135a inhibition reversed the promoting effect of DANCR silence on paclitaxel sensitivity in prostate cancer cells. Downregulation of DANCR increased paclitaxel sensitivity in prostate cancer cells by negatively regulating the expression of miR-135a.

Abstract 4014: Clinical validation of Lantern Pharma’s Response Algorithm for Drug Positioning and Rescue (RADRTM)

Abstract Lantern Pharma has developed a technology platform termed RADRTM that can be used to predict true responders before conducting a clinical trial in order to achieve higher success rates. RADRTM is an Artificial Intelligence (Al)-based machine learning approach for complex biomarker identification and patient stratification. RADRTM is a combination of three automated modules working sequentially to generate drug- and tumor-specific gene signatures predictive of response. RADRTM integrates biological knowledge, data-driven feature selection, and robust Al algorithms to facilitate hypothesis-free drug- and cancer-specific biomarker development. We present retrospective analyses performed as part of RADRTM validation using at least 9 independent datasets of patients from selected cancer types treated with approved drugs including chemotherapy, targeted therapy and immune-oncology agents. Pre-treatment patient gene expression profiles along with corresponding treatment outcomes were used as algorithm inputs. Model training was typically performed using an initial set of genes derived from cancer cell line data when available, and further applied to a subset of patient data for model tuning and final gene signature development. Model testing and performance computation were carried out on patient records held out as blinded datasets. The response prediction accuracy, true positive rate (TPR), true negative rate (TNR) false discovery rate, positive predictive value and Matthew’s Correlation Coefficient were among the model performance metrics calculated. On average, RADRTM achieved a response prediction accuracy of 80% during clinical validation. For instance, in an analysis of 92 breast cancer patients, RADRTM generated a signature of 18 genes whose expression level was predictive of Paclitaxel treatment response at an overall accuracy of 78% and 81% TPR/ 76% TNR. The above results imply that the application of the RADRTM program to this Paclitaxel trial in breast cancer patients could have potentially reduced the number of patients in the treatment arm from 92 unselected patients to 24 biomarker-selected patients to produce the same number of responders. Moreover, we cite published evidence correlating genes from this 18-gene signature with increased Paclitaxel sensitivity in breast cancer. The value of the platform architecture is derived from its validation through the analysis of about 6 million oncology-specific clinical data points, more than 120 drug-cancer interactions, and over 600 patient records. Thus, by implementing unique biological, statistical and machine learning workflows, Lantern Pharma's RADRTM technology is capable of deriving robust biomarker panels for pre-selecting true responders for recruitment into clinical trials which may improve the success rate of oncology drug approvals. Citation Format: Yuvanesh Vedaraju, Umesh Kathad, Aditya Kulkarni, Barry Henderson, Gregory Tobin, Panna Sharma, Arun Asaithambi. Clinical validation of Lantern Pharma’s Response Algorithm for Drug Positioning and Rescue (RADRTM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4014.

Development and clinical validation of Lantern Pharma’s AI engine: Response algorithm for drug positioning and rescue (RADR).

3114 Background: The Response Algorithm for Drug positioning and Rescue (RADR) technology is Lantern Pharma's proprietary Artificial Intelligence (Al)-based machine learning approach for biomarker identification and patient stratification. RADR is a combination of three automated modules working sequentially to generate drug- and tumor type-specific gene signatures predictive of response. Methods: RADR integrates genomics, drug sensitivity and systems biology inputs with supervised machine learning strategies and generates gene expression-based responder/ non-responder profiles for specific tumor indications with high accuracy, in addition to identification of new correlations of genetic biomarkers with drug activity. Pre-treatment patient gene expression profiles along with corresponding treatment outcomes were used as algorithm inputs. Model training was typically performed using an initial set of genes derived from cancer cell line data when available, and further applied to patient data for model tuning, cross-validation and final gene signature development. Model testing and performance computation were carried out on patient records held out as blinded datasets. Response prediction accuracy and sensitivity were among the model performance metrics calculated. Results: On average, RADR achieved a response prediction accuracy of 80% during clinical validation. We present retrospective analyses performed as part of RADR validation using more than 10 independent datasets of patients from selected cancer types treated with approved drugs including chemotherapy, targeted therapy and immunotherapy agents. For an instance, the application of the RADR program to a Paclitaxel trial in breast cancer patients could have potentially reduced the number of patients in the treatment arm from 92 unselected patients to 24 biomarker-selected patients to produce the same number of responders. Also, we cite published evidence correlating genes from RADR derived biomarkers with increased Paclitaxel sensitivity in breast cancer. Conclusions: The value of RADR platform architecture is derived from its validation through the analysis of about ~17 million oncology-specific clinical data points, and ~1000 patient records. By implementing unique biological, statistical and machine learning workflows, Lantern Pharma's RADR technology is capable of deriving robust biomarker panels for pre-selecting true responders for recruitment into clinical trials which may improve the success rate of oncology drug approvals.

Investigation of the potential theranostic role of KDM5B/miR-29c signaling axis in paclitaxel resistant endometrial carcinoma

ObjectiveEndometrial cancer (EC) is one of the most common female reproductive system tumors. In this study, we explored the clinical significance of Histone demethylase KDM5B gene and its effects on paclitaxel (PTX) sensitivity in EC. MethodFirst, we found that KDM5B expression significantly higher in EC tissues and cell lines. The elevated KDM5B expression was associated with high pathological grade and low PTX sensitivity. The functional role of KDM5B in PTX-resistant Ishikawa-R and HEC1A-R cells were examined by gene silencing experiments. ResultsKnockdown of KDM5B resulted in the re-sensitization towards paclitaxel in both resistant cell lines. In addition, we also identified that microRNA-29c-3p (a tumor suppressor) was significantly lower in the EC cells and linked to the low PTX sensitivity of EC. The up-regulation of miR-29c-3p using exogenous mimic molecules markedly increased PTX sensitivity in both cell lines and reduced expression of KDM5B while the inhibitor of miR-29-3p resulted in the opposite effects. Notably, we also demonstrated that the level of miR-29c-3p was inversely correlated to the invasive, colony-forming abilities and PTX resistance in both cell lines. We also identified that miR-29c-3p has a binding site in the 3′UTR of the KDM5B gene, establishing a link of this signaling axis. In conclusion, high-expression of KDM5B is associated with the poor response to PTX in EC patients. Silencing of KDM5B led to the reversal of PTX resistance through miR-29c-3p/KDM5B pathway in EC. ConclusionOur findings provide new insights into the mechanism by which EC cells acquire paclitaxel resistance and potential this signaling as a theronostic marker for this malignancy.

Abstract IA08: Kinase-mediated modulation of paclitaxel sensitivity in ovarian cancer

Abstract Epithelial ovarian cancer is the fourth most common cause of cancer death among women in the developed world. Currently only 30% of ovarian cancer patients remain free from disease long term. Paclitaxel is an integral component of primary therapy for ovarian cancer, but less than half of ovarian cancers respond to the drug. Enhancing the response to primary therapy with paclitaxel could improve outcomes for women with the disease. In recent years, we have identified several kinases that regulate the sensitivity of cancer cells to paclitaxel by modulating cancer metabolism, enhancing microtubule stability, inhibiting centrosome splitting or blocking AKT/survivin signaling. We performed siRNA kinome-screens to identify molecular targets whose decreased expression overcomes paclitaxel resistance and increases paclitaxel sensitivity in ovarian cancer cells. Upon assessing cell viability, we showed that 20% of the potential kinase targets whose knockdown modulates paclitaxel sensitivity participate in glucose and energy metabolism. Among these, a leading candidate was 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2), an isoform of the glycolytic enzyme phosphofructokinase (PFK2). Many cancer cells depend on glucose for survival. Glycolysis involves ten metabolic reactions catalyzed by enzymes whose expression is frequently increased during malignant transformation. Many reports document the role of oncogenic signaling in regulating the activity of metabolic enzymes to support the enhanced macromolecule synthesis required for rapid proliferation of cancer cells. Cancer cells express altered levels of the different PFK-2/FBPase-2 isoenzymes and even modulate their relative kinase and bisphosphatase activities according to metabolic needs in a spatial and/or temporal manner. PFKFB2 is a bifunctional glycolytic enzyme that regulates the level of fructose-2,6-bisphosphate (Fru-2,6-BP) and is overexpressed in a fraction of ovarian and breast cancers. We found that knockdown of PFKFB2 inhibited clonogenic growth and enhanced paclitaxel sensitivity in ovarian and breast cancer cell lines with wild-type TP53 (wtTP553). Additionally, PFKFB2 siRNA or PFKFB2 shRNA significantly inhibited tumor growth and enhanced sensitivity to paclitaxel in xenografts derived from two ovarian cancer cell lines. Knockdown of PFKFB2 increased the rate of glycolysis, but decreased the flow of intermediates through the pentose-phosphate pathway in cancer cell lines with wtTP53, decreasing NADPH. Reactive oxygen species (ROS) accumulated after PFKFB2 knockdown, which stimulated phosphorylation of Janus kinase (JNK), induced G1 cell cycle arrest, and initiated apoptosis that depended upon upregulation of p21Cip1 and Puma. Thus, PFKFB2 is a glycolytic enzyme that drives tumor cell growth and enhances paclitaxel resistance by inhibiting TP53-dependent G1 cell cycle arrest and apoptosis. These findings highlight the interaction of cancer-altered metabolism with cell proliferation and chemosensitivity, which may provide a novel target in patients with ovarian and breast cancers where TP53 function remains intact. We also found that knockdown of kinases that regulate microtubule stability could sensitize ovarian cancer cells to paclitaxel treatment. In previous studies, baseline microtubule stability correlated with response to paclitaxel in ovarian cancer cell lines and both parameters could be enhanced by knockdown of individual kinases. Using the initial 14 kinase targets, we performed assays of microtubule stability, apoptosis, and cell cycle arrest with all possible pairs of kinase siRNA which enhance paclitaxel sensitivity across multiple cell lines and found that dual knockdown of IKBKB/STK39 had the greatest effect on enhancing paclitaxel stability. Our study documents the impact of sequentially silencing IKBKB and STK39 on paclitaxel sensitivity, providing a rationale for siRNA-based therapy. Different siRNAs are in the developmental pipeline for a variety of diseases including cancer, and more than a dozen are in phase I or II clinical trials. One of the most promising candidates to emerge from our kinase screen was salt inducible kinase 2 (SIK2), a serine/threonine kinase that is required for bipolar mitotic spindle formation and normal mitotic progression. With Dr. Ahmed Ahmed, we previously found knockdown of SIK2 induces polyploidy by blocking centrosome splitting and inhibits PI3K, sensitizing cells to paclitaxel. We demonstrated that knockdown of SIK2 inhibits xenograft growth and potentiates paclitaxel activity in vivo. Subsequently, we have partnered with Arrien Pharmaceuticals to test a small-molecule inhibitor of SIK2. We have found that novel SIK2 inhibitors, ARN-3236 and ARN3261, block the activity of SIK2 kinase and inhibit ovarian cancer cell growth, enhancing paclitaxel sensitivity in cultured cells and in xenografts. ARN-3261 will enter first-in-human trials at MD Anderson early next year. Taken together, our results support the development of small-molecule kinase inhibitors that modulate glycolysis, enhance microtubule stability, induce polyploidy, and inhibit AKT/survivin signaling, providing novel routes to enhance primary paclitaxel-based therapy for ovarian cancer and to improve patient outcomes. Citation Format: Zhen Lu. Kinase-mediated modulation of paclitaxel sensitivity in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr IA08.

Sperm associated antigen 9 (SPAG9) a promising therapeutic target of ovarian carcinoma.

SPAG9 is a novel tumor associated antigen, expressed in variety of malignancies. However, its role in ovarian cancer remains unexplored. SPAG9 expression was validated in ovarian cancer cells by real time PCR and Western blot. SPAG9 involvement in cell cycle, DNA damage, apoptosis, paclitaxel sensitivity and epithelial- mesenchymal transition (EMT) was investigated employing RNA interference approach. Combinatorial effect of SPAG9 ablation and paclitaxel treatment was evaluated in in vitro. Quantitative PCR and Western blot analysis revealed SPAG9 expression in A10, SKOV-3 and Caov3 compared to normal ovarian epithelial cells. SPAG9 ablation resulted in reduced cellular proliferation, colony forming ability and enhanced cytotoxicity of chemotherapeutic agent paclitaxel. Effect of ablation of SPAG9 on cell cycle revealed S phase arrest and showed decreased expression of CDK1, CDK2, CDK4, CDK6, cyclin B1, cyclin D1, cyclin E and increased expression of tumor suppressor p21. Ablation of SPAG9 also resulted in increased apoptosis with increased expression of various pro- apoptotic molecules including BAD, BID, PUMA, caspase 3, caspase 7, caspase 8 and cytochrome C. Decreased expression of mesenchymal markers and increased expression of epithelial markers was found in SPAG9 ablated cells. Combinatorial effect of SPAG9 ablation and paclitaxel treatment was evaluated in in vitro assays which showed that ablation of SPAG9 resulted in increased paclitaxel sensitivity and caused enhanced cell death. In vivo ovarian cancer xenograft studies showed that ablation of SPAG9 resulted in significant reduction in tumor growth. Present study revealed therapeutic potential of SPAG9 in ovarian cancer.

MicroRNA-22 Suppresses Breast Cancer Cell Growth and Increases Paclitaxel Sensitivity by Targeting NRAS.

In recent study, microRNAs have various important functions in diverse biological processes and progression of cancer. In human breast cancer, microRNA-22 has been reported to be downregulated. However, molecular mechanism of microRNA-22 in breast cancer progression and chemosensitivity has not been well studied. In our study, these results demonstrated that microRNA-22 expression levels were significantly reduced in 40 pairs of human breast cancer tissues when compared to normal tissues. Enforced expression of microRNA-22 inhibited activity of cell proliferation and cell migration in breast cancer cells. Furthermore, microRNA-22 targeted NRAS proto-oncogene, GTPase (NRAS) in breast cancer cells. The expression levels of NRAS in human clinical specimens were higher in breast cancer tissues when compared to normal tissues. Moreover, microRNA-22 sensitized breast cancer cells to paclitaxel by regulation of NRAS. Our results then demonstrated that microRNA-22 functioned as a tumor suppressor microRNA and indicated potential application for the diagnosis and treatment of cancer in the future.

Abstract B138: Prognostic value of PTEN loss for second-line paclitaxel treatment efficacy in metastatic gastric cancer

Abstract Introduction: For recurrent and metastatic gastric cancer, systemic chemotherapy is used for palliative purposes, and a paclitaxel- or irinotecan-based regimen is typically used in the second-line setting. Recent advances in research including the TCGA project now enable gastric cancer to be divided into four molecular subgroups, yet whether there are differences in chemotherapy response for each molecular subgroup remain largely unknown. Our aim in this study is to retrospectively compare the efficacy of paclitaxel as second-line chemotherapy according to molecular subtype and to evaluate prognostic value for these markers. Method: A total of 235 metastatic gastric cancer (mGC) patients who have been treated with second-line paclitaxel-containing regimen (paclitaxel monotherapy, FL/paclitaxel) between 2005-2017 were assessed. Archived formalin-fixed, paraffin-embedded tissue blocks obtained before the start of paclitaxel treatment were examined for EGFR, HER2, and PTEN status using immunohistochemistry (IHC). HER2 positivity was defined as either IHC 3+ or 2+ with amplification confirmed by FISH/SISH, and EGFR positivity was defined as IHC 3+. PTEN loss was defined as having complete loss of PTEN expression. MSI-H and EBV-positive group was excluded in this study. Treatment response was based on CT images taken every 2 to 3 cycles of chemotherapy and evaluated according to the RECIST criteria. Overall survival (OS) and progression-free survival (PFS) were calculated based on the starting point of second-line chemotherapy until death or disease progression, respectively. Those negative for all three markers (HER2, EGFR, PTEN loss) were categorized as genome stable (GS)-like molecular group. Result: Patients were classified into subgroups based on HER2, EGFR, and PTEN expression; 19.6% (46/235) were HER2 positive, 15.0% (26/173) were EGFR positive, and PTEN loss was observed in 11.6% (20/173). Expression status for the three markers showed tendency towards mutual exclusivity. When coexpression rates were taken into account, 35.3% (61/173) showed positivity in at least one of the markers, and 64.7% (112/173) were negative for all three. This latter group, categorized as GS-like molecular group, showed a tendency towards poor overall survival compared with patients positive for at least one molecular marker, with median survival of 5.7 vs 8.8 months (P=0.012). There were no significant differences in OS or PFS in terms of HER2 and EGFR status. However, patients with PTEN loss had significantly longer overall survival than those with intact PTEN expression, with median survival time of 12.0 months (95% confidence interval [CI], 7.0 - 21.2) and 6.2 months (95% CI, 4.7-7.2), respectively (P=0.003). Conclusion: In mGC patients treated with second-line paclitaxel chemotherapy, HER2 and EGFR status had no predictive or prognostic value. Contrary to the general association of PTEN loss with poor prognosis in many solid cancers including gastric cancer, PTEN loss group showed better survival outcome for second-line paclitaxel treatment in our study. Indirect association of PTEN with other pathways of multidrug resistance or cell cycle-related molecules that ultimately affect the PI3K/Akt pathway may play a role. Further investigation is needed for specific mechanisms and factors that lead to increased paclitaxel sensitivity in the PTEN loss group. Citation Format: Kyoo Hyun Kim, Jeong Hyun Jo, Sejung Park, Minkyu Jung, Woo Sun Kwon, Jee Hung Kim, Hyo Song Kim, Seung-Hoon Beom, Hyun Cheol Chung, Sun Young Rha. Prognostic value of PTEN loss for second-line paclitaxel treatment efficacy in metastatic gastric cancer [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 B138.

Abstract 3564: Silencing pfkfb2 enhances paclitaxel sensitivity by modulating metabolism of p53 wt ovarian and breast cancer cells and xenografts

Abstract Breast cancer is the most common cause and epithelial ovarian cancer the fourth most common cause of cancer death among women in the developed world. While breast cancer can be cured in 70% of cases, only 30% of ovarian cancer patients remain free from disease long term. Paclitaxel is an integral component of primary therapy for both forms of cancer, but less than half of breast and ovarian cancers respond to the drug. Enhancing the response to primary therapy with paclitaxel could improve outcomes for women with both diseases. In recent years several kinases have been identified that regulate the sensitivity of cancer cells to paclitaxel by inhibiting centrosome splitting or enhancing microtubule stability. Much less attention has been given to kinases that affect paclitaxel sensitivity by modulating cancer cell metabolism. We previously performed siRNA kinome-screens to identify molecular targets whose decreased expression overcomes paclitaxel resistance and increases paclitaxel sensitivity in ovarian cancer cells. We showed that 20% of the potential kinase targets whose knockdown modulates paclitaxel sensitivity participate in glucose and energy metabolism. Among these, a leading candidate was 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2), an isoform of the glycolytic enzyme phosphofructokinase (PFK2). PFKFB2 is overexpressed in a fraction of ovarian and breast cancers. Knockdown of PFKFB2 inhibited clonogenic growth of ovarian and breast cancer cell lines and enhanced paclitaxel sensitivity in the cell lines with wt TP53. Liposome encapsulated PFKFB2 siRNA significantly inhibited tumor growth and enhanced sensitivity to paclitaxel in xenografts derived from ovarian cancer cell lines. Knockdown of PFKFB2 increased glycolysis, decreasing the flow of glycolytic intermediates to the pentose-phosphate pathway with reduced G6PD activity in wt TP53 cancer cell lines. With decreased NADPH, ROS accumulated after PFKFB2 knockdown, stimulating phosphorylation of JNK, inducing G1 cell cycle arrest, and initiating apoptosis dependent upon upregulation of p21Cip1 and Puma which are downstream targets of TP53. Our studies have shown for the first time that PFKFB2, a glycolytic enzyme, drives tumor cell growth and regulates paclitaxel sensitivity by inducing apoptosis and G1 cell cycle arrest. These findings highlight a remarkable degree of coordination between cancer metabolism with cell proliferation and chemo-sensitivity, which may provide a novel target in patients with ovarian cancers and breast cancers where TP53 function remains intact. Citation Format: Hailing Yang, Shu Zhang, Weiqun Mao, Ahmed A. Ahmed, Nicholas B. Jennings, Cristian Rodriguez-Aguayo, Gabriel Lopez-Berestein, Anil K. Sood, Xiao-Feng Le, Zhen Lu, Robert C. Bast. Silencing pfkfb2 enhances paclitaxel sensitivity by modulating metabolism of p53 wt ovarian and breast cancer cells and xenografts [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 3564. doi:10.1158/1538-7445.AM2017-3564

Kuguacin J isolated from bitter melon leaves modulates paclitaxel sensitivity in drug-resistant human ovarian cancer cells.

We previously reported the multidrug resistance-reversing ability of kuguacin J (KJ) in cervical cancer cells via the inhibition of P-glycoprotein (P-gp) function. This study investigated whether KJ could promote cisplatin- and paclitaxel (PTX)-induced cancer cell death in drug-resistance human ovarian cancer cells (SKOV3). Cytotoxicity testing showed that SKOV3 was more resistant to cisplatin and PTX compared to drug-sensitive human ovarian cancer cells (A2780). The cytotoxicity of PTX was significantly increased in SKOV3 cells when co-treated with KJ. We found that enhancement of PTX toxicity in the cells was not related to P-gp inhibition. To elucidate the mechanism by which KJ increases PTX sensitivity, the expression of cell death involving proteins was analyzed by Western blot analysis. The results showed that PTX treatment increased the level of an anti-apoptotic protein, survivin, which may be involved in drug resistance in SKOV3. The co-treatment with PTX and KJ dramatically decreased the level of survivin and markedly induced cleavage of PARP and caspase-3, which are apoptotic-induced molecules. These findings may support the use of KJ as an effective chemosensitizer in combination with conventional chemotherapy to promote PTX sensitization in ovarian cancer patients.

Circadian Clock Gene Bmal1 Inhibits Tumorigenesis and Increases Paclitaxel Sensitivity in Tongue Squamous Cell Carcinoma.

Circadian clock genes regulate cancer development and chemotherapy susceptibility. Accordingly, chronotherapy based on circadian phenotypes might be applied to improve therapeutic efficacy. In this study, we investigated whether the circadian clock gene Bmal1 inhibited tumor development and increased paclitaxel sensitivity in tongue squamous cell carcinoma (TSCC). Bmal1 expression was downregulated and its rhythmic pattern of expression was affected in TSCC samples and cell lines. Ectopic Bmal1 inhibited cell proliferation, migration and invasion in vitro, and tumor growth in mouse xenograft models of TSCC. After exposure to paclitaxel, Bmal1-overexpressing cells displayed a relative increase in apoptosis and were more susceptible to paclitaxel treatment in vivo Mechanistic investigations suggested a regulatory connection between Bmal1, TERT, and the oncogenic transcriptional repressor EZH2 (enhancer of zeste homolog 2), the recruitment of which to the TERT promoter increased paclitaxel-induced apoptosis and cell growth inhibition. Clinically, paclitaxel efficacy correlated positively with Bmal1 expression levels in TSCC. Overall, our results identified Bmal1 as a novel tumor suppressor gene that elevates the sensitivity of cancer cells to paclitaxel, with potential implications as a chronotherapy timing biomarker in TSCC. Cancer Res; 77(2); 532-44. ©2016 AACR.

Abstract 3100: Hsp90 inhibition results in GR, AR and JAK protein degradation, decreased triple-negative breast cancer proliferation and increased paclitaxel sensitivity

Abstract Glucocorticoid receptor (GR) activity in triple-negative breast cancer (TNBC) is associated with cell survival and chemotherapy resistance. Targetable molecular drivers for TNBC have been difficult to identify and therefore standard treatment remains limited to chemotherapy. Recently, new-generation small molecule Hsp90 inhibitors (e.g. ganetespib and NVP-AUY922) have proven to be both potent inhibitors of Hsp90 client proteins and far less toxic than previous Hsp90 inhibitors. Moreover, favorable clinical responses have been observed in some TNBC patients enrolled in a phase II clinical trial of ganetespib monotherapy. A ganetespib and paclitaxel clinical trial is ongoing in breast cancer; however, it is not known which Hsp90 client proteins contribute to increased tumor chemotherapy sensitivity. Recent studies in our lab found that inhibition of glucocorticoid receptor (GR) activity plays an important role in Hsp90 inhibitor-mediated sensitization of TNBC cells to paclitaxel-induced cytotoxicity. However, we also observed that following significant shRNA-mediated GR depletion, ganetespib still provided some sensitization of TNBC cells to paclitaxel. This suggests that inactivation of additional anti-apoptotic Hsp90 client proteins play a role in mediating ganetespib effects. Proteins closely associated with GR function that are also key Hsp90 inhibitor client proteins include the androgen receptor (AR, implicated in TNBC proliferation and apoptosis) and JAK, a component of JAK/STAT signaling pathway implicated in TNBC tumorigenesis and chemoresistance. Here we hypothesize that AR and JAK/STAT signaling have overlapping but distinct roles in Hsp90-mediated chemotherapy sensitization of TNBCs. Western blot analysis of TNBC xenografts treated with ganetespib +/- paclitaxel showed depletion of AR as well as JAK1 and 2. Ganetespib treatment also led to decreased levels of TMPRSS2 gene expression, an AR target gene. Additionally Western blot analysis showed decreased levels of activated phosphorylated STAT3 transcriptional factor, a downstream mediator of JAK, following ganetespib treatment of TNBC cell lines. These results suggest the involvement of AR and JAK in mediating ganetespib mechanism of action. We are currently beginning to determine how AR and JAK/STAT signaling pathways may interact with GR signaling by establishing CRISPR/Cas9 knockouts of GR, AR, and/or JAK in TNBC cells treated with ganetespib +/- paclitaxel. Understanding the relative roles of Hsp90 client proteins in TNBC is predicted to lead to a more rational selection of patients with TNBCs likely to benefit from Hsp90-inhibitor treatment. Citation Format: Abena S. Agyeman, Wesley J. Jun, Suzanne D. Conzen. Hsp90 inhibition results in GR, AR and JAK protein degradation, decreased triple-negative breast cancer proliferation and increased paclitaxel sensitivity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3100.