Paclitaxel Chemosensitivity Research Articles

Downregulation of long noncoding RNA PVT1 attenuates paclitaxel resistance in glioma cells

Drug resistance in clinical cancer treatment has become an issue. We focus on abnormally expressed lncRNAs in glioma and investigating the function of PVT1. The paclitaxel-resistant glioma cells SHG-44 RE was obtained through screening the SHG44 cells that were cultured in medium containing a certain concentration of paclitaxel. Cell survival of SHG44 RE and SHG44 cells under the treatment of paclitaxel was detected by MTT assay. The aberrant expressed lncRNAs were screened out with microarray analysis. Further qRT-PCR was utilized to validate the expression of lncRNA PVT1 in the two cells. After manipulating the expression of PVT1, cell viability and apoptosis were measured by MTT and flow cytometry respectively. LncRNA PVT1 was overexpressed in glioma cells SHG-44 RE compared with parent SHG-44 cells. Down-regulation of lncRNA PVT1 inhibited the SHG-44 RE cell viability and increased glioma SHG-44 RE cells apoptosis after paclitaxel treatment, suggesting that inhibition of lncRNA PVT1 improved paclitaxel sensibility in human glioma cells. Down-regulation of PVT1 could enhance chemosensitivity of paclitaxel, induce apoptosis of glioma cells and noteworthy inhibit glioma cells proliferation. Our findings of PVT1 could contribute to attenuate paclitaxel resistance in clinical medicine.

Network Pharmacology-Based Validation of Caveolin-1 as a Key Mediator of Ai Du Qing Inhibition of Drug Resistance in Breast Cancer.

Chinese formulas have been paid increasing attention in cancer multidisciplinary therapy due to their multi-targets and multi-substances property. Here, we aim to investigate the anti-breast cancer and chemosensitizing function of Ai Du Qing (ADQ) formula made up of Hedyotis diffusa, Curcuma zedoaria (Christm.) Rosc., Astragalus membranaceus (Fisch.) Bunge, and Glycyrrhiza uralensis Fisch. Our findings revealed that ADQ significantly inhibited cell proliferation in both parental and chemo-resistant breast cancer cells, but with little cytotoxcity effects on the normal cells. Besides, ADQ was found to facilitate the G2/M arresting and apoptosis induction effects of paclitaxel. Network pharmacology and bioinformatics analysis further demonstrated that ADQ yielded 132 candidate compounds and 297 potential targets, and shared 22 putative targets associating with breast cancer chemoresponse. Enrichment analysis and experimental validation demonstrated that ADQ might improve breast cancer chemosensitivity via inhibiting caveolin-1, which further triggered expression changes of cell cycle-related proteins p21/cyclinB1 and apoptosis-associated proteins PARP1, BAX and Bcl-2. Besides, ADQ enhanced in vivo paclitaxel chemosensitivity on breast cancer. Our study not only uncovers the novel function and mechanisms of ADQ in chemosensitizing breast cancer at least partly via targeting caveolin-1, but also sheds novel light in utilizing network pharmacology in Chinese Medicine research.

Expression of C-fos in nasopharyngeal carcinoma and the relationship with chemosensitivity and prognosis

Objective:To investigate the expression of C-fos in patients with nasopharyngeal carcinoma(NPC), and analyze the relationship between the expression of C-fos and the clinical characteristics, chemosensitivity and prognosis. Method:Clinical and follow-up data of 75 NPC patients was analyzed retrospectively. The expression of C-fos was detected by immunohistochemical assay, and chemosensitivity was detected by ATP bioluminescent anticancer drug sensitivity detection technology. The relationship between them was studied.Result:The expression of C-fos in NPC was statistically higher than that in the control nasopharyngeal mucosa(P<0.001). It was found that C-fos had no statistical relationship with the gender, age, pathologic type, clinical stage of tumor classification, lymph node status, metastasis status and overall stage of NPC patients(P>0.05). NPC had different chemosensitivity with 8 anticancer drugs(P<0.001).There was a significant difference in chemosensitivity of paclitaxel between the high expression of C-fos group and the low expression of C-fos group(P=0.036). The rate of tumor progression was significantly higher in NPC patients with high expression of C-fos than in the low expression group(P=0.014).There was no significant difference in overall survival between the two groups(P=0.076). Conclusion:C-fos is highly expressed in NPC tissues, and the high expression of C-fos in NPC tissues may be related to tumor progression and resistance to paclitaxel.

Targeting miR-21 decreases expression of multi-drug resistant genes and promotes chemosensitivity of renal carcinoma.

Renal cell carcinoma, the most common neoplasm of adult kidney, accounts for about 3% of adult malignancies and is usually highly resistant to conventional therapy. MicroRNAs are a class of small non-coding RNAs, which have been previously shown to promote malignant initiation and progression. In this study, we focused our attention on miR-21, a well described oncomiR commonly upregulated in cancer. Using a cohort of 99 primary renal cell carcinoma samples, we showed that miR-21 expression in cancer tissues was higher than in adjacent non-tumor tissues whereas no significant difference was observed with stages, grades, and metastatic outcome. In vitro, miR-21 was also overexpressed in renal carcinoma cell lines compared to HK-2 human proximal tubule epithelial cell line. Moreover, using Boyden chambers and western blot techniques, we also showed that miR-21 overexpression increased migratory, invasive, proliferative, and anti-apoptotic signaling pathways whereas opposite results were observed using an anti-miR-21-based silencing strategy. Finally, we assessed the role of miR-21 in mediating renal cell carcinoma chemoresistance and further showed that miR-21 silencing significantly (1) increased chemosensitivity of paclitaxel, 5-fluorouracil, oxaliplatin, and dovitinib; (2) decreased expression of multi-drug resistance genes; and (4) increased SLC22A1/OCT1, SLC22A2/OCT2, and SLC31A1/CTR1 platinum influx transporter expression. In conclusion, our results showed that miR-21 is a key actor of renal cancer progression and plays an important role in the resistance to chemotherapeutic drugs. In renal cell carcinoma, targeting miR-21 is a potential new therapeutic strategy to improve chemotherapy efficacy and consequently patient outcome.

Genetic and therapeutic inhibition of the receptor tyrosine kinase AXL restores paclitaxel chemosensitivity in uterine serous cancer

Genetic and therapeutic inhibition of the receptor tyrosine kinase AXL restores paclitaxel chemosensitivity in uterine serous cancer

A Novel Compound ARN-3236 Inhibits Salt-Inducible Kinase 2 and Sensitizes Ovarian Cancer Cell Lines and Xenografts to Paclitaxel.

Purpose: Salt-inducible kinase 2 (SIK2) is a centrosome kinase required for mitotic spindle formation and a potential target for ovarian cancer therapy. Here, we examine the effects of a novel small-molecule SIK2 inhibitor, ARN-3236, on sensitivity to paclitaxel in ovarian cancer.Experimental Design: SIK2 expression was determined in ovarian cancer tissue samples and cell lines. ARN-3236 was tested for its efficiency to inhibit growth and enhance paclitaxel sensitivity in cultures and xenografts of ovarian cancer cell lines. SIK2 siRNA and ARN-3236 were compared for their ability to produce nuclear-centrosome dissociation, inhibit centrosome splitting, block mitotic progression, induce tetraploidy, trigger apoptotic cell death, and reduce AKT/survivin signaling.Results: SIK2 is overexpressed in approximately 30% of high-grade serous ovarian cancers. ARN-3236 inhibited the growth of 10 ovarian cancer cell lines at an IC50 of 0.8 to 2.6 μmol/L, where the IC50 of ARN-3236 was inversely correlated with endogenous SIK2 expression (Pearson r = -0.642, P = 0.03). ARN-3236 enhanced sensitivity to paclitaxel in 8 of 10 cell lines, as well as in SKOv3ip (P = 0.028) and OVCAR8 xenografts. In at least three cell lines, a synergistic interaction was observed. ARN-3236 uncoupled the centrosome from the nucleus in interphase, blocked centrosome separation in mitosis, caused prometaphase arrest, and induced apoptotic cell death and tetraploidy. ARN-3236 also inhibited AKT phosphorylation and attenuated survivin expression.Conclusions: ARN-3236 is the first orally available inhibitor of SIK2 to be evaluated against ovarian cancer in preclinical models and shows promise in inhibiting ovarian cancer growth and enhancing paclitaxel chemosensitivity. Clin Cancer Res; 23(8); 1945-54. ©2016 AACR.

SH003 enhances paclitaxel chemosensitivity in MCF-7/PAX breast cancer cells through inhibition of MDR1 activity.

Paclitaxel is an anti-cancer drug for treating cancer, but paclitaxel resistance is reported in cancer cells. Multidrug resistance (MDR) is related with the epithelial-to-mesenchymal transition (EMT) mechanism, which plays a key role in cancer metastasis. Moreover, EMT mechanism is connected to tamoxifen resistance in breast cancer cells. Consequently, oncologists are interested in finding new MDR1 inhibitors originating from herbal medicines to have less side-effect. Here, we investigated an inhibition effect of SH003 on MDR1 activity in paclitaxel-resistant MCF-7/PAX breast cancer cells. Our results showed that paclitaxel did not inhibit a proliferation in paclitaxel-resistant MCF-7 breast cancer cells. Paclitaxel-resistant MCF-7 cells showed an increase of MDR1 activity, which was confirmed by measuring an amount of accumulated rhodamine 123 in the cells. Also, qRT-PCR and Western blot assays confirmed that paclitaxel-resistant MCF-7 cells exhibited high MDR1 expression level. Furthermore, paclitaxel-resistant MCF-7 cells showed mesenchymal morphology with alterations of EMT markers, and acquired tamoxifen resistance with a decrease of ERα expression. We also found that a combinatorial treatment of SH003 and paclitaxel in paclitaxel-resistant MCF-7 cells caused apoptosis in synergistic manner, which was due to SH003 inhibition of MDR1 expression. Therefore, SH003 could be a potential agent for overcoming MDR in drug-resistant cancer cells.

Proton Pump Inhibition Enhances the Cytotoxicity of Paclitaxel in Cervical Cancer.

PurposeThis study was conducted to investigate whether a proton pump inhibitor (PPI) could enhance chemosensitivity via the inhibition of vacuolar-type H+ ATPase (V-ATPase) in cervical cancer.Materials and MethodsThe expression of V-ATPase was evaluated in 351 formalin-fixed, paraffin-embedded human cervical cancer tissues using immunohistochemistry and compared with clinicopathologic risk factors for disease prognosis. The influence of cell proliferation and apoptosis following V-ATPase siRNA transfection or esomeprazole pretreatment was assessed in cervical cancer cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and enzyme-linked immunosorbent assay, respectively.ResultsImmunohistochemical analysis revealed that V-ATPase was expressed in about 60% of cervical cancer tissue samples (211/351), and the expression was predominantly found in adenocarcinoma histology (p=0.016). Among patients with initially bulky cervical cancer (n=89), those with V-ATPase expression had shorter disease-free survival (p=0.005) and overall survival (p=0.023). Co-treatment with V-ATPase siRNA or esomeprazole with paclitaxel significantly decreased the cell proliferation of cervical cancer cell lines, including HeLa and INT407, compared to cell lines treated with paclitaxel alone (p < 0.01). Moreover, V-ATPase siRNA or esomeprazole followed by paclitaxel significantly increased the expression of active caspase-3 in these cells compared to cells treated with paclitaxel alone (both, p < 0.05).ConclusionV-ATPase was predominantly expressed in cervical adenocarcinoma, and the expression of V-ATPases was associated with poor prognosis. The inhibition of V-ATPase via siRNA or PPI (esomeprazole) might enhance the chemosensitivity of paclitaxel in cervical cancer cells.

Linking omentum and ovarian cancer: NO.

Ovarian cancers are one of the most lethal gynecological cancers because they are usually diagnosed in advanced stages when metastasis has already occurred in the peritoneal cavity. Interestingly, ovarian cancers frequently invade the fatty pad of adipose tissue along the stomach called the omentum [1]. Recent studies have shown that omentum contains a population of stem cell-like cells such as adipose stromal cells (ASCs) that engraft in tumors and encourage cancer progression [2]. Omentum-derived ASCs (O-ASCs) have been demonstrated to contribute to the formation of a hospitable environment for the development of ovarian cancer metastasis [2]. Previous studies have shown that O-ASCs enhance proliferation and migration of ovarian cancer cells while reducing their response to chemotherapy and radiation [2]. However, the mechanistic underpinnings of O-ASCs’ role in tumor progression and growth are still unclear. Recently we demonstrated that nitric oxide (NO)-mediated metabolic coupling between O-ASCs and ovarian cancer cells contributes to ovarian cancers’ growth and increased chemosensitivity [3]. Our study revealed that O-ASCs secreted arginine, which was used by cancer cells for NO generation. NO is generated endogenously by an enzymatic conversion of arginine into citrulline through nitric oxide synthase (NOS). NOS has been found to be differentially expressed in obese and non-obese individuals and has been shown to be overexpressed in aggressive ovarian tumors [4]. We revealed that O-ASCs secreted arginine could rescue reduced proliferation rates of ovarian cancers caused by arginine deprivation [3]. Notably, overweight O-ASCs were able to rescue the reduced proliferation of cancer cells more than their lean counterparts. However, more studies are required to shed light on the role of obesity in ovarian cancer development and progression. Several studies indicate that NO is a double-edged molecule. It is a tumor promoter at lower concentrations (less than 500 nM), but damages DNA and induces apoptosis at higher concentrations (millimolar range) [5]. We found that ovarian cancer cells in cocultures with O-ASCs had significantly higher NO levels compared to their non-coculture counterparts. We previously showed that NO synthesis upregulates Warburg effect in ovarian cancers [5]. Interestingly, coculture of cancer cells with O-ASCs reduced oxygen consumption rates (OCR) in cancer cells. We confirmed this shift in ovarian cancers’ metabolism from oxidative phosphorylation (OXPHOS) to glycolysis by comparing the contributions of both pathways towards cellular ATP generation and our data elucidated that O-ASCs increased NO synthesis in cancer cells, resulting in suppression of mitochondrial respiration. Previously, we showed that O-ASCs induced chemo-resistance in cancer cells [2]. Interestingly, in recent studies we found that O-ASCs-mediated chemoresistance can be deregulated by disrupting NO homeostasis [3]. We added L-arginase in direct-contact cocultures of O-ASCs and cancer cells. Both L-arginase (which depletes any secreted arginine by O-ASCs and thereby blocks NO synthesis in cancer cells) and L-NAME (a NO synthase inhibitor) in direct-contact cocultures of O-ASCs and ovarian cancer cells, increased chemosensitivity of paclitaxel in cancer cells. Our results suggest that combined approach of depleting arginine using L-arginase, along with inhibiting NO synthesis in cancer cells using L-NAME, may be a viable therapeutic approach for targeting ovarian cancers, to disrupt the communication between cancer cell and O-ASCs. Arginine is not the only player in the metabolic coupling of O-ASCs and ovarian cancers. Surprisingly, O-ASCs-secreted arginine when is used for NO synthesis in cancer cells, generates citrulline as a byproduct, which is secreted by cancer cells and in turn is ingested by O-ASCs. We hypothesized that this high amount of secreted citrulline concentrations may be beneficial for NO metabolism in O-ASCs. As mentioned before, O-ASCs are multipotent population of mesenchymal stem cells that can differentiate into adipocytes. Surprisingly, we found that secreted citrulline by cancer cells significantly increased adipogenesis capacity of O-ASCs and stimulated the lipid droplet accumulation within O-ASCs. Studies have shown that NO inhibits lipolysis of lipid depots in subcutaneous adipose tissue [6]. This highlights the metabolic symbiosis between omentum-derived ASCs and cancer cells in maintaining NO homeostasis in tumors. Future studies are needed to investigate the mechanism behind the modulation of ovarian tumor's metabolism by NO. The post-translational modification of metabolic enzymes caused by binding of NO with their cysteine residue (s-nitrosylation) may play a crucial role. S-nitrosylation has been known to alter the functions of enzymes in fatty acid metabolism as well as other central energy pathways [7]. The impact of obesity in ovarian cancer initiation and progression has been studied and further studies are needed to explore the role of NO metabolism in this modulation. Furthermore, the link between omentum and other hormonal tumors such prostate cancers needs to be further studied. Even as new mechanisms implicating omentum for ovarian cancer metastasis keep emerging, many aspect of its biology still remains to be unveiled [8]. Figure 1 Crosstalk between ovarian cancers and O-ASCs in tumor microenvironment

Therapeutic effect of anti CEACAM6 monoclonal antibody against lung adenocarcinoma by enhancing anoikis sensitivity

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) plays a crucial role in tumorigenesis of lung cancer. However, the therapeutic potential for anti CEACAM6 monoclonal antibody (mAb) has only been limitedly explored. Here, we evaluate the therapeutic potential of naked anti CEACAM6 mAb against lung adenocarcinoma. Clone 8F5, recognizing B domain of CEACAM6, is established by immunizing A549 cells and screening for clones double positive for A549 and CEACAM6-Fc recombinant protein. We found that 85.7% of 70 resected lung adenocarcinoma tissue sections were positive for CEACAM6, whereas all squamous cell carcinoma examined were negative. A549 cells with high levels of CEACAM6 demonstrated more aggressive growth nature and showed increased paclitaxel chemosensitivity upon 8F5 binding. Treatment with 8F5 to A549 decreased cellular CEACAM6 expression and reversed anoikis resistance. 8F5 also decreased cellular status of Akt phosphorylation and increased apoptosis via caspase activation. In a mouse model of lung adenocarcinoma with xenotransplanted A549 cells, 8F5 treatment alone demonstrated 40% tumor growth inhibition. When combined with paclitaxel treatment, 8F5 markedly enhanced tumor growth inhibition, up to 80%. In summary, we demonstrate that anti CEACAM6 mAb is an effective therapeutic treatment for lung adenocarcinoma whose effect is further enhanced by combined treatment with paclitaxel.

Association between ABCB1 G2677T/A polymorphisms and chemosensitivity of paclitaxel in advanced gastric cancer

To investigate the association between ABCB1 polymorphisms and chemosensitivity of paclitaxel in Chinese advanced gastric cancer(AGC) patients. Clinical data and peripheral blood prior to chemotherapy of 412 AGC patients treated with first-line capecitabine plus paclitaxel(pactitaxel group, n=268) or cisplatin(cisplatin group, n=268) in Peking University Cancer Hospital from December 2008 to April 2013 were retrospectively collected. ABCB1 G2677T/A polymorphisms were determined using PCR amplification and Sanger Sequencing. Clinical response evaluation and survival analysis were performed using RECIST1.1 criteria and Kaplan-Meier curve, respectively. The associations of ABCB1 G2677T/A polymorphisms with clinical response and survival were analyzed statistically. The genotypes of ABCB1 were detected in all the patients and the frequency of wild type(G2677G), single allele variants(G2677T+G2677A), and two allele variants (T2677T+T2677A+A2677A) was 22.8%(94/412), 49.8%(205/412), and 27.4%(113/412), respectively. In paclitaxel group, the disease control rate(DCR)[89.9%(116/129)] and median progression-free survival(PFS)(190 days) of patients with single allele variants of G2677T/A were significantly higher than those of wild type patients[76.1%(51/67) and 110 days, all P<0.05], and did not differ statistically from those with two allele variants. In cisplatin group, no significant differences were observed among patients with different genotypes of ABCB1 in terms of the DCR or PFS(all P>0.05). ABCB1 G2677T/A polymorphisms are associated with chemosensitivity of paclitaxel in gastric cancer.

Upregulation of microRNA-200a associates with tumor proliferation, CSCs phenotype and chemosensitivity in ovarian cancer.

Ovarian cancer is alethal gynecologic malignancy and always has apoor prognosis. Despite new treatments modalities, the long term outcomes had not been significantly improved in the past 30 years. Although microRNA-200a (miR-200a) has been reported to be aprognostic marker in ovarian cancer, it's exact role in ovarian cancer remain unclear. In this study, we inserted the response element of miR-200a in ovarian cancer cell line via lentivirus-mediated transgene in vitro, and qRT-PCR (real time quantitative reverse transcription PCR) assay confirmed that miR-200a was up regulated compared with control. Then colony-formation assay, cell cycle analysis, CCK8 assays in vitro and xenograft experiments in vivo were performed and verified that miR-200a promoted proliferation, while blocked the formation of tumor spheroids and reduced the ratio of SP (side population) cells in ovarian cancer. Finally, we invalidated that miR-200a significantly enhanced the chemosensitivity of paclitaxel but not cisplatin in both adherent culture and sphere culture. Taken together, we demonstrated that upregulation miR-200a promoted proliferation and inhibited CSCs phenotype in OVCAR-3 ovarian cancer cell line, combined with cell cycle-targeting drug paclitaxel could effectively eliminate the "side effects" of proliferation, and showed evidences that this strategy may be promising for ovarian cancer treatment.

Combination of microtubule associated protein-tau and β-tubulin III predicts chemosensitivity of paclitaxel in patients with advanced gastric cancer

AimTo investigate the role of microtubule associated protein-tau (MAP-tau) and β-tubulin III (TUBB3) in predicting the chemosensitivity of paclitaxel in patients with advanced gastric cancer (GC). MethodsMAP-tau and TUBB3 expressions were detected using immunohistochemistry in 244 advanced GC patients prior to chemotherapy. The associations of MAP-tau and TUBB3 expressions with paclitaxel sensitivity were assessed using in vitro and in vivo xenograft analysis. ResultsA total of 149 patients receiving paclitaxel plus capecitabine (cohort 1) and 95 patients receiving cisplatin plus capecitabine (cohort 2) were included in this study. In cohort 1, the clinical benefit rate (CBR), median progression-free survival (PFS) and overall survival (OS) were found to be significantly higher in patients with low levels of MAP-tau and TUBB3 expressions and were significantly higher than those in patients with high levels of MAP-tau and TUBB3 expressions (CBR: 72.2% versus 35.9%; PFS: 271 versus 102days; OS: 394 versus 173days; all P<0.05). This was not observed in cohort 2. In in vitro studies, the sensitivity of paclitaxel in human gastric cancer cells was inversely correlated with the expression levels of MAP-tau and TUBB3, as in in vivo animal xenografts. ConclusionsThe combination of MAP-tau and TUBB3 was found to predict chemosensitivity to paclitaxel in gastric cancer in vitro and in vivo. This merits further study and may help guide individual therapy.

Association between CHFR methylation and chemosensitivity of paclitaxel in advanced gastric cancer

Aberrant methylation of CHFR (Checkpoint with forkhead associated and ring finger) gene was frequently happened in cancer and had been reported to be associated with the sensitivity of microtubule inhibitors in several cancer cells containing gastric cancer cells in vitro experiments [1]. However, the clinical significance of CHFR methylation and chemosensitivity of microtubule inhibitors (paclitaxel or docetaxel) in gastric cancer patients was investigated only in few studies with limited samples, and the conclusion was controversial [2, 3]. In this study, we aimed to investigate the association between CHFR methylation and the sensitivity of paclitaxel in Chinese advanced gastric cancer (AGC) patients to identify the potential predictor of paclitaxel. Tumor or matched non-tumor biopsies prior chemotherapy from 117 AGC patients receiving paclitaxel plus capecitabine (cohort 1, n = 59) or cisplatin plus capecitabine (cohort 2, n = 58) were retrospectively collected and analyzed for CHFR methylation status by MethyLight assay. We further investigated the association between CHFR methylation and the sensitivity of paclitaxel in AGC patients to identify its predictive significance. This study was approved by the Ethics Committee of Peking University Cancer Hospital and was performed according to the Declaration of Helsinki Principles. The median age of patients was 59 years with all patients had been evaluable for clinical response. The percentage of responders and non-responders was 36.8 and 63.2 %, respectively. Among all 117 patients, 46 patients had tumor tissues and matched non-tumor tissues. CHFR methylation occurred frequently in gastric cancer tissues (35/117, 29.9 %) compared to nontumor tissues (0/46, 0 %; P\ 0.001). For all patients, no significant differences were found between CHFR methylation and age, gender, histological differentiation, Lauren classification, metastasis, or HER2 status (all P[ 0.05). We analyzed the correlation between CHFR methylation and chemosensitivity of paclitaxel in clinical cohorts. However, no significant differences were seen between CHFR methylation and clinical response in all patients or any clinical cohort (P[ 0.05) as shown in Table 1. Up to August 2013, the median PFS and OS for all patients were 143 and 288 days, respectively. There were no differences of PFS or OS between patients with positive and negative methylation of CHFR in all patients (Table 1). For cohort 1, the median PFS and OS in patients with positive methylation of CHFR were 165 and 283 days, which had no difference with those (154 and 335 days) in patients with negative methylation of CHFR (Table 1). Also, the same results were seen in cohort 2 (Table 1). This study demonstrated that CHFR methylation was frequent and specific in gastric cancer tissues, while absent in non-tumor tissues. Our results verified that no correlation was found between CHFR methylation and paclitaxel chemosensitivity in clinical cohorts, which helped to disabuse the inconclusive results. The correlation between CHFR methylation and prognosis of patients needed to be further validated in large samples. M. Wang L. Shen (&) Department of GI Oncology, Peking University Cancer Hospital and Institute, No. 52, Fucheng Road, Haidian District, Beijing 100142, China e-mail: lin100@medmail.com.cn

The overexpression of P21-activated kinase 5 (PAK5) promotes paclitaxel-chemoresistance of epithelial ovarian cancer

P21-activated kinase 5 (PAK5) is the recently identified member of the group II p21-activated kinases (PAKs) family, which is characterized by a highly conserved amino-terminal Cdc42/Rac interactive binding domain and a carboxyl terminal kinase domain. However, the role of PAK5 in gynecological cancers has not been evaluated so far. It is remarkable that we found PAK5 was overexpressed in epithelial ovarian cancer (EOC), which is faced with an obstacle of paclitaxel resistance. Therefore, in this study, we aim to examine the PAK5 expression during EOC progression, the role of PAK5 in malignant progression of EOC and the probable relationship between PAK5 and EOC paclitaxel resistance. By immunohistochemistry, our results showed that PAK5 expression was increased with EOC progression through the adenoma to carcinoma sequence, with the highest expression level in invasive and metastatic EOCs. Furthermore, the expression level of PAK5 was also found to increase in accordance with the development of EOC Federation International of Gynecology and Obstetrics stages (P = 0.038) and differentiation grades (P = 0.008). Remarkably, those patients who recurred within 6 months after accepting tumor reductive surgery and the following carboplatin + paclitaxel chemotherapy had the highest PAK5 expression (P = 0.015). Moreover, in in vitro studies, we found that SK-OV-3 cell growth was decreased while paclitaxel chemosensitivity was correspondingly increased with the down-regulation of PAK5. Taken together, our study demonstrated that PAK5 is correlated to human EOC and increased PAK5 expression promotes EOC progression, and PAK5 regulates EOC cell paclitaxel chemoresistance.

Overexpression of centrosomal protein Nlp confers breast carcinoma resistance to paclitaxel

Nlp (ninein-like protein), an important molecule involved in centrosome maturation and spindle formation, plays an important role in tumorigenesis and its abnormal expression was recently observed in human breast and lung cancers. In this study, the correlation between overexpression of Nlp and paclitaxel chemosensitivity was investigated to explore the mechanisms of resistance to paclitaxel and to understand the effect of Nlp upon apoptosis induced by chemotherapeutic agents. Nlp expression vector was stably transfected into breast cancer MCF-7 cells. With Nlp overexpression, the survival rates, cell cycle distributions and apoptosis were analyzed in transfected MCF-7 cells by MTT test and FCM approach. The immunofluorescent assay was employed to detect the changes of microtubule after paclitaxel treatment. Immunoblotting analysis was used to examine expression of centrosomal proteins and apoptosis associated proteins. Subsequently, Nlp expression was retrospectively examined with 55 breast cancer samples derived from paclitaxel treated patients. Interestingly, the survival rates of MCF-7 cells with Nlp overexpressing were higher than that of control after paclitaxel treatment. Nlp overexpression promoted G2-M arrest and attenuated apoptosis induced by paclitaxel, which was coupled with elevated Bcl-2 protein. Nlp expression significantly lessened the microtubule polymerization and bundling elicited by paclitaxel attributing to alteration on the structure or dynamics of β-tubulin but not on its expression. The breast cancer patients with high expression of Nlp were likely resistant to the treatment of paclitaxel, as the response rate in Nlp negative patients was 62.5%, whereas it was 58.3 and 15.8% in Nlp (+) and Nlp (++) patients respectively (p = 0.015). Nlp expression was positively correlated with those of Plk1 and PCNA. These findings provide insights into more rational chemotherapeutic regimens in clinical practice, and more effective approaches might be developed through targeting Nlp to increase chemotherapeutic sensitivity.

PL2-1: Macrophages as Novel Targets for Therapy in Breast Cancer.

Abstract While BC has not historically been linked to underlying inflammation or infection, it exhibits tumor-associated inflammation marked by infiltration of innate and adaptive immune cells into developing tumors. In BC, macrophages are one of the most abundant innate immune cells present. BC-associated macrophages are regulated in part by colony stimulating factor 1 (CSF1), a key cytokine involved in monocyte/macrophage maturation, recruitment and activation, and its cognate receptor CSF1R. Macrophage presence in BC correlates with increased CSF1, increased vascular density, and worse clinical outcome. We reported that CD4+ T cells promote invasion and metastasis of mammary adenocarcinomas by directly regulating macrophage phenotype that in turn fosters invasive tumor growth, presence of circulating tumor cells and pulmonary metastasis. This preclinical data implied that women with BC heavily infiltrated by macrophages would have a worse clinical outcome as compared to tumors not heavily infiltrated with macrophages. We evaluated survival outcomes in 698 women with invasive BC treated with surgery alone and found that recurrence-free survival could be stratified based upon macrophage and T cell infiltration. Thus, we investigated CSF1 and CSF1R antagonists, in combination with standard-of-care chemotherapy (CTX) in mouse models of mammary carcinogenesis. We found that when macrophage infiltration in mammary adenocarcinomas was blocked, paclitaxel (PTX) chemosensitivity was increased, accompanied by development of productive anti-tumor immune responses and CD8+ cytotoxic T cell (CTL) infiltration. The combined effects of these changes were reduced primary tumor growth, 85% reduction in metastases and increased survival. In collaboration with clinical colleagues, we are currently evaluating the clinical benefit of macrophage modulation in preclinical models of BC to facilitate biomarker identification, and inform clinical trials of CTX in combination with macrophage-antagonists. Based on our preliminary data, we hypothesize that components of macrophage responses in BC can be identified to serve as biomarkers for risk stratification. And, that these components can be effectively targeted for therapeutic intervention, resulting in reduced late-stage BC development and metastasis when combined with CTX. LMC acknowledges generous support from the NIH/NCI (R01CA130980, R01CA13256, R01CA140943, R01CA15531), the Department of Defense (W81XWH-09-1-0342, W81XWH-10-BCRP-EOHS-EXP) and the Susan G Komen Foundation (KG111084) Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PL2-1.

IASPP and Chemoresistance in Ovarian Cancers: Effects on Paclitaxel-Mediated Mitotic Catastrophe

iASPP is a specific regulator of p53-mediated apoptosis. Herein, we provided the first report on the expression profile of iASPP in ovarian epithelial tumor and its effect on paclitaxel chemosensitivity. Expression and amplification status of iASPP was examined in 203 clinical samples and 17 cell lines using immunohistochemistry, quantitative real-time PCR, and immunoblotting, and correlated with clinicopathologic parameters. Changes in proliferation, mitotic catastrophe, apoptosis, and underlying mechanism in ovarian cancer cells of different p53 status following paclitaxel exposure were also analyzed. The protein and mRNA expression of iASPP was found to be significantly increased in ovarian cancer samples and cell lines. High iASPP expression was significantly associated with clear cell carcinoma subtype (P = 0.003), carboplatin and paclitaxel chemoresistance (P = 0.04), shorter overall (P = 0.003), and disease-free (P = 0.001) survival. Multivariate analysis confirmed iASPP expression as an independent prognostic factor. Increased iASPP mRNA expression was significantly correlated with gene amplification (P = 0.023). iASPP overexpression in ovarian cancer cells conferred resistance to paclitaxel by reducing mitotic catastrophe in a p53-independent manner via activation of separase, whereas knockdown of iASPP enhanced paclitaxel-mediated mitotic catastrophe through inactivating separase. Both securin and cyclin B1/CDK1 complex were involved in regulating separase by iASPP. Conversely, overexpressed iASPP inhibited apoptosis in a p53-dependent mode. Our data show an association of iASPP overexpression with gene amplification in ovarian cancer and suggest a role of iASPP in poor patient outcome and chemoresistance, through blocking mitotic catastrophe. iASPP should be explored further as a potential prognostic marker and target for chemotherapy.

ATP‐binding cassette B1 gene polymorphisms, mRNA expression and chemosensitivity to paclitaxel in non‐small cell lung cancer cells

The adenosine triphosphate (ATP)-binding cassette, sub-family B, member 1 (ABCB1) gene encodes P-glycoprotein (Pgp), which plays an important role in drug disposition by limiting intracellular uptake of paclitaxel. ABCB1 gene polymorphisms may alter the expression and function of Pgp, thereby influencing the response to chemotherapy. A panel of 17 non-small cell lung cancer (NSCLC) cell lines was used to investigate whether alterations in the ABCB1 gene or its mRNA expression correlated with in vitro chemosensitivity to paclitaxel. Polymorphisms in the ABCB1 gene were evaluated by direct sequencing. mRNA expression levels were assessed by quantitative real-time reverse transcription PCR. In vitro chemosensitivity to paclitaxel was expressed as half-maximal inhibitory concentration values, using a tetrazolium (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)-based colorimetric assay. The variant allele frequencies for four ABCB1 gene polymorphisms were 14.71% for 2677G>T/A, 32.35% for 2734T>C, 23.53% for 3396C>T and 76.47% for 3435C>T. There was a significant positive correlation between ABCB1 mRNA expression and half-maximal inhibitory concentration values for paclitaxel (r=0.5322, P=0.0279). None of the four ABCB1 gene polymorphisms were associated with paclitaxel chemosensitivity or ABCB1 mRNA expression in the 17 cell lines. These in vitro results suggest that high ABCB1 mRNA expression may be a predictive biomarker for poor chemosensitivity to paclitaxel. The panel of NSCLC cell lines may provide clues and indications for establishing clinically useful relationships between a given polymorphism or level of gene expression and chemosensitivity to an anti-cancer agent.

Prediction of nonsmall cell lung cancer sensitivity to cisplastin and paclitaxel based on marker gene expression

The goal of the present study was to define gene expression signatures that predict a chemosensitivity of non-small cell lung cancer (NSCLC) to cisplatin and paclitaxel. To generate set of candidate genes likely to be predictive a current knowledge of the pathways involved in resistance and sensitivity to individual drugs was used. Forty four genes coding proteins belonging to following categories: ATP-dependent transport proteins, detoxification system proteins, reparation system proteins, tubulin and proteins responsible for its synthesis, cell cycle and apoptosis proteins were considered. Eight NSCLC cell lines (A549, Calul, H1299, H322, H358, H460, H292, and H23) were used in our study. For each NSCLC cell line a cisplatin and paclitaxel chemosensitivity as well as an expression level of 44 candidate genes were evaluated. To develop a chemosensitivity prediction model based on selected genes expression level a multiple regression analysis was performed. The model based on the expression level of 11 genes (TUBB3, TXR1, MRP5, MSH2, ERCC1, STMN, SMAC, FOLR1, PTPN14, HSPA2, GSTP1) allowed us to predict the paclitaxel cytotoxic concentration with high level of correlation (r = 0.91, p < 0.01). However, none model developed was able to reliably predict a sensitivity of the NSCLC cells to cisplatin.