Platinum-resistant Cell Lines Research Articles

Abstract 3642: Phospho-CHK1 as a prognostic biomarker in ovarian cancer and a potential target in platinum-resistant disease

Abstract Purpose: Ovarian cancer frequently relapses due to inherent or acquired resistance to platinum-based therapy. To increase therapeutic response, the checkpoint kinases 1 and 2 (CHK1, CHK2) are being targeted in early clinical trials in combination with DNA damaging agents due to their roles in sensing DNA damage and inducing cell cycle arrest. Due to the limited studies on the use of biomarkers to overcome platinum resistance, we set out to characterize potential targets in a cohort of ovarian tumors and paired platinum sensitivity ovarian cancer cell lines. Methods: Total and phospho-protein expression were measured in a panel of 12 ovarian cancer cell lines using antibody arrays in order to characterize signaling pathways. Differentially expressed proteins were measured in a cohort of 128 pre-treatment malignant ovarian tumour lysates using reverse phase protein arrays. Sulforhodamine B cytotoxicity assays were used to assess the DNA damage response relevance of potential targets in paired platinum-sensitive and -resistant ovarian cancer cell lines. Results: Cell line expression clustering identified two groups that differed in patient history: predominantly platinum-based chemotherapy or chemotherapy-naïve background. The overexpressed proteins in the former cluster comprised mainly DNA damage response proteins including p-CHK1 (Ser317) and p-CHK2 (Ser516) implicating these proteins as potential mediators of platinum resistance. Though p-CHK2 had no prognostic value, high p-CHK1 levels were associated with poor overall survival in univariate analysis (medians 21 vs 38 months; corrected P=0.03). In multivariate analysis using a Cox proportional hazard model with other significant factors from univariate analysis (platinum sensitivity, stage, grade, residual disease, and histology), high p-CHK1 tumors had a relative risk of 3.0 (95% CI 1.1 - 8.0, P=0.03), 5.6 for platinum sensitivity (95% CI 2.8-11.1, P<0.001), 1.9 for tumor stage (95% CI 1.2-3.1, P=0.005), and 1.5 for histology (95% CI 1.1-1.9, P=0.004). CHK1 is phosphorylated at Ser317 during DNA damage and accordingly the DNA damage marker p-H2AX (Ser139) was highly expressed in the high p-CHK1 tumor group (P=0.008). The CHK1/2 inhibitor AZD7762 addition had variable effects on the cisplatin dose response when comparing the platinum-sensitive to the paired platinum-resistant cell lines. 50 nM AZD7762 (5.7% growth inhibition) and cisplatin treatment of the platinum resistant PEO4 cells induced a cisplatin concentration response (combination IC50=1.7μM vs cisplatin IC50=9.8μM) similar to that of its paired platinum sensitive PEO1 cell line (cisplatin IC50=2.7μM). Conclusion: This is the first study to identify p-CHK1 as an independent prognostic ovarian cancer biomarker and supports CHK1 as a therapeutic target in platinum-resistant ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3642. doi:1538-7445.AM2012-3642

Abstract A21: Tissue platinum concentration and tumor response in non-small cell lung cancer

Abstract Background: Platinum resistance is a major limitation in the treatment of advanced non-small cell lung cancer (NSCLC). Reduced intracellular drug accumulation is one of the most consistently identified features of platinum-resistant cell lines, but clinical data are limited. We assessed effects of tissue platinum concentrations on tumor response, time to recurrence, progression-free survival (PFS) and overall survival (OS) in patients with early-stage NSCLC following neoadjuvant platinum-based chemotherapy. Methods: We measured total platinum concentrations in 44 archived fresh frozen NSCLC specimens from patients who received neoadjuvant platinum-based chemotherapy. Samples were analyzed by flameless atomic absorption spectrophotometry to assess absorbance reading associated with platinum concentration. Four specimens from patients who underwent surgery only and one patient who received pemetrexed as neoadjuvant chemotherapy were analyzed as negative controls. Absorbance value per mg of tissue was correlated with percent reduction in tumor size on post- vs prechemotherapy CT scans. Kaplan-Meier curves and log-rank tests were used to evaluate differences in time to recurrence and survival between two groups divided by median platinum concentration. Results: Platinum absorbance values in 44 neoadjuvant specimens were easily detectable and ranged from 0.6 to 6.7 × 10−3 per mg of tissue while five negative controls demonstrated absorbance readings similar to 0.1N HCL. Platinum absorbance correlated significantly with percent reduction in tumor size (R2 = 0.48, P<0.00001). The same correlations were seen in cisplatin (P = 0.0003), carboplatin (P = 0.0013), adenocarcinoma (P = 0.0003) and squamous cell carcinoma (P = 0.019) groups. Furthermore, there was no significant impact of potential variables such as the type of platinum compound, number of cycles, pre-treatment tumor diameter and time lapse from last chemotherapy on platinum concentration. Patients with lower Pt concentration also had shorter time to recurrence (P = 0.0494), progression free survival (P = 0.0366) and overall survival (P = 0.035). Conclusion: This is the first tissue-based study to establish a relationship between tissue platinum concentrations and response in NSCLC. Reduced intratumoral platinum accumulation may constitute a significant mechanism of platinum resistance even in clinical specimens. Further studies investigating factors that modulate intracellular platinum concentration are warranted (Supported by National Foundation of Cancer Research 90088436, Department of Defense W81XWH-07-1-0306, National Institute of Health CA127263, CA160687 and CA16672).

Epigenetic regulation and tumorigenicity of ovarian cancer cells.

e15546 Background: Ovarian cancer typically has an excellent response to chemotherapy but has a high rate of recurrence with patients ultimately dying from chemoresistance. The objective of this study was to investigate the relationship between histone modification, DNA methylation and DNA damage repair response that occur in platinum resistant ovarian cancer cell lines treated with epigenetic modifiers in combination with low dose cisplatin to develop new approaches for chemotherapy resistant ovarian cancer. Methods: We selected two common epigenetic modifiers Trichostain A (TSA), a histone deacetylase inhibitor and 5-aza-2'-deoxycytidine (decitabine), a demethylating agent which were evaluated alone and in combination with low dose cisplatin for their tumorigencity in SCID mice and their spheroid formation in serum-free medium with two ovarian cancer cell lines SKOV3 and Hey. We also measured the effect of these drugs on tumor suppression pathways, AKT pathway, and expression of cadherins as well as histone modification. Results: All combinations of TSA, decitabine and cisplatin inhibited both tumorigenicity and spheroid formation in both cell lines with Hey exhibiting more resistance to treatment. E-cadherin expression was suppressed in SKOV3 cells while N-caderin expression was suppressed in Hey cells with both TSA and cisplatin and decitabine and cisplatin. In the Hey cell line, p53 expression was increased by sequential treatment with low dose decitabine and low dose cisplatin and by low dose TSA and low dose cisplatin. The profiling of histone code revealed distinctly different chromatin regulation between Hey cells and SKOV3 cells. The expression patterns and characteristics of cell adhesion, mutiple-drug transporter, apoptosis are distinctly different in these two ovarian cancer cell lines, which may be due to the different origins and stages of these cell lines. Conclusions: The combinations of decitabine, TSA and cisplatin suppressed the formation and growth of xenograft and tumor spheroid, altered the molecular mechanism of cell adhesion, chemosensitivity, and chromatin modification which may provide a new and promising treatment strategy for chemoresistant ovarian cancer.

Abstract 4949: microRNA (miR) analysis as a tool for discovering platinum resistance mechanisms in non-small cell lung cancer (NSCLC)

Abstract Background: NSCLC is the most common cause of cancer-related death in the world. Platinum-based chemotherapy is the mainstay of treatment both in the adjuvant and the metastatic settings, but platinum resistance is common. A variety of mechanisms have been reported to underlie platinum-resistance. miRs are short non-coding RNA molecules, each regulating a large number of genes. We hypothesized that in platinum-resistant NSCLC cells, a small number of differentially expressed miRs modulating an abundance of gene products, activate in concert a large number of platinum-resistance mechanisms. Aim: To uncover mechanisms of platinum-resistance in NSCLC, by indentifying miRs differentially expressed in platinum resistant cells. Methods: Platinum-resistant cell lines were created by cisplatin selection of Calu6 and NCI-H23, NSCLC cell lines. RNA was hybridized to Affymetrix miR arrays, followed by TargetScan algorithm analysis. Levels of specific miRs were quantified by RT-PCR (ABI) and were modulated by miR inhibitors (Ambion). Results: Two sets of sensitive (S) and platinum-resistant (R) cell lines were created, each set consisting of a parental S line and several R sub-lines. miR array analysis was performed on the S and R lines. miRs that were significantly more abundant in each of four R lines compared to the corresponding S lines were compiled. Genes predicted to be targeted by each of those miRs with a p value < .05 were tabulated. Surprisingly, only 16 genes were common to those four gene lists, including CDK6, an important G1-S cell cycle regulator. mRNA and protein levels of CDK6 were higher in S compared to R lines, and accordingly, growth rate of R lines was slower. miR145, one of the miRs upregulated in the R lines, is predicted to target CDK6. Transfection of a miR145 inhibitor resulted in a mild increase in mRNA levels of CDK6, supporting its role as a regulator of CDK6. Conclusions: Utilizing a number of biological systems (sets of cells lines) that model the same phenotype (platinum resistance) allows for a robust analysis of the involved molecules. miR profiling of multiple systems can identify genes that are commonly regulated in different systems by different miRs. Using this approach we discovered upregulation of miRs, including miR145, resulting in downregulation of CDK6, as a potential mechanism of platinum resistance in NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4949. doi:10.1158/1538-7445.AM2011-4949

Abstract 3517: Epigenetic interactive regulation and synergistic action of Trichostatin A, 5-aza-2′-deoxycytidine and Cisplatin in ovarian cancer

Abstract Objective: To investigate the relationship between histone modification, DNA methylation and DNA damage repair response that occur in platinum resistant ovarian cancer cell lines treated with epigenetic modifiers in combination with low dose chemotherapy to develop new approaches for chemotherapy resistant ovarian cancer. Materials and Methods: We selected two common epigenetic modifiers Trichostatin A (TSA), a histone deacetylase inhibitor and 5-aza-2′-deoxycytidine (decitabine), a demethylating agent which were evaluated alone and in combination with low dose Cisplatin for their tumorigencity in SCID mice and their spheroid formation in serum-free medium with two ovarian cancer cell lines SKOV3 and Hey. We also measured the effect of these drugs on tumor suppression pathways, AKT pathway, and expression of cadherins as well as histone modification. Results: All combinations of TSA, decitabine and Cisplatin inhibited both tumorigenicity and spheroid formation in both cell lines with Hey exhibiting more resistance to treatment. E-cadherin expression was suppressed in SKOV3 cells while N-caderin expression was suppressed in Hey cells with both TSA and Cisplatin and decitabine and Cisplatin. In the Hey cell line, p53 expression was increased by sequential treatment with low dose decitabine and low dose cisplatin and by low dose TSA and low dose cisplatin. The profiling of histone code revealed distinctly different chromatin regulation between Hey cells and SKOV3 cells. The expression patterns and characteristics of cell adhesion, mutiple-drug transporter, apoptosis are distinctly different in these two ovarian cancer cell lines, which may be due to the different origins and stages of these cell lines. Conclusions: The combinations of decitabine, TSA and Cisplatin suppressed the formation and growth of xenograft and tumor spheroid, altered the molecular mechanism of cell adhesion, chemosensitivity, and chromatin modification which may provide a new and promising treatment strategy for chemoresistant ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3517. doi:10.1158/1538-7445.AM2011-3517

Abstract 1729: Connecting Gli1 with chemoresistance in small cell lung cancer

Abstract Background. Small-cell lung cancer (SCLC) accounts for ∼15% of all lung cancers, and is the most aggressive form of lung cancer. Drug resistance to chemotherapeutics is particularly crippling in SCLC as there are limited treatment options. We previously examined gene expression in platinum-resistant SCLC cell lines, and reported aberrant expression of a subset of hedgehog pathway related genes, among which Gli1 was particularly significant. Gli1 is a known transcription factor that is involved with regulation of a number of key genes in cell fate determination and proliferation. In recent years, its role in oncogenesis and cancer progression has been studied. Herein, we test the hypothesis that increased Gli1 expression contributes to cisplatin and etoposide resistance in SCLC. Methods. Two SCLC cell lines, H69 and H526, were transduced with a Gli1 over-expressing (o/e) vector (Kasper et al, 2007) to create two lines expressing the gene: H69-Gli1 and H526-Gli1. Gli1 expression was verified by qRT-PCR. Cellular proliferation was measured over 96 hours. To study the drug dose response, Gli1 o/e and parental lines were treated with single-agent or combination dosing of cisplatin and etoposide (standard first-line SCLC therapy), and cell viability was assessed after 72 hours. Relative Gli1 expression was compared with cisplatin response in four additional SCLC cell lines (H146, H187, H345, H1688). To further understand aberrant cellular signaling in Gli1 o/e cells, gene expression analysis was performed on H69-Gli1 and H69 parental cells (2 replicates) using Agilent 44K expression arrays. Data was normalized and analyzed using Genespring software. Changes greater than 2 fold were considered significant. Results. Compared to parental cells, Gli1 expression was 11 fold and 149 fold higher in H69-Gli1 and H526-Gli1, respectively. Proliferation assays showed no significant differences in parental vs. Gli1 o/e cells. Drug dose response experiments showed at least a 1.5 fold shift in cisplatin resistance in H69-Gli1 compared to H69. The same trend was also observed in combination dosing of cisplatin and etoposide. H526-Gli1 showed no significant change in cisplatin resistance compared to H526. In the four additional SCLC lines a positive trend was observed where increased Gli1 expression correlated with increased cisplatin resistance. Gene expression analysis showed that there are over 1200 genes significantly differentially-expressed in H69-Gli1 cells as compared to parental cells. Conclusions. Gli1 over-expression leads to increased platinum resistance in SCLC cells. We are currently in the process of performing pathway analysis on the gene expression data to study the pathways affected by Gli1 over-expression in H69 cells, leading to increased platinum resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1729. doi:10.1158/1538-7445.AM2011-1729

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Targeting Aldehyde Dehydrogenase Cancer Stem Cells in Ovarian Cancer

Aldehyde dehydrogenase-1A1 (ALDH1A1) expression characterizes a subpopulation of cells with tumor-initiating or cancer stem cell properties in several malignancies. Our goal was to characterize the phenotype of ALDH1A1-positive ovarian cancer cells and examine the biological effects of ALDH1A1 gene silencing. In our analysis of multiple ovarian cancer cell lines, we found that ALDH1A1 expression and activity was significantly higher in taxane- and platinum-resistant cell lines. In patient samples, 72.9% of ovarian cancers had ALDH1A1 expression in which the percentage of ALDH1A1-positive cells correlated negatively with progression-free survival (6.05 vs. 13.81 months; P < 0.035). Subpopulations of A2780cp20 cells with ALDH1A1 activity were isolated for orthotopic tumor-initiating studies, where tumorigenicity was approximately 50-fold higher with ALDH1A1-positive cells. Interestingly, tumors derived from ALDH1A1-positive cells gave rise to both ALDH1A1-positive and ALDH1A1-negative populations, but ALDH1A1-negative cells could not generate ALDH1A1-positive cells. In an in vivo orthotopic mouse model of ovarian cancer, ALDH1A1 silencing using nanoliposomal siRNA sensitized both taxane- and platinum-resistant cell lines to chemotherapy, significantly reducing tumor growth in mice compared with chemotherapy alone (a 74%-90% reduction; P < 0.015). These data show that the ALDH1A1 subpopulation is associated with chemoresistance and outcome in ovarian cancer patients, and targeting ALDH1A1 sensitizes resistant cells to chemotherapy. ALDH1A1-positive cells have enhanced, but not absolute, tumorigenicity but do have differentiation capacity lacking in ALDH1A1-negative cells. This enzyme may be important for identification and targeting of chemoresistant cell populations in ovarian cancer.

F3-Targeted Cisplatin-Hydrogel Nanoparticles as an Effective Therapeutic That Targets Both Murine and Human Ovarian Tumor Endothelial Cells In vivo

Recent studies indicate that ovarian cancer may be highly responsive to antivascular therapeutics. We have developed an antivascular tumor therapeutic using the F3 peptide to target cisplatin-loaded nanoparticles (F3-Cis-Np) to tumor vessels. We show that although F3-Cis-Np bind with high specificity to both human ovarian tumor cells and tumor endothelial cells in vitro, they only show cytotoxic activity against the tumor endothelial cells. In vivo these nanoparticles bind primarily to tumor endothelial cells. Therapeutic studies in both flank and orthotopic i.p. murine ovarian tumor models, as well as human tumor xenograft models, show rapid tumor regression with treatment. Treatment was associated with significant vascular necrosis consistent with an antivascular effect. Furthermore, treatment was active in both platinum-sensitive and platinum-resistant cell lines. Importantly, we show that F3-Cis-Np bind to human tumor endothelial cells in vitro and to human tumor vessels in vivo. Therapy targeting human vasculature in vivo with F3-Cis-Np led to near complete loss of all human tumor vessels in a murine model of human tumor vasculature. Our studies indicate that F3-targeted vascular therapeutics may be an effective treatment modality in human ovarian cancer.

Mitochondrial comparative proteomics of human ovarian cancer cells and their platinum‐resistant sublines

Resistance to platinum-based chemotherapy is the major obstacle to successful treatment of ovarian cancer. It is evident that mitochondrial defects and the dysfunctions of oxidative phosphorylation and energy production in ovarian cancer cells were directly related to their resistance to platinum drugs. Using 2-D DIGE, we compared mitochondrial proteins from two platinum-sensitive human ovarian cancer cell lines (SKOV3 and A2780) with that of four platinum-resistant sublines (SKOV3/CDDP, SKOV3/CBP, A2780/CDDP, and A2780/CBP). Among the 236 differentially expressed spots, five mitochondrial proteins (ATP-α, PRDX3, PHB, ETF, and ALDH) that participate in the electron transport respiratory chain were identified through mass spectrometry. All of them are downregulated in one or two of the platinum-resistant cell lines. Three proteins (ATP-α, PRDX3, and PHB) were validated by using western blot and immunohistochemistry. There is a significant decrease of PHB in tumor tissues from ovarian cancer patients who were resistant to platinum-based chemotherapies. This is the first direct mitochondrial proteomic comparison between platinum-sensitive and resistant ovarian cancer cells. These studies demonstrated that 2-D DIGE-based proteomic analysis could be a powerful tool to investigate limited mitochondrial proteins, and the association of PHB expression with platinum resistance indicates that mitochondria defects may contribute to platinum resistance in ovarian cancer cells.

Aberrant methylation of breast and ovarian cancer susceptibility gene 1 in chemosensitive human ovarian cancer cells does not involve the phosphatidylinositol 3′‐kinase–Akt pathway

Methylation is an important silencing mechanism of breast and ovarian cancer susceptibility gene 1 (BRCA1) expression in sporadic ovarian cancer. However, the role of BRCA1 methylation in chemotherapy in sporadic ovarian cancer and the related pathways have not been understood completely. This study has determined the roles of BRCA1 hypermethylation in chemotherapy of sporadic ovarian cancer and its related signaling pathways. We used bisulfite sequencing, real-time polymerase chain reaction, and western blotting to check the methylation state and expression levels of BRCA1 of the following cell lines: platinum-sensitive human ovarian cancer cell line COC1, platinum-resistant cell line COC1/DDP, SKOV-3, and 5-Aza-dC treated COC1. The cisplatin sensitivity of ovarian cancer cells was examined by MTS (methyl-thiazol tetrazolium) assay. Tumorigenicity in vivo and DDP-based chemosensitivity were compared among the above cells. Phosphatidylinositol 3'-kinase (PI3K)-Akt pathway activation in ovarian cancer cells was studied by western blotting. The frequency of BRCA1 methylation in the COC1 cell line was higher than in COC1/DDP and SKOV-3 cell lines, whereas the mRNA and protein expression of BRCA1 were lower than in the COC1/DDP and SKOV-3 cell lines. DNA demethylation decreased the chemosensitivity of COC1 cells and partially increased the expression levels of BRCA1. The activation of the PI3K-Akt pathway was low in ovarian cancer cells. Our results indicate that hypermethylation of BRCA1 might play an important role in the chemosensitivity of ovarian cancer, and that the PI3K-Akt pathway is not involved in this response.

Abstract 4142: The effects of SBHA on Notch 1 expression and platinum sensitivity in ovarian cancer cells

Abstract Objective: Despite advances in radical surgical debulking and intraperitoneal chemotherapy, ovarian cancer remains the most deadly gynecologic malignancy. The Notch signaling pathway plays an important role in cellular differentiation, proliferation, and apoptosis. We have previously found Notch 1 intracellular domain (NICD) to be highly expressed in the most platinum resistant ovarian cancer cell lines. Recent evidence has shown that histone deacetylase inhibitors (HDACi's) decrease platinum resistance in oral squamous cell cancers. In addition, the HDACi suberoyl bis-hydroxamic acid (SBHA) has been found to modify Notch 1 expression in other cancers. We hypothesized that SBHA would reduce ovarian cancer cell proliferation through inhibition of the Notch 1 signaling pathway and help sensitize ovarian cancer cells to platinum. Methods: Following treatment with SBHA, NICD expression in the ovarian carcinoma cell lines OVCAR3, SKOV3, and CaOV3 was analyzed by Western blot. The effects of SBHA and cisplatin on ovarian cancer cell growth were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) growth assay. Results: NICD expression by Western blot was consistently reduced in all 3 cell lines following treatment with SBHA. Dose-dependent growth inhibition of ovarian cancer cells was demonstrated with SBHA treatment. Furthermore, the addition of SBHA to cisplatin resulted in additional reduction of NICD and further growth suppression in SKOV3, the most platinum resistant cell line tested. Conclusions: SBHA treatment of ovarian cancer cells led to decreased NICD expression and dose dependent growth reduction in ovarian cancer cells. In addition, we found that SBHA in combination with platinum further reduced NICD expression and growth in SKOV3 cells. These findings provide a compelling argument for further study into the role that Notch 1 signaling may be playing in platinum resistance and ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4142.

Abstract 2570: Novel mechanisms of taxane and platinum resistance in esophagogastric cancer cells by gene expression profiling

Abstract BACKGROUND Platinum analogues and taxanes are clinically effective in esophagogastric (EG) cancers and demonstrate significant non-cross resistance. Nevertheless, resistance (primary or acquired) remains a major therapeutic problem. We have developed platinum and docetaxel resistant cell line models and aim to use gene expression profiling for hypothesis generation to identify; predictive biomarkers; novel mechanisms of action and resistance; therapeutic targets and to provide insight into mechanisms of cross resistance. This will inform optimal cytotoxic and targeted therapy combination strategies. METHODS Resistant daughter cell lines were generated from 3 parental EG cancer cell lines (OE33, OE21, AGS) by exposure to increasing concentrations of oxaliplatin, cisplatin and docetaxel for at least 12 months. Cell viability was measured at 72h by the MTT assay. IC50 values were calculated in Prism Software and P&amp;lt;0.05 were statistically significant (Student's t-test). Affymetrix GeneChip Exon 1.0 were used to measure gene expression. Gene expression data were normalized in GeneSpring GX v10.0.1 using RMA for summarisation and baseline transformation of log transformed data to the median. The microarrays passed QC analysis, including hybridisation and labelling controls, Pos_vs_Neg_AUC and all probe Set RLE mean. RESULTS Initial data available compared daughter oxaliplatin resistant OE21ox4 line to OE21wt. Of the 17881 core genes present on the array, 14727 were expressed above background (20th −100th percentile) and of these the expression of 528 transcripts was altered by ≥ 2 fold (3 fold, n=113 and 5 fold, n=26). MMP9 demonstrated the highest change (14.3 fold decrease in resistant cells). Interestingly, ABCC2/ MRP, known to confer resistance to many cytotoxics, including taxanes but not platinums, was reduced by 6.3 fold in resistant cells. Gene ontology analysis identified transcripts whose products are associated with the extracellular region as significantly represented in the 538 entity gene set (P=6.971 × 10−7). Seven biological pathways were significantly altered in oxaliplatin-resistant compared to the sensitive parental OE21 cells (2fold geneset, n=528): alpha VI beta IV integrin (P=0.004), EGFR1 (P=0.003), ID (P=2.41^-4), IL1 (P=0.013), IL2 (P=0.027), IL6 (P=0.021), TCR (P=0.039). The αVIβIV and IL1 pathways were also significantly altered after chemotherapy in a parallel study of serial biopsies from EG cancer patients in our group. CONCLUSION We have a panel of EG cancer cell lines representative of clinical subgroups of the disease and have developed resistant daughter lines to oxaliplatin, cisplatin and docetaxel. A preliminary analysis of the OE21 oesophageal squamous carcinoma cell line and its oxaliplatin resistant daughter line is presented. A comparative gene expression data analysis of the entire panel of cell lines will be presented at the meeting. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2570.

Decitabine effect on human tumor expression of various transporters

2540 Background: We reported that immunohistochemistry (IHC) expression of the platinum/copper transporter CTR1 in human tumors correlated inversely with DNA methylation and was increased by the DNA demethylating agent decitabine (D. Stewart et al, Proc ASCO 2008). Decitabine also increased expression of folate transporters in platinum-resistant cell lines that have decreased expression of folate transporters and of small GTPases such as RHOA that regulate endocytosis (D Shen et al. Br J Cancer 2004). Methods: Tumors were biopsied pre decitabine and on cycle 1 day 12 in 31 patients with refractory malignancies in a phase I study of decitabine given days 1–5 ± 8–12 each cycle. We used IHC to assess the folate carriers FOLR1 and RFC1, the glucose transporter GLUT4 and the endocytosis regulating small GTPase RHOA. Scores of 0–300 were calculated by multiplying staining intensity (0–3) by % cells staining. LINE assays were used to assess % global DNA methylation. Results: DNA methylation did not correlate with FOLR1, RFC1 and GLUT4 scores but did correlate inversely with RHOA (r= -0.58, p=0.006). Median tumor IHC scores post vs pre decitabine were 80 vs 80 (range, 0–270 vs 0–210, p=0.89) for FOLR1, 90 vs 90 (range, 15–300 vs 0–300, p=0.17) for RFC1, 0 vs 0 (range 0- 140 vs 0–70, p=0.61) for GLUT4, and 77.5 vs 50 (range, 0–210 vs 0–210, p=0.03) for RHOA. If only tumors with pre decitabine scores &lt;150 were included, post vs pre RFC1 differences were significant (95 vs 80, p=0.004). Outcomes were similar whether or not 4 melanoma patients (in whom melanin pigment decreased the reliability of IHC scoring) were included. Conclusions: As previously noted with the copper/platinum transporter CTR1, treatment with the DNA demethylating agent decitabine was associated with a significant increase in tumor expression of the endocytosis regulator RHOA in resistant human tumors. Expression of the folate transporter RFC1 was also increased if only patients with levels that were initially low were included. This suggests that decitabine should be tested for its ability to increase uptake of chemotherapy in resistant tumors. Promoter methylation for these transporters has not yet been assessed. Supported in part by NIH grant UO1 CA062461–10 &amp; R21 CA112895–01A1. No significant financial relationships to disclose.

Impact of Platinum on the Whole Mitochondrial Genome of Ovarian Carcinomas Both In Vivo and In Vitro

To investigate somatic mitochondrial DNA mutation in primary and recurrent ovarian carcinoma tissues as well as that in drug-resistant cell lines to illuminate the impact of chemotherapeutic drugs on mitochondrial DNA (mtDNA). Complete mtDNA genomes of 20 pairs of ovarian carcinomas and their matched normal tissues together with 2 ovarian carcinoma cell lines and their 4 platinum-resistant cell lines were sequenced. Mitochondrial DNA alterations, consequent amino acid alterations were compared between the 2 groups of patients and the 2 types of cell lines. A large number of mtDNA new polymorphisms (55) and mutations (18) were identified in 20 ovarian carcinoma samples. Platinum-based chemotherapy did not increase the number of new polymorphisms (P = 0.094), mutations (P = 0.688), and consequent amino acid alterations (P = 0.202 and 0.795). Data gained from the cell lines also indicated that platinum had some effect on the mitochondrial genome but not specific to particular positions. What we found suggested that mtDNA damage could be made by chemotherapeutic drugs but not as much as imagined in ovarian carcinomas. Some of the mtDNA defects might be part of the disease processes and cell properties as well as a consequence of treatment.

Gefitinib Maintenance in Stage III Non–Small-Cell Lung Cancer

■■■ TOTHEEDITOR:Kellyetal 1 recentlyreportedinJournalofClinical Oncology that gefitinib maintenance after chemoradiotherapy and consolidation chemotherapy was associated with a significant decreaseinoverallsurvival(butonlyaslightdecreaseinprogression-free survival)comparedwithplaceboinstageIIInon‐small-celllungcancer(NSCLC).Theyofferpotentialexplanationsforthis,asdoKeedyet al 2 in an accompanying editorial. We would like to suggest an additional possible reason for this outcome. The flattening of the NSCLC dose-response curve at higher chemotherapy doses suggests that the most important factor in chemotherapy resistance is deficiency of factors required for efficacy. 3 Platinum-resistant cell lines may have decreased expression of a range of transporters that are important for cellular uptake of chemotherapy and nutrients. 4 In patients with a variety of resistant tumor types, we assessed tumor expression (by immunohistochemistry) of the copper transporter CTR1, which is also important in platinum uptake. CTR1 expression was significantly lower in tumors of patients who had received either chemotherapy or targeted therapies withintheprevious3monthsthanintumorsofpatientswithalonger interval off therapy. 5 The correlation with time from last chemotherapy or targeted therapy was stronger than the correlation with time from last cytotoxic therapy alone. This leads us to hypothesize that either chemotherapy or targeted therapy may result in a broad reduction in membrane transporters and that this, in turn, may generate broad cross resistance. This could, in part, explain why adding tyrosine kinase inhibitors to chemotherapy did not improve outcome in chemotherapy-naive advanced NSCLC patients 6 and might also predict that monoclonal antibodies (that do not require uptake across membranes) might add to chemotherapy even if small molecules did not. If correct, this would also predict that most other currentlyavailablesmallmoleculestargetingtumorcellswilladdlittle

Phase I and pharmacokinetic study of NC-6004, a new platinum entity of cisplatin-conjugated polymer forming micelles

2573 Background: NC-6004 contains cisplatin derivatives in micellar nanoparticles. Preclinical activity was seen in platinum resistant cell lines. We report a phase I trial of NC-6004 with doses from 10–120mg/m2 cisplatin equivalent. Methods: NC-6004 dose was escalated in patients with refractory solid tumors according to toxicity with 1 patient per dose level until grade 2 toxicity observed then 3–6 evaluable patients per dose level. Eligible patients had adequate bone marrow, hepatic and renal function. NC-6004 was administered as a 1 hour intravenous infusion on day 1 of a 21 day cycle (C). Patients were monitored for usual dose-limiting toxicities (DLT) and renal function assessed by EDTA GFR. Pharmacokinetics (PK) of cisplatin were characterized by atomic absorption spectrometry for total platinum, micellar component by gel-filtration, inductively-coupled plasma mass spectrometry for free platinum species. Patients were restaged every 2 cycles and treatment continued if clinical benefit and acceptabl...

Clinical relevance of a novel EGF pathway directly linked to E2F3a in ovarian cancer

5588 Background: Over the last decade the retinoblastoma protein and its down-stream effectors have been shown to be involved in tumorigenesis and progression in various cancer entities. Especially the E2F family of transcription factors, are regarded to be fundamental players in the deregulation of the cell cycle control. Proliferation-promoting E2F1 to E2F3a are responsible for promoting the cell cycle at the G1/S phase checkpoint, E2F3b to E2F8 seem to act as inhibitors of cell proliferation. By our knowledge, there are no data considering a direct interplay between the EGF-R dependent pathways and the E2F family. Methods: The functional relationship between E2F3 isoforms and EGF-R was evaluated in EGF stimulated ovarian cancer cell lines by the use of RT-PCR. Protein expression was estimated by immunoblot analyses. In vitro data and expression of miRNA-34a were confirmed in a collective of 128 ovarian cancer patients. Results: Herein we show that in ovarian cancer cell lines EGF and TGF-alpha treatment selectively increase the transcription of E2F3a, whereas all other members of the E2F-family remain unaffected upon EGF-R stimulation. E2F3a transcription was completely abolished by simultaneous treatment with EGF-R inhibitors such as Cetuximab, Erlotinib and Lapatinib. Furthermore, the E2F3a peak observed after 3 to 12 hrs after EGF treatment was associated with the initiation of cell proliferation. In addition, our investigations reveal, that EGF treatment also reduced the transcription of miRNA-34a, a specific negative epigenetic regulator of E2F3a, in ovarian cancer cells. E2F3a and E2F3b expression are shown to be elevated in 128 ovarian cancer tissue samples compared to healthy controls. Both isoforms were significantly associated with FIGO stage and histological grading. However, only the proliferation-promoting E2F3a was associated with immunohistochemically demonstrated phosphorylated EGF-R and turned out to be an independent prognosticator for progression free and overall survival. In line with this, E2F3a was also the only member of the E2F-family which was constitutively up-regulated in platinum resistant ovarian cancer cell lines. Conclusion: Taken together, this so far un-described EGF-E2F3a axis seems to play a pivotal role in the tumor biology of ovarian cancer. No significant financial relationships to disclose.

Characterization of Oxaliplatin−DNA Adduct Formation in DNA and Differentiation of Cancer Cell Drug Sensitivity at Microdose Concentrations

(trans-R, R)-1,2-diaminocyclohexaneoxalatoplatinum(II) (oxaliplatin) is a recently approved platinum analogue for use in the chemotherapy of metastatic colorectal cancer. Like many cytotoxic drugs, oxaliplatin exerts its antitumor effects by covalent modification of DNA. We report an accelerator mass spectrometry (AMS) assay to measure the kinetics of oxaliplatin-induced DNA damage and repair. We determined the apparent rate of oxaliplatin adduction to salmon sperm DNA. The oxaliplatin-DNA adduct distribution was further investigated at the nucleoside level by HPLC-AMS. Cultured platinum-sensitive testicular (833K) and platinum-resistant breast and bladder (MDA-MB-231 and T24, respectively) cancer cells were incubated with a subpharmacological concentration of oxaliplatin (0.2 microM). Both cellular and DNA radiocarbon contents in the drug-sensitive testicular cells had approximately twice the area under the curve as compared to the more platinum-resistant cell lines, implying that differential accumulation of the drug may be responsible for the sensitivity of cancer cells to platinum treatment. The lowest concentration of radiocarbon measured was approximately 1+/-0.1 amol/microg of DNA, when assaying 1 microg of DNA. This sensitivity for measuring oxaliplatin-DNA adducts is the highest reported to date. The sensitivity offered by this method may be applicable to other DNA-damaging drugs, metabolisms studies, and diagnostics development.

Novel cytotoxic agents from an unexpected source: Bile acids and ovarian tumor apoptosis

Objective Unique biologic activities have been identified for the 4 different bile acids: cholic acid (CA, chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), and ursodeoxycholic acid (UDCA). The aim of this study was to examine and compare the effects of these 4 bile acids on the human ovarian cancer cell lines A2780 and A2780-CP-R(cisplatin-resistant) and to evaluate mechanisms of action. Methods Antiproliferative effects were determined by the cytotoxic MTT assay. Cells undergoing apoptosis were identified by morphologic analysis of cells stained using Diff-Quick and nuclear staining with DAPI and by quantitative nucleosome ELISA assay. Cells were lysed in buffer after 24 h of exposure to three different concentrations of bile acid (50 mM, 200 mM, and 400 mM) and protein concentrations were determined. Cell extracts containing 25 mg of protein were assayed for protein kinase C (PKC) enzyme activity. Results None of the bile acids stimulated proliferation of ovarian cancer cells. CA and UDCA had only minimal cytotoxic effect even at maximum concentrations. In contrast, DCA and CDCA administration resulted in statistically significant dose-dependent cytotoxicity in both platinum sensitive and platinum-resistant cell lines ( p < 0.05). Cells incubated with DCA and CDCA exhibited morphologic features characteristic of apoptosis. The quantitative nucleosome ELISA assay demonstrated over 10 times increased nucleosome levels after cells were treated for 24 h by DCA and CDCA at 200 mM and 400 mM as compared to CA or UDCA treatment and to untreated controls ( p < 0.01). All 4 bile acids reduced PKC activity at concentrations of 200 and 400 mM ( p < 0.01). Conclusions CDCA and DCA have significant cytotoxic activity in ovarian cancer cells via induction of apoptosis. The mechanism of apoptosis appears to be mediated by alternative kinases distinct from PKC. CDCA and DCA may have clinical utility in the treatment of ovarian cancer pending in vivo confirmation of activity especially in cisplatin-resistant disease.