Michael D. Mueller

Correlations among ERCC1, XPB, UBE2I, EGF, TAL2 and ILF3 revealed by gene signatures of histological subtypes of patients with epithelial ovarian cancer

The aim of this study was to better understand the mechanisms of tumor development and disease progression in human epithelial ovarian cancer. Fifty genes were screened for gene signature; 20 expressed genes were assessed in tumor and normal samples of EOC patients by RT-PCR. Expression of UBE2I, EGF, TAL2 and ILF3 was validated by qPCR on the ABI Prism 7000 Detection System. ERCC1 and XPB expression was previously determined by RT-PCR in these specimens. Statistical analyses include two-sided Kruskal-Wallis test, pairwise comparison, Pearson correlation coefficient and paired t-test. In comparison to normal samples, 6 genes demonstrated distinct expression patterns in tumor tissues, with high expression observed for ERCC1, XPB and ILF3 (p=0.001, 0.0007 and 0.002, respectively) and low expression observed for TAL2 and EGF (both p<0.0001). This differential expression pattern between normal and tumor tissues may reflect in part the development of ovarian cancer. Significant differences in expression patterns of these genes in clear cell, endometrioid, mucinous and serous ovarian cancer were observed. Comparison of expression of any two EOC subtypes revealed multiple gene involvement in histopathological differentiation and cancer progression. A positive association was found between ERCC1 and XPB expression (r=0.53, p<0.0001) and between TAL2 and EGF expression (r=0.817, p<0.0001) suggesting the existence of gene linkage in these tumors. The differences in expression patterns of studied genes between tumors and normal specimens, between histological subtypes and correlations among studied genes, may indicate their involvement in tumor growth and disease progression in human epithelial ovarian cancer. Further investigation of these genes may enable better understanding of the molecular mechanism of tumorigenesis and identification of potential biomarkers.

The Role of Wild-Type p53 in Cisplatin-Induced Chk2 Phosphorylation and the Inhibition of Platinum Resistance with a Chk2 Inhibitor

The major obstacle in platinum chemotherapy is the repair of platinum-damaged DNA that results in increased resistance, reduced apoptosis, and finally treatment failure. Our research goal is to determine and block the mechanisms of platinum resistance. Our recent studies demonstrate that several kinases in the DNA-repair pathway are activated after cells are exposed to cisplatin. These include ATM, p53, and Chk2. The increased Chk2 phosphorylation is modulated by p53 in a wild-type p53 model. Overexpression of p53 by cDNA transfection in wt-p53 (but not p53 deficient) cells doubled the amount of Chk2 phosphorylation 48 hours after cisplatin treatment. p53 knockdown by specific siRNA greatly reduced Chk2 phosphorylation. We conclude that wild-type p53, in response to cisplatin stimulation, plays a role in the upstream regulation of Chk2 phosphorylation at Thr-68. Cells without normal p53 function survive via an alternative pathway in response to the exogenous influence of cisplatin. We strongly suggest that it is very important to include the p53 mutational status in any p53 involved studies due to the functional differentiation of wt p53 and p53 mutant. Inhibition of Chk2 pathway with a Chk2 inhibitor (C3742) increased cisplatin efficacy, especially those with defective p53. Our findings suggest that inhibition of platinum resistance can be achieved with a small-molecule inhibitor of Chk2, thus improving the therapeutic indices for platinum chemotherapy.

CHK2 and ERCC1 in the DNA Adduct Repair Pathway that Mediates Acquired Cisplatin Resistance

Increased DNA-adduct repair is a leading mechanism of acquired cisplatin resistance. Our previous studies show that overexpression of ERCC1, the essential component of nucleotide excision repair, is associated with enhanced repair of cisplatin-induced DNA-adduct and with clinical resistance to platinum chemotherapy. Current investigations provide extensive data on the mechanism of cisplatin resistance via the DNA-adduct repair pathway. In a study of cisplatin-induced molecular signature in human ovarian cancer A2780 cells, activation of ATM, p53, Chk2, P48, and P21 were observed, with Chk2 identified as an upstream regulator of the ERCC1 recognition/repair pathway. Our data demonstrate that Chk2 is activated and regulated by p53 in wild-type p53-replete cells. We also found that activated Chk2 can be dephosphorylated by PP2A. In other words, PP2A negatively regulates Chk2 by dephosphorylating phosphorylated Chk2. Previous findings by our group suggested that ovarian cancer A2780/CP70 cells, in response to cisplatin exposure, showed an increase of ERCC1 mRNA, with increased transcription and prolonged ERCC1 mRNA half-life. Functional analysis of the ERCC1 promoter by CAT assay indicates that the region from -220 to -110 appears essential to constitutive expression of ERCC1 gene and a more forward upstream region is responsible for cisplatininduced ERCC1 overexpression. Identification of a functional cis-element in the drug-responsive region by EMSA revealed that activator AP1 and repressor MZF1 responded to cisplatin stimulation. Overexpression of MZF1 repressed the ERCC1 promoter activity in cisplatin treated cells, indicating that MZF1 is a repressor in regulation of ERCC1 transcription. After cisplatin exposure, the mRNA level of MZF1 decreased and mRNA levels of c-jun and c-fos increased, suggesting that MZF1 and AP1 coordinately mediate cisplatin-invoked gene expression in these cells. Taken together, in response to cisplatin treatment, decreased MZF1 and increased AP1 binding activities within the drug-responsive region of the ERCC1 promoter appear to be the leading mechanism of up-regulation of ERCC1 expression. In conclusion, our investigations reveal two key factors—Chk2 and ERCC1—that participate in the DNA-adduct repair pathway that mediates acquired cisplatin resistance. Down-regulation of these two critical genes may antagonize cisplatin resistance in the treatment of human ovarian cancer.

Abstract 569: Dicycloplatin, a platinum compound appears effective in treating prostate cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Dicycloplatin (DCP) is a new platinum-based drug developed in China. Due to its special structure, DCP possesses superior stability and water-solubility, compared to cisplatin and carboplatin. Experimental findings in China demonstrate low in vitro cytotoxicity of dicycloplatin against a variety of human cancer cell lines. DCP toxicity profile of 210 mg/kg of LD50 compares favorably to 14.27 mg/kg for cisplatin and 164 mg/kg for carboplatin. In vivo studies indicate that IV administration of 20 mg/kg DCP in rats is followed by 3.64 μg/g of drug distribution in the prostate. Histoculture Drug Response Assay (HDRA) confirms clinical efficacy of chemotherapy for various cancer types, including prostate cancer. Pre-clinical and clinical studies in China suggest that DCP produces fewer side effects with a maximum tolerated dose of 650 mg/m2. Patients with prostate cancer treated by DCP alone demonstrated persistent benefits. In one case, the prostate-specific antigen (PSA) level returned to normal after 5 consecutive dosages of 600mg administered orally every two weeks. No significant side effects were observed. Investigations of molecular mechanisms at WVU, USA suggest that DCP induces gene signature profile in human ovarian cancer cells through mechanisms similar to other platinum drugs. In conclusion, dicycloplatin may be an effective chemotherapeutic agent for patients with prostate cancer and further investigations in animal and preclinical tests are warranted. 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 569.

Abstract B71: DNA damage responses to cisplatin and dicycloplatin, a new platinum analog.

Background: Dicycloplatin (DCP), a new platinum compound approved by the Chinese FDA in March of 2012, shows better water solubility, greater stability, and lower toxicity, compared to cisplatin and carboplatin. Preclinical in vitro and in vivo studies and a Phase I clinical trial demonstrated that DCP possesses strong antitumor activity and lower adverse events than carboplatin. Phase II clinical trials using dicycloplatin plus paclitaxel in chemotherapy-naive patients with advanced NSCLC found that efficacy and safety of DCP plus paclitaxel regimen were comparable to those of carboplatin plus paclitaxel regimen, with slightly better tolerance. Materials and Methods: Pharmacokinetic study of DCP characteristics was conducted using AP-4000TM LC-MS/MS System in patient blood samples. Molecular mechanism studies of dicycloplatin-induced gene-signature profiling were determined by Immunoblotting and compared to cisplatin profiling in human ovarian cancer cells. Results: Pharmacokinetic mass spectrometry shows different spectrums of dicycloplatin and of carboplatin in plasma. Two hours after administration, plasma concentration of dicycloplatin prototype is still high (17.1 μg/ml), following a peak concentration of 26.9 µg/ml at 0.5-h. Molecular mechanism investigation showed DCP-induced activation of several kinases including phosphorylations of Chk2 at threonine 68, p53 at serine 15 and BRCA1 at serine 1497. The increases in p-Chk2 and p-BRCA1 showed that the amount of DCP-induced phosphorylation doubled at 48-h and tripled for p-p53 at 24-h, compared to controls. Conclusion: Dicycloplatin appears to activate DNA damage-repair pathways through mechanisms similar to cisplatin. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B71. Citation Format: Jing Jie Yu, Xuqing Yang, Qinhua Song, Michael D. Mueller, Scot C. Remick. DNA damage responses to cisplatin and dicycloplatin, a new platinum analog. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B71.

Abstract 2427: Molecular and pharmacokinetic studies of dicycloplatin, a new platinum analogue, suggest it is a promising chemotherapeutic agent.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Dicycloplatin (DCP), a new platinum compound recently approved by the Chinese FDA, is a platinum-based supramolecule. The host-part (carboplatin) and a guest-part (cyclobutane-1,1-dicarboxylic acid or CBDC) are linked by hydrogen bonds. DCP has a stable chemical structure and good water solubility. Pharmacokinetic studies show that the prototype DCP concentration 2-h after administration is still high (17.1 μg/ml), after 26.9 μg/ml at 0.5 hour. In comparison, 5.01 μg/ml of carboplatin prototype is obtained 2-h after administration. Preclinical in vitro and in vivo studies and a phase I clinical trial demonstrated that DCP possesses significant antitumor activity, with fewer adverse effects than carboplatin. Molecular investigation of DCP-treated ovarian cancer A2780 cells showed activation of several kinases, including Chk2, p53, BRCA1 and other downstream factors. The increase in activated p-Chk2, p-p53 and p-BRCA1 after 1-h DCP exposure at IC50 dose occurred in a time-dependent manner. Quantitative analysis of phosphorylated Chk2 and BRCA1 expression revealed that the amount of dicycloplatin-induced phosphorylation of the two factors doubled at 48-h and tripled for p53 at 24-h, compared to controls. We observed that the DCP-induced activation profile and cisplatin-activated mechanisms are similar. We conclude that dicycloplatin is a promising platinum chemotherapeutic compound. Citation Format: Xuqing Yang, Qinhua Song, Michael D. Mueller, Jing Jie Yu. Molecular and pharmacokinetic studies of dicycloplatin, a new platinum analogue, suggest it is a promising chemotherapeutic agent. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2427. doi:10.1158/1538-7445.AM2013-2427

Abstract 3644: The PSP94αprostasin pathway and early detection of ovarian cancer

Objective: Our long-term goal is to better understand the mechanism of tumor development in human epithelial ovarian cancer. The short-term goal is to identify a biomarker for early detection of this malignancy. Methods: qPCR was performed for PSP94 expression using SYBR® Green reagents kit in 3 pairs of tumor and normal samples of ovarian cancer patients and 4 ovarian cancer cell lines on ABI 7500 Fast Real-Time PCR System. Overexpression of PSP94 or prostasin by cDNA transfection was performed in ovarian cancer cell lines. The expression levels of PSP94 and prostasin after cDNA transfection were determined by western blot analysis. Results: In gene expression analysis by qPCR, we found that PSP94 expresses in tumor tissues of ovarian cancer patients and cancer cell lines but not in normal ovarian specimens. Our investigation of the relationship between PSP94 and prostasin by cDNA transfection assay demonstrated that expression of prostasin was increased in the PSP94 overexpressed cells. In contrast, PSP94 expression did not change in the prostasin-forced-expression cells. This suggests that the two genes most likely share part of a regulation network and that PSP94 appears to be an upstream regulator of prostasin. Conclusions: We conclude that PSP94 expression occurs only in tumor samples and PSP94 and prostasin share a signaling pathway. We hypothesize that alteration of PSP94-prostasin pathway may be an early event in the development of epithelial ovarian cancer; PSP94 appears to be an upstream regulator that modulates prostasin expression. Thus, PSP94 seems to play an important role in tumorigenesis and may hold great promise as a biomarker for early detection of human epithelial 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 3644. doi:1538-7445.AM2012-3644

Abstract 1697: Gene signature, histopathological classification, and cisplatin-activated expression profiles in human epithelial ovarian cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL PURPOSES: To identify biomarkers in epithelial ovarian cancer (EOC) and investigate mechanism of cisplatin-induced activation and expression, 50 genes were screened from 131 ovarian cancer patient samples. Twenty expressed genes were assessed in 30 samples by qPCR. Ovarian cancer cells were treated with cisplatin and studied for expression of cisplatin-activated genes. RESULTS: Studies of gene signature of histopathological classification TAL2, EGF, ILF3 and UBE21 shown unique expression patterns in EOC histological types. The expression of ILF3 in tumor tissues was significantly higher than normal samples, and especially high in serous carcinomas, compared to mucinous, endometrial and clear cell carcinomas. ILF3 and UBE21 showed overexpression in advanced stage and poorly differentiated patient samples. Investigation of cisplatin-activated signaling indicated increased mRNA expression of ILF3, UBE21 and other kinases involved in cell cycle checkpoint, DNA repair, and apoptosis such as p53, Chk2, c-jun, and hMLH1. CONCLUSIONS: Overexpression of ILF3 and UBE21 in advanced stage and advanced grade of EOC specimens suggests that these two genes may play important roles in tumorgenesis, tumor progression and pathological differentiation of the disease. Results from in vitro studies demonstrate that activated genes p53, Chk2, c-jun and hMLH1 play a role in chemo-resistance to cisplatin therapy. Further investigation of the molecular mechanisms of the identified genes is warranted. 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 1697. doi:10.1158/1538-7445.AM2011-1697

Abstract 2982: Activation of checkpoint kinase Chk2 by cisplatin and its role in cisplatin resistance

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC One of the mechanisms of platinum resistance in cancer chemotherapy is the activation of DNA damage checkpoint pathways. Chk2 is a multifunctional key component of the DNA replication-monitoring S/G2 checkpoint system, which mediates DNA damage signaling following genotoxic stress and leads to DNA repair or apoptosis. Developing checkpoint kinase inhibitors for better treatment of cancer has been a major target in drug discovery over the past decade. In our study, flow cytometric experiments show that cisplatin induces an initial accumulation of cells at S phase of cell cycle followed by an increase of cell fraction at G2 phase, suggesting cisplatin-induced cell cycle arrest. Treatment of cisplatin in human ovarian carcinoma cells resulted in Chk2 phosphorylation at Thr-68 and p53 phosphorylation at Ser-15. The observed cisplatin-induced increase in p53 phosphorylation preceded (by about 12 hours) the observed increase in Chk2 phosphorylation. Elevated protein levels of ATM, p53 and P21 by cisplatin in these cells appear in a dose-dependent manner. In addition, overexpression of p53 by cDNA transfection in cells with wild type p53-function doubled the amount of Chk2 phosphorylation 48 hours after cisplatin treatment, whereas p53 knock-down by specific siRNA greatly reduced Chk2 phosphorylation. Furthermore, selective Chk2 Inhibitor (C3742) potentiated the cell-killing effect of cisplatin in both sensitive and resistant ovarian cancer cells. In conclusion, our results suggest that after cisplatin exposure the DNA damage response pathway is activated with greatly increased Chk2 phosphorylation which is modulated by p53. Inhibition of Chk2 with a small-molecule inhibitor may overcome cisplatin resistance in cancer chemotherapy. 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 2982.

Abstract A172: Dicycloplatin in cancer

Dicycloplatin (DCP) is a new platinum‐based drug developed in China. It possesses superior stability and water‐solubility compared to cisplatin and carboplatin. Experimental findings in China demonstrate in vitro cytotoxicity of dicycloplatin against a variety of human cancer cell lines and significant in vivo antitumor activity in mouse tumor models. DCP toxicity profile of 210 mg/kg of LD50 compares favourably to 14.27 mg/kg for cisplatin and 164 mg/kg for carboplatin. Histoculture Drug Response Assay (HDRA) indicates clinical efficacy of chemotherapy for various cancer types including brain cancer. Pre‐clinical and clinical studies in China suggest that DCP produces fewer side effects with a maximum tolerated dose of 650 mg/m2. In one case, DCP with radiotherapy successfully treated a lung cancer patient with brain metastasis. Polar surface area (PSA) and LogP calculations for estimating solubility and ability to cross the BBB indicate that DCP possesses the largest PSA (132) and CLogP (1), compared to carboplatin (106.2; −0.34) and cisplatin (53.6; −2.5). Investigations of molecular mechanisms at WVU suggest that DCP induces gene signature profile in human ovarian cancer cells through mechanisms similar to other platinum drugs. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A172.