Jing Jie Yu

A nucleotide polymorphism in ERCC1 in human ovarian cancer cell lines and tumor tissues.

We studied the DNA sequence of the entire coding region of ERCC1 gene, in five cell lines established from human ovarian cancer (A2780, A2780/CP70, MCAS, OVCAR-3, SK-OV-3), 29 human ovarian cancer tumor tissue specimens, one human T-lymphocyte cell line (H9), and non-malignant human ovary tissue (NHO). Samples were assayed by PCR-SSCP and DNA sequence analyses. A silent mutation at codon 118 (site for restriction endonuclease MaeII) in exon 4 of the gene was detected in MCAS, OVCAR-3 and SK-OV-3 cells, and NHO. This mutation was a C-->T transition, that codes for the same amino acid: asparagine. This transition converts a common codon usage (AAC) to an infrequent codon usage (AAT), whereas frequency of use is reduced two-fold. This base change was associated with a detectable band shift on SSCP analysis. For the 29 ovarian cancer specimens, the same base change was observed in 15 tumor samples and was associated with the same band shift in exon 4. Cells and tumor tissue specimens that did not contain the C-->T transition, did not show the band shift in exon 4. Our data suggest that this alteration at codon 118 within the ERCC1 gene, may exist in platinum-sensitive and platinum-resistant ovarian cancer tissues.

Oncocytic, focally anaplastic, thyroid cancer responding to erlotinib††

Objective. To highlight the molecular findings and clinical response of a patient with rapidly progressing, focally anaplastic, oncocytic thyroid carcinoma (OTC) treated with erlotinib. Case Summary. A 69-year-old woman with recurrent, focally anaplastic OTC was given a therapeutic trial of erlotinib, a small molecule inhibitor of epidermal growth factor receptor (EGFR). Formalin-fixed, paraffin-embedded portions of the tumor were analyzed for EGFR expression, and tumor genomic DNA was amplified by polymerase chain reaction (PCR) and subjected to EGFR mutation analysis. Discussion. An early and dramatic response was achieved with erlotinib. The tumor was focally positive for EGFR by immunostaining and two point mutations were identified, one on exon 18 and one on exon 20 in the tyrosine kinase (TK) domain. Conclusion. Erlotinib or other novel protein kinase pathway inhibitors should be evaluated further in patients with aggressive thyroid cancer variants, who may exhibit these and perhaps other tyrosine kinase mutations. J Oncol Pharm Practice (2009) 15: 111—117.

An ERCC1 splicing variant involving the 5'-UTR of the mRNA may have a transcriptional modulatory function

Human ovarian cancer cells and tissues were examined for the presence or absence of a 42-bp splicing variant of ERCC1 gene, and for a possible functional role of this 42-bp sequence. This specific sequence exists in exon I, the 5′-UTR of the gene. Loss of this 42-bp sequence was associated with increased ERCC1 mRNA expression, in an assessment of 121 ovarian cancer specimens (p2<10−6). In cells in tissue culture, the absence of the 42-bp segment was associated with a twofold increased ability to drive transcription in a Luciferase reporter system. Protein can be demonstrated in ovarian cancer cells based on EMSA analysis. Computer analysis shows that this 42-bp sequence contains several binding sites, including a core-binding domain for protein RFX1, transcriptional repressor. These preliminary results lay the groundwork in determination of potential roles for a negative regulatory element in NER repair pathway.

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.

COMPARATIVE ANALYSES OF RELATIVE ERCC3 AND ERCC6 MRNA LEVELS IN GLIOMAS AND ADJACENT NON-NEOPLASTIC BRAIN

Nucleotide excision repair (NER) is an ordered process in nonmalignant cells, in both human and nonhuman systems. We previously reported that in human brain, there is discordant mRNA expression of excision repair cross‐complementing (ERCC) 1 and ERCC2 in malignant tissues, concurrent with excellent concordance of these genes in nonmalignant tissues from the same patients. Here we have extended these studies to compare low‐grade tumors to high‐grade tumors and to include ERCC3 (which links DNA repair with DNA transcription) and ERCC6 (which is essential for gene‐specific repair). Glial tumor and adjacent normal brain specimens from 19 individuals were studied. Paired malignant and nonmalignant tissues were obtained from 12 of these patients. For ERCC3, there was excellent concordance of mRNA expression between malignant and nonmalignant tissues from the same individuals (P = 0.003). For ERCC6, no concordance was observed (P = 0.314). Tumor tissue from patients with high‐grade gliomas exhibited marked discordance of mRNA expression patterns in situations in which good concordance was observed in tumor tissue from low‐grade gliomas. We previously established that malignant brain tumors show increased disorder of genes in the NER process, as compared with nonmalignant tissues. These data suggest that increasing disorder in the NER process may occur as cells move from low‐grade to high‐grade malignancy.

Absence of evidence for allelic loss or allelic gain for ERCC1 or for XPD in human ovarian cancer cells and tissues

We have previously reported on mRNA expression of ERCC1, XPA and XPD in human ovarian cancer cells and tissues. Several factors can influence mRNA expression for any given gene. Alterations in gene copy number for ERCC1 and/or XPD have been reported to occur in malignant glioma specimens. Human ovarian cancer cell lines and tissues were therefore examined for evidence of altered gene copy number in selected genes within the nucleotide excision repair (NER) pathway. Six ovarian cancer cell lines were studied: A2780, A2780/CP70, SKOV3, MCAS, QvCar3 and Caov4. Cellular sensitivity to cisplatin varies by more than 1 log between some of these cells. In each of these cell lines, the genes examined included ERCC1, XPA, XPB, XPD, XPG, CSB and p53. Genomic DNA was also extracted from ovarian cancer specimens taken from 22 patients and assessed for evidence of allelic loss and/or allelic gain for ERCC1 and XPD. Twelve of the clinical specimens were from patients with platinum-sensitive tumors and ten were from patients with platinum-resistant tumors. In no case could we demonstrate a reproducible variation in gene copy number in any cell line. Among the human tissues studied, there was one case of allelic gain out of 22 specimens. We therefore conclude that alterations in gene copy number is not a common event in human ovarian cancer. Other mechanisms must be invoked to explain differences in mRNA expression for these genes.

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.