Treatment-Related Protein Biomarker Expression Differs between Primary and Recurrent Ovarian Carcinomas
ABSTRACT The molecular characteristics of recurrent ovarian cancers following chemotherapy treatment have been poorly characterized. Such knowledge could impact salvage therapy selection. Since 2008, we have profiled 168 patients' ovarian cancers to determine the expression of proteins that may predict chemotherapy response or are targets for drugs that are in clinical trials for ovarian cancer treatment. Expression of epidermal growth factor receptor (EGFR), HER2, VEGF, ER, c-Met, IGF1R, Ki67, COX2, PGP/MDR1, BCRP, MRP1, excision repair complementation group 1 (ERCC1), MGMT, TS, RRM1, TOPO1, TOP2A, and SPARC was measured by immunohistochemical analyses at Clinical Laboratory Improvement Amendments-certified laboratories. Our univariate analysis of 56 primary and 50 recurrent tumors from patients with advanced stage ovarian serous carcinoma revealed that PGP and ERCC1 were significantly upregulated in recurrent lesions (P < 0.05). To determine whether these or any of the other markers were differentially expressed in specimens obtained from the same individual at diagnosis and at recurrence, we analyzed 43 matched tumor specimens from 19 advanced stage ovarian carcinoma patients. We confirmed the expression differences in PGP and ERCC1 that were observed in the cohort analysis but discovered that the expression levels of BCRP, RRM1, and COX2 were also discordant in more than 40% of the matched tumor specimens. These results may have implications both for the use of biomarkers in therapy selection as well as for their discovery and validation. Expression of these and other candidate response biomarkers must be evaluated in much larger studies and, if confirmed, support the need for profiling of recurrent tumor specimens in future clinical trials.
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ABSTRACT: P-glycoprotein (P-gp), a member of the ATP-binding cassette transporter family, is overexpressed in a number of different cancers and some studies show that P-gp overexpression can be correlated to poor prognosis or therapeutic resistance. Here we sought to elucidate if PF-3758309 (PF-309), a novel p-21 activated kinase inhibitor, efficacy was influenced by tumor P-gp. Based on in vitro proliferation data, a panel of colorectal cancer cell lines were ranked as sensitive or resistant and ABCB1 (P-gp) expression was evaluated by microarray for these cell lines. P-gp expression was determined by western blot and activity determined by rhodamine efflux assay. Knock down of P-gp and pharmacologic inhibition of P-gp to restore PF-309 activity was performed in vitro. PF-309 activity was evaluated in vivo in cell line xenograft models and in primary patient derived tumor xenografts (PDTX). Mice were treated with 25 mg/kg PF-309 orally, twice daily. On the last day of treatment, tumor and plasma were collected for PF-309 analysis. Here we show that ABCB1 gene expression correlates with resistance to PF-309 treatment in vitro and the expression and activity of P-gp was verified in a panel of resistant cells. Furthermore, inhibition of P-gp increased the sensitivity of resistant cells, resulting in a 4-100-fold decrease in the IC50s. Eleven cell line xenografts and 12 PDTX models were treated with PF-309. From the cell line xenografts, we found a significant correlation between ABCB1 gene expression profiles and tumor response. We evaluated tumor and plasma concentrations for eight tumor models (three cell line xenografts and five PDTX models) and a significant correlation was found between tumor concentration and response. Additionally, we show that tumor concentration is approximately fourfold lower in tumors that express P-gp, verified by western blot. Our in vitro and in vivo data strongly suggests that PF-309 efficacy is influenced by the expression of tumor P-gp.Frontiers in Pharmacology 03/2013; 4:22. DOI:10.3389/fphar.2013.00022
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ABSTRACT: Cisplatin and paclitaxel are standard chemotherapy for metastatic ovarian cancer, but with limited efficacy. Cancer stem/progenitor cells (or tumor-initiating cells, TICs) are hypothesized to be chemoresistant, and the existence of TICs in ovarian cancer has been previously demonstrated. However, the key signals and molecular events regulating the formation and expansion of ovarian tumor-initiating cells (OTICs) remain elusive. Here, we show that c-Kit is not just a marker of OTICs, but also a critical mediator of the phenotype that can be a viable target for the treatment of ovarian cancer. In contrast to non-OICs, c-Kit was overexpressed in OTICs. Moreover, the use of small interfering RNA to inhibit c-Kit expression markedly attenuated the number and size of OTIC subpopulations, inhibited the expression of stem cell markers and decreased the tumorigenic capabilities of OTICs. Imatinib (Gleevec), a clinical drug that blocks c-Kit kinase activity, also demonstrated its inhibition potency on OTICs. In addition, cisplatin/paclitaxel, which killed non-OTICs, with c-Kit knockdown or imatinib revealed that this was critically required for intervening ovarian cancer progression and recurrence in vitro and in xenograft tumors in vivo. Similar results were obtained with OTICs derived from ovarian carcinoma patients. Studies into the mechanisms suggest an important role for the activation of Wnt/β-catenin and ATP-binding cassette G2 downstream of c-Kit. The tumor-promoting microenvironment, such as hypoxia, could promote OTICs via upregulation of c-Kit expression. These results unravel an integral role for c-Kit in ovarian neoplastic processes and shed light on its mechanisms of action.Oncogene advance online publication, 16 July 2012; doi:10.1038/onc.2012.290.Oncogene 07/2012; DOI:10.1038/onc.2012.290 · 8.56 Impact Factor
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ABSTRACT: Background An antibody with cross-reactivity can create unexpected side effects or false diagnostic reports if used for clinical purposes. ERCC1 is being explored as a predictive diagnostic biomarker for cisplatin-based chemotherapy. High ERCC1 expression is linked to drug resistance on cisplatin-based chemotherapy. 8F1 is one of the most commonly used monoclonal antibodies for evaluating ERCC1 expression levels in lung cancer patient tissues, but it has been noted that this antibody cross-reacts with an unknown protein. Results By using a high density protein microarray chip technology, we discovered that 8F1 not only reacts with its authentic target, ERCC1, but also cross-reacts with an unrelated nuclear membrane protein, PCYT1A. The cross-reactivity is due to a common epitope presented on these two unrelated proteins. Similar to the subcellular localization of ERCC1, IHC tests demonstrated that PCYT1A is localized mainly on nuclear membrane. In this study, we also discovered that the PCYT1A gene expression level is significantly higher than the ERCC1 gene expression level in a certain population of lung cancer patient tissue samples. To develop the best monoclonal antibody for ERCC1 IHC analysis, 18 monoclonal antibodies were generated and 6 of them were screened against our protein microarray chip. Two clones showed high mono-specificity on the protein microarray chip test and both worked for the IHC application. Conclusion In summary, the 8F1 clone is not suitable for ERCC1 IHC assay due to its cross-reactivity with PCYT1A protein. Two newly generated monoclonal antibodies, 4F9 and 2E12, demonstrated ultra-specificity against ERCC1 protein and superior performance for IHC analyses.BMC Biotechnology 11/2012; 12(1):88. DOI:10.1186/1472-6750-12-88 · 2.59 Impact Factor