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Acquisition of platinum resistance following first line platinum/taxane therapy is commonly observed in ovarian cancer patients and prevents clinical effectiveness. There are few options to prevent platinum resistance; however, demethylating agents have been shown to resensitize patients to platinum therapy thereby demonstrating that DNA methylation is a critical contributor to the development of platinum resistance. We previously reported the Epidermal Growth Factor Receptor (EGFR) is a novel regulator of DNA methyltransferase (DNMT) activity and DNA methylation. Others have shown that EGFR activation is linked to cisplatin treatment and platinum resistance. We hypothesized that cisplatin induced activation of the EGFR mediates changes in DNA methylation associated with the development of platinum resistance. To investigate this, we evaluated EGFR signaling and DNMT activity after acute cisplatin exposure. We also developed an in vitro model of platinum resistance to examine the effects of EGFR inhibition on acquisition of cisplatin resistance. Acute cisplatin treatment activates the EGFR and downstream signaling pathways, and induces an EGFR mediated increase in DNMT activity. Cisplatin resistant cells also showed increased DNMT activity and global methylation. EGFR inhibition during repeated cisplatin treatments generated cells that were more sensitive to cisplatin and did not develop increases in DNA methylation or DNMT activity compared to controls. These findings suggest that activation of EGFR during platinum treatment contributes to the development of platinum resistance. Furthermore, EGFR inhibition may be an effective strategy at attenuating the development of platinum resistance thereby enhancing the effectiveness of chemotherapeutic treatment in ovarian cancer.

Acquisition of platinum resistance following first line platinum therapy is commonly observed in ovarian cancer patients and is a major obstacle to clinical effectiveness. Currently there are no options available to prevent platinum resistance; however, studies show that demethylating agents may resensitize patients to platinum therapy thereby demonstrating DNA methylation as a critical contributor to the development of platinum resistance. Using a cellular model of platinum resistance we have previously shown that resistant cells have increased DNA methyltransferase (DNMT) activity as well as increased global DNA methylation. These observations were dependent on cisplatin induced activation of the Epidermal Growth Factor Receptor (EGFR) which we previously identified as a novel a regulator of DNMT activity and DNA methylation. Inhibition of the EGFR conferred during a platinum resistance paradigm decreased DNMT activity, decreased global methylation and attenuated resistance in our platinum resistant model. We hypothesized that further genome wide analysis of DNA methylation in resistant, parental and EGFR inhibited cell lines at specific CG sites would reveal targets of methylation important for the development of platinum resistance. Using the Illumina 450K methylation array, we found few significant alterations in DNA methylation for given genes between control and cisplatin resistant (CPR) cells. However, additional DNA methylation analysis with the 450K methylation array of patient tumor samples at initial diagnosis as well as after relapse, while not significant, showed similar trends in the data for certain genes namely CSRNP3, LOC400904, LYPD6, MTMR9L, NIPAL4 and RBP7. We are still evaluating the effects of EGFR inhibition in vitro on DNA methylation patterns. We suggest that while the in vitro model of platinum resistance corresponds to patient samples in some aspects, ovarian cancer cells display a great deal of heterogeneity in DNA methylation as described by others and a much larger sample size is necessary to draw concrete conclusions. Current studies are evaluating gene expression in control and resistant cells to validate whether alterations in DNA methylation coincide with changes in expression. Citation Format: Michaela L. Granados, Laurie G. Hudson, Li Luo, Sabrina L. Samudio-Ruiz. DNA methylation analysis in cisplatin resistant ovarian cancer cells and recurrent ovarian cancer patient samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4437.

Acquisition of platinum resistance following first line platinum-taxane therapy occurs in a large portion of ovarian cancer patients and continues to be a major challenge in clinical effectiveness. To date, there are limited interventions available to prevent or reverse platinum resistance; however, there has been some advances in the use of demethylating agents in the resensitization of patients to platinum based therapy. This highlights epigenetic alterations, in particular DNA methylation, as critical contributors to the acquisition of drug resistance in ovarian cancer. The Epidermal Growth Factor Receptor (EGFR) has also been linked to chemoresistance and is known to be activated by cisplatin treatment. We previously published that activation of the EGFR can increase DNA methyltransferase (DMNT) activity and, over extended activation, can significantly increase global DNA methylation in ovarian cancer cells. We hypothesized that cisplatin induced activation of the EGFR could mediate changes in DNA methylation associated with the development of platinum resistance. To investigate this, we used an in vitro model of platinum resistance and evaluated EGFR signaling and DNMT activity after acute cisplatin and in platinum resistant cells. We found that cisplatin treatment activates the EGFR and downstream signaling pathways JAK and AKT. Cisplatin also increased DNMT activity and this increase was dependent on EGFR activation. Over repeated sequential treatments of cisplatin, we successfully developed platinum resistant cells that possessed significant increases in DNMT activity and had moderate, but significant, increases in global DNA methylation. Preliminary data using the EGFR inhibitor Erlotinib during the development of platinum resistant cells suggest that the EGFR plays an integral role in the acquisition of platinum resistance. This work implicates EGFR driven alterations to DNMT activity and DNA methylation as a novel mechanism for development of platinum resistance. Further studies are currently being done to evaluate potential alterations in activation of downstream signaling pathways, levels of DNA methyltransferases and other changes in expression associated with platinum resistance. Citation Format: Sabrina L. Samudio-Ruiz, Michaela L. Granados, Laurie G. Hudson. Epidermal growth factor receptor mediated alterations in DNA methylation associated with the development of platinum resistance in ovarian cancer cells [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1427.

The 5-year survival rate for women diagnosed with ovarian cancer remains dismally unchanged at 44%. Resistance to platinum compounds in ovarian cancer imparts a major obstacle to increasing patient survival. The epidermal growth factor receptor (EGFR) plays a role in the regulation of growth and differentiation in normal cells, and proliferation in malignant cells. It is known that disregulation of the EGFR pathway, including prolonged receptor activation, is associated with platinum resistance in cellular models and our lab revealed a novel mechanism by which constitutive EGFR activation regulates DNA methyltransferase (DNMT) activity and DNA methylation. Platinum resistance is associated with epigenetic downregulation (via DNA methylation) of genes involved in platinum drug response, particularly genes important for drug uptake. Given the established link between platinum resistance and alterations to DNA methylation, our studies investigated the effects of acute cisplatin treatment on EGFR activation and DNMT activity. We hypothesized that acquired chemoresistance resulting from cisplatin treatment is initially triggered by cisplatin-induced EGFR activation and subsequent alterations to DNMT activity. We show activation of EGFR upon treatment of OVCA433 cells with cisplatin. Functional EGFR activation following cisplatin treatment was also observed by evaluating activation of downstream targets, Jak2 and Akt. Hence, cisplatin facilitates activation of EGFR and its downstream signaling pathways. Moreover, there was also a significant increase in DNMT activity in cisplatin treated cells when compared to untreated passage controls. These observations unmask a potential mechanism by which initial exposure to cisplatin can activate EGFR signaling and cause epigenetic alterations that may contribute to the development of platinum resistance. By understanding the underlying mechanism for development of platinum resistance and discovering targets of methylation, we may be able to enhance sensitivity to platinum based chemotherapeutics. Citation Format: Michaela Lee Granados, Laurie G. Hudson, Sabrina Leigh Samudio-Ruiz. Cisplatin induced activation of the epidermal growth factor receptor as a mechanism for epigenetic silencing in ovarian cancer cell platinum resistance. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B14.

Platinum resistance in ovarian cancer continues to be an impediment to clinical effectiveness. While multiple alterations associated with platinum resistance have been proposed, mechanisms responsible for those alterations, particularly epigenetic alterations, are still under investigation. Epigenetic alterations, specifically epigenetic silencing induced by DNA methylation, to genes involved in drug response is a common occurrence in platinum resistance. We have previously published that activation of the Epidermal Growth Factor Receptor (EGFR) in ovarian cancer cells increases activity of DNA methyltransferases (DNMTs) and extended EGFR activation can also increase global DNA methylation. In a concurrent abstract, we show that acute cisplatin treatment activates the EGFR and increases DNMT activity. Our overarching hypothesis for this work is that the EGFR is a key player in the development of platinum resistance by its ability to regulate DNMT activity and DNA methylation and thereby could be a therapeutic target for preventing acquisition of platinum resistance during chemotherapeutic treatment. Here, we show data obtained using a cellular model of platinum resistance that we have generated in the lab. We show that OVCA 433 cells treated repeatedly with increasing doses of cisplatin acquire resistance and demonstrate resistance in a 3D model which is more indicative of the ovarian cancer cell microenvironment. These cisplatin resistant cells (CPR) also display significantly increased DNMT activity compared to passage control (non-resistant) cells, with modest increases in global DNA methylation. While hyperactivation of the EGFR is not observed in this chronic model of cisplatin treatment, we do see a significant decrease in Copper transporter 1 (CTR1) levels in CPR cells. CTR1 is particularly important in cisplatin drug uptake, thus we believe that platinum resistance in CPR cells is due to downregulation of CTR1 potentially by DNA methylation. Current studies are evaluating DNA methylation of CTR1 and other genes that may be contributing to the development of platinum resistance. Future studies inhibiting the EGFR during cisplatin treatment will be imperative in linking cisplatin induced EGFR activation to the changes in DNMT activity and methylation that we are observing in our model of platinum resistance. This work implicates EGFR driven alterations to DNMT activity and DNA methylation as a novel mechanism for development of platinum resistance. Citation Format: Sabrina L. Samudio-Ruiz, Michaela L. Granados, Laurie G. Hudson. Epigenetic alterations associated with platinum resistance in an ovarian cancer cell model. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B46.