Sabrina L. Samudio-Ruiz

Fetal Alcohol Spectrum Disorder-associated depression: evidence for reductions in the levels of brain-derived neurotrophic factor in a mouse model

Prenatal ethanol exposure is associated with an increased incidence of depressive disorders in patient populations. However, the mechanisms that link prenatal ethanol exposure and depression are unknown. Several recent studies have implicated reduced brain-derived neurotrophic factor (BDNF) levels in the hippocampal formation and frontal cortex as important contributors to the etiology of depression. In the present studies, we sought to determine whether prenatal ethanol exposure is associated with behaviors that model depression, as well as with reduced BDNF levels in the hippocampal formation and/or medial frontal cortex, in a mouse model of fetal alcohol spectrum disorder (FASD). Compared to control adult mice, prenatal ethanol-exposed adult mice displayed increased learned helplessness behavior and increased immobility in the Porsolt forced swim test. Prenatal ethanol exposure was associated with decreased BDNF protein levels in the medial frontal cortex, but not the hippocampal formation, while total BDNF mRNA and BDNF transcripts containing exons III, IV or VI were reduced in both the medial frontal cortex and the hippocampal formation of prenatal ethanol-exposed mice. These results identify reduced BDNF levels in the medial frontal cortex and hippocampal formation as potential mediators of depressive disorders associated with FASD.

Hippocampal N-Methyl-D-Aspartate Receptor Subunit Expression Profiles in a Mouse Model of Prenatal Alcohol Exposure

BACKGROUND Although several reports have been published showing prenatal ethanol exposure is associated with alterations in N-methyl-D-aspartate (NMDA) receptor subunit levels and, in a few cases, subcellular distribution, results of these studies are conflicting. METHODS We used semi-quantitative immunoblotting techniques to analyze NMDA receptor NR1, NR2A, and NR2B subunit levels in the adult mouse hippocampal formation isolated from offspring of dams who consumed moderate amounts of ethanol throughout pregnancy. We employed subcellular fractionation and immunoprecipitation techniques to isolate synaptosomal membrane- and postsynaptic density protein-95 (PSD-95)-associated pools of receptor subunits. RESULTS We found that, compared to control animals, fetal alcohol-exposed (FAE) adult mice had: (i) increased synaptosomal membrane NR1 levels with no change in association of this subunit with PSD-95 and no difference in total NR1 expression in tissue homogenates; (ii) decreased NR2A subunit levels in hippocampal homogenates, but no alterations in synaptosomal membrane NR2A levels and no change in NR2A-PSD-95 association; and (iii) no change in tissue homogenate or synaptosomal membrane NR2B levels but a reduction in PSD-95-associated NR2B subunits. No alterations were found in mRNA levels of NMDA receptor subunits suggesting that prenatal alcohol-associated differences in subunit protein levels are the result of differences in post-transcriptional regulation of subunit localization. CONCLUSIONS Our results demonstrate that prenatal alcohol exposure induces selective changes in NMDA receptor subunit levels in specific subcellular locations in the adult mouse hippocampal formation. Of particular interest is the finding of decreased PSD-95-associated NR2B levels, suggesting that synaptic NR2B-containing NMDA receptor concentrations are reduced in FAE animals. This result is consistent with various biochemical, physiological, and behavioral findings that have been linked with prenatal alcohol exposure.

Prenatal ethanol exposure persistently impairs NMDA receptor‐dependent activation of extracellular signal‐regulated kinase in the mouse dentate gyrus

The dentate gyrus (DG) is the central input region to the hippocampus and is known to play an important role in learning and memory. Previous studies have shown that prenatal alcohol is associated with hippocampal‐dependent learning deficits and a decreased ability to elicit long‐term potentiation (LTP) in the DG in adult animals. Given that activation of the extracellular signal‐regulated kinase 1/2 (ERK1/2) signaling cascade by NMDA receptors is required for various forms of learning and memory, as well as LTP, in hippocampal regions, including the DG, we hypothesized that fetal alcohol‐exposed adult animals would have deficits in hippocampal NMDA receptor‐dependent ERK1/2 activation. We used immunoblotting and immunohistochemistry techniques to detect NMDA‐stimulated ERK1/2 activation in acute hippocampal slices prepared from adult fetal alcohol‐exposed mice. We present the first evidence linking prenatal alcohol exposure to deficits in NMDA receptor‐dependent ERK1/2 activation specifically in the DG of adult offspring. This deficit may account for the LTP deficits previously observed in the DG, as well as the life‐long cognitive deficits, associated with prenatal alcohol exposure.

Increased DNA methyltransferase activity and DNA methylation following epidermal growth factor stimulation in ovarian cancer cells

Ovarian cancer progression is correlated with accumulation of aberrant CpG island methylation. In ovarian cancer, ascites fluid contains numerous Epidermal-Growth-Factor-Receptor (EGFR) activators, which could result in a tumor microenvironment of constant EGFR activation. Signaling pathways downstream of EGFR, such as Ras, regulate DNA methylation. We hypothesized that chronic EGFR activation could alter DNA methylation. We found that EGFR activation increased DNA methyltransferase (DNMT) activity acutely, as well as after long-term EGF treatment or expression of a mutationally activated EGFR. Furthermore, this increase in DNMT activity was dependent on EGFR catalytic activity and resulted in increased global DNA methylation. Additionally, treatment with the DNMT inhibitor/hypomethylating agent 5-Aza-2’-deoxycytidine (AZA) inhibited the EGF induced increase of both DNMT activity and global methylation. These data support a role for EGFR in the process of accumulated DNA methylation during ovarian cancer progression and suggest that epigenetic therapy may be beneficial for the treatment of ovarian cancer.

Contributions of the Epidermal Growth Factor Receptor to Acquisition of Platinum Resistance in Ovarian Cancer Cells

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.

Persistent N-cadherin Expression Induced by Extended Epidermal GrowthFactor Exposure Regulates Multicellular Aggregate Compaction andSensitivity to Cisplatin

Objective: In ovarian cancer, activation of the epidermal growth factor receptor (EGFR) is associated with poor prognosis. The presence of EGFR activators in patient ascites fluid may cause constitutive EGFR activation thereby contributing to metastasis and/or resistance to therapy. Our goal was to identify alterations resulting from constitutive EGFR activation that influence cell behavior and drug sensitivity. Methods: We used an in vitro model (OVCA 433 cells) to evaluate changes in mesenchymal marker levels and multicellular aggregate (MCA) formation following long term epidermal growth factor (EGF) treatment. We determined sensitivity to cisplatin following EGF treatment and evaluated the role of the mesenchymal marker, N-cadherin, in aggregate formation and sensitivity using siRNA. Results: We found that EGFR activation led to phenotypic and functional changes in ovarian tumor cells that were retained after ligand was withdrawn (removal). Expression levels of the mesenchymal markers N-cadherin and vimentin were elevated in EGF treated and removal cells. This persistent increase in mesenchymal markers was associated with a significant increase in (MCA) compaction and cell spreading when MCAs were plated on collagen. N-cadherin silencing decreased MCA compaction and spreading in cells following extended exposure to EGF or in cells with high endogenous levels of N-cadherin. Furthermore, the compact MCA structure in EGF treated cells with increased N-cadherin expression conferred resistance to cisplatin and N-cadherin silencing largely restored cisplatin sensitivity. Conclusion: Our results indicate that prolonged EGFR activation causes a persistent change in mesenchymal marker expression, which regulates compaction and drug sensitivity. The findings implicate N-cadherin as a key regulator of the EGFR-dependent functional changes in MCAs such as compaction, spreading and sensitivity to platinum-based chemotherapeutics. These findings suggest a mechanism by which persistent EGFR activation in the microenvironment may drive changes in ovarian cancer cells that contribute to the poor prognosis associated with EGFR activation.

Abstract 4437: DNA methylation analysis in cisplatin resistant ovarian cancer cells and recurrent ovarian cancer patient samples

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.

Abstract B14: Cisplatin induced activation of the epidermal growth factor receptor as a mechanism for epigenetic silencing in ovarian cancer cell platinum resistance

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.

Abstract B46: Epigenetic alterations associated with platinum resistance in an ovarian cancer cell model

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.

Abstract POSTER-THER-1427: Epidermal growth factor receptor mediated alterations in DNA methylation associated with the development of platinum resistance in ovarian cancer cells

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.