<i>EPS8</i> Inhibition Increases Cisplatin Sensitivity in Lung Cancer Cells

Cisplatin, a commonly used chemotherapeutic, is associated with ototoxicity, renal toxicity and neurotoxicity, thus identifying means to increase the therapeutic index of cisplatin may allow for improved outcomes. A SNP (rs4343077) within EPS8, discovered through a genome wide association study of cisplatin-induced cytotoxicity and apoptosis in lymphoblastoid cell lines (LCLs), provided impetus to further study this gene. The purpose of this work was to evaluate the role of EPS8 in cellular susceptibility to cisplatin in cancerous and non-cancerous cells. We used EPS8 RNA interference to determine the effect of decreased EPS8 expression on LCL and A549 lung cancer cell sensitivity to cisplatin. EPS8 knockdown in LCLs resulted in a 7.9% increase in cisplatin-induced survival (P = 1.98×10−7) and an 8.7% decrease in apoptosis (P = 0.004) compared to control. In contrast, reduced EPS8 expression in lung cancer cells resulted in a 20.6% decrease in cisplatin-induced survival (P = 5.08×10−5). We then investigated an EPS8 inhibitor, mithramycin A, as a potential agent to increase the therapeutic index of cisplatin. Mithramycin A decreased EPS8 expression in LCLs resulting in decreased cellular sensitivity to cisplatin as evidenced by lower caspase 3/7 activation following cisplatin treatment (42.7%±6.8% relative to control P = 0.0002). In 5 non-small-cell lung carcinoma (NSCLC) cell lines, mithramycin A also resulted in decreased EPS8 expression. Adding mithramycin to 4 NSCLC cell lines and a bladder cancer cell line, resulted in increased sensitivity to cisplatin that was significantly more pronounced in tumor cell lines than in LCL lines (pEPS8, such as mithramycin A, could improve cisplatin treatment by increasing sensitivity of tumor relative to normal cells.</p

Genome-Wide Local Ancestry Approach Identifies Genes and Variants Associated with Chemotherapeutic Susceptibility in African Americans

Chemotherapeutic agents are used in the treatment of many cancers, yet variable resistance and toxicities among individuals limit successful outcomes. Several studies have indicated outcome differences associated with ancestry among patients with various cancer types. Using both traditional SNP-based and newly developed gene-based genome-wide approaches, we investigated the genetics of chemotherapeutic susceptibility in lymphoblastoid cell lines derived from 83 African Americans, a population for which there is a disparity in the number of genome-wide studies performed. To account for population structure in this admixed population, we incorporated local ancestry information into our association model. We tested over 2 million SNPs and identified 325, 176, 240, and 190 SNPs that were suggestively associated with cytarabine-, 5′-deoxyfluorouridine (5′-DFUR)-, carboplatin-, and cisplatin-induced cytotoxicity, respectively (p≤10−4). Importantly, some of these variants are found only in populations of African descent. We also show that cisplatin-susceptibility SNPs are enriched for carboplatin-susceptibility SNPs. Using a gene-based genome-wide association approach, we identified 26, 11, 20, and 41 suggestive candidate genes for association with cytarabine-, 5′-DFUR-, carboplatin-, and cisplatin-induced cytotoxicity, respectively (p≤10−3). Fourteen of these genes showed evidence of association with their respective chemotherapeutic phenotypes in the Yoruba from Ibadan, Nigeria (pTP53I11, COPS5 and GAS8, which are known to be involved in tumorigenesis. Although our results require further study, we have identified variants and genes associated with chemotherapeutic susceptibility in African Americans by using an approach that incorporates local ancestry information.</p

Differential contribution of cis -regulatory elements to higher order chromatin structure and expression of the CFTR locus

Higher order chromatin structure establishes domains that organize the genome and coordinate gene expression. However, the molecular mechanisms controlling transcription of individual loci within a topological domain (TAD) are not fully understood. The cystic fibrosis transmembrane conductance regulator (CFTR) gene provides a paradigm for investigating these mechanisms. CFTR occupies a TAD bordered by CTCF/cohesin binding sites within which are cell-type-selective cis-regulatory elements for the locus. We showed previously that intronic and extragenic enhancers, when occupied by specific transcription factors, are recruited to the CFTR promoter by a looping mechanism to drive gene expression. Here we use a combination of CRISPR/Cas9 editing of cis-regulatory elements and siRNA-mediated depletion of architectural proteins to determine the relative contribution of structural elements and enhancers to the higher order structure and expression of the CFTR locus. We found the boundaries of the CFTR TAD are conserved among diverse cell types and are dependent on CTCF and cohesin complex. Removal of an upstream CTCF-binding insulator alters the interaction profile, but has little effect on CFTR expression. Within the TAD, intronic enhancers recruit cell-type selective transcription factors and deletion of a pivotal enhancer element dramatically decreases CFTR expression, but has minor effect on its 3D structure

Genome-Wide Local Ancestry Approach Identifies Genes and Variants Associated with Chemotherapeutic Susceptibility in African Americans

Chemotherapeutic agents are used in the treatment of many cancers, yet variable resistance and toxicities among individuals limit successful outcomes. Several studies have indicated outcome differences associated with ancestry among patients with various cancer types. Using both traditional SNP-based and newly developed gene-based genome-wide approaches, we investigated the genetics of chemotherapeutic susceptibility in lymphoblastoid cell lines derived from 83 African Americans, a population for which there is a disparity in the number of genome-wide studies performed. To account for population structure in this admixed population, we incorporated local ancestry information into our association model. We tested over 2 million SNPs and identified 325, 176, 240, and 190 SNPs that were suggestively associated with cytarabine-, 5′-deoxyfluorouridine (5′-DFUR)-, carboplatin-, and cisplatin-induced cytotoxicity, respectively (p≤10−4). Importantly, some of these variants are found only in populations of African descent. We also show that cisplatin-susceptibility SNPs are enriched for carboplatin-susceptibility SNPs. Using a gene-based genome-wide association approach, we identified 26, 11, 20, and 41 suggestive candidate genes for association with cytarabine-, 5′-DFUR-, carboplatin-, and cisplatin-induced cytotoxicity, respectively (p≤10−3). Fourteen of these genes showed evidence of association with their respective chemotherapeutic phenotypes in the Yoruba from Ibadan, Nigeria (p<0.05), including TP53I11, COPS5 and GAS8, which are known to be involved in tumorigenesis. Although our results require further study, we have identified variants and genes associated with chemotherapeutic susceptibility in African Americans by using an approach that incorporates local ancestry information

Effect of mithramycin on sensitivity of molecularly distinct non-small-cell lung carcinoma cell lines.

<p>+ : Wild-type mt : Mutant.</p><p>*Mutation status of each cell line was provided from ATCC (Manassas, Virginia).</p

<i>EPS8</i> Inhibition Increases Cisplatin Sensitivity in Lung Cancer Cells

<div><p>Cisplatin, a commonly used chemotherapeutic, is associated with ototoxicity, renal toxicity and neurotoxicity, thus identifying means to increase the therapeutic index of cisplatin may allow for improved outcomes. A SNP (rs4343077) within <i>EPS8</i>, discovered through a genome wide association study of cisplatin-induced cytotoxicity and apoptosis in lymphoblastoid cell lines (LCLs), provided impetus to further study this gene. The purpose of this work was to evaluate the role of <i>EPS8</i> in cellular susceptibility to cisplatin in cancerous and non-cancerous cells. We used <i>EPS8</i> RNA interference to determine the effect of decreased <i>EPS8</i> expression on LCL and A549 lung cancer cell sensitivity to cisplatin. <i>EPS8</i> knockdown in LCLs resulted in a 7.9% increase in cisplatin-induced survival (<i>P</i> = 1.98×10⁻⁷) and an 8.7% decrease in apoptosis (<i>P</i> = 0.004) compared to control. In contrast, reduced <i>EPS8</i> expression in lung cancer cells resulted in a 20.6% decrease in cisplatin-induced survival (<i>P</i> = 5.08×10⁻⁵). We then investigated an <i>EPS8</i> inhibitor, mithramycin A, as a potential agent to increase the therapeutic index of cisplatin. Mithramycin A decreased <i>EPS8</i> expression in LCLs resulting in decreased cellular sensitivity to cisplatin as evidenced by lower caspase 3/7 activation following cisplatin treatment (42.7%±6.8% relative to control <i>P</i> = 0.0002). In 5 non-small-cell lung carcinoma (NSCLC) cell lines, mithramycin A also resulted in decreased <i>EPS8</i> expression. Adding mithramycin to 4 NSCLC cell lines and a bladder cancer cell line, resulted in increased sensitivity to cisplatin that was significantly more pronounced in tumor cell lines than in LCL lines (p<0.0001). An EGFR mutant NSCLC cell line (H1975) showed no significant change in sensitivity to cisplatin with the addition of mithramycin treatment. Therefore, an inhibitor of <i>EPS8</i>, such as mithramycin A, could improve cisplatin treatment by increasing sensitivity of tumor relative to normal cells.</p></div

LCL caspase 3/7 activity for 5 µM cisplatin treatment alone compared to cisplatin plus 0.01 µM mithramycin.

<p>Each LCL experienced lower cisplatin-induced apoptotic activity with the added mithramycin relative to a no drug treatment control. Mithramycin treated 10859, 11830, 11840, and 12156 resulted in a 47.1, 40.8, 48.9, and 34.0% decrease from cisplatin alone, respectively. Data represents two separate experiments, each done in triplicate with standard error of the mean.</p

NSCLC cell lines and bladder cell line, HTB9, are shown for various concentrations of cisplatin with the presence and absence of mithramycin.

<p>Square shape represents cisplatin concentrations alone, while the triangle represents cisplatin with the addition of mithramycin (0.01 µM). Curves represent two independent experiments done in triplicate with standard error of the mean.</p

Percent of <i>EPS8</i> expression compared to scrambled control with standard error of the mean is shown for 5 LCLs tested at 5, 29 and 53 hrs after nucleofection and cell line A549 at 29 and 53 hrs after nucleofection (A).

<p>Values include 2 independent experiments with qRT-PCR run in duplicate. Cell line changes in percent survival (<i>P</i> = 1.98×10⁻⁷) and caspase 3/7 activity (<i>P</i> = 0.004) for all LCLs and A549 (<i>P</i> = 5.08×10⁻⁵) at 5 µM cisplatin due to <i>EPS8</i> knockdown are shown with standard error of the mean using 6 replicates from 2 independent experiments (B).</p