Jimena Perez

Differential promoter methylation of kinesin family member 1a in plasma is associated with breast cancer and DNA repair capacity

Methylation alterations of CpG islands, CpG island shores and first exons are key events in the formation and progression of human cancer, and an increasing number of differentially methylated regions and genes have been identified in breast cancer. Recent studies of the breast cancer methylome using deep sequencing and microarray platforms are providing a novel insight on the different roles aberrant methylation plays in molecular subtypes of breast cancer. Accumulating evidence from a subset of studies suggests that promoter methylation of tumor-suppressor genes associated with breast cancer can be quantified in circulating DNA. However, there is a paucity of studies that examine the combined presence of genetic and epigenetic alterations associated with breast cancer using blood-based assays. Dysregulation of DNA repair capacity (DRC) is a genetic risk factor for breast cancer that has been measured in lymphocytes. We isolated plasma DNA from 340 participants in a breast cancer case control project to study promoter methylation levels of five genes previously shown to be associated with breast cancer in frozen tissue and in cell line DNA: MAL, KIF1A, FKBP4, VGF and OGDHL. Methylation of at least one gene was found in 49% of the cases compared to 20% of the controls. Three of the four genes had receiver characteristic operator curve values of ≥0.50: MAL (0.64), KIF1A (0.51) and OGDHL (0.53). KIF1A promoter methylation was associated with breast cancer and inversely associated with DRC. This is the first evidence of a significant association between genetic and epigenetic alterations in breast cancer using blood-based tests. The potential diagnostic utility of these biomarkers and their relevance for breast cancer risk prediction should be examined in larger cohorts.

Genome-wide methylation profiling reveals Zinc finger protein 516 (ZNF516) and FK-506-binding protein 6 (FKBP6) promoters frequently methylated in cervical neoplasia, associated with HPV status and ethnicity in a Chilean population

Cervical cancer is a major health concern among women in Latin America due to its high incidence and mortality. Therefore, the discovery of molecular markers for cervical cancer screening and triage is imperative. The aim of this study was to use a genome wide DNA methylation approach to identify novel methylation biomarkers in cervical cancer. DNA from normal cervical mucosa and cervical cancer tissue samples from Chile was enriched with Methylated DNA Immunoprecipitation (MeDIP), hybridized to oligonucleotide methylation microarrays and analyzed with a stringent bioinformatics pipeline to identify differentially methylated regions (DMRs) as candidate biomarkers. Quantitative Methylation Specific PCR (qMSP) was used to study promoter methylation of candidate DMRs in clinical samples from two independent cohorts. HPV detection and genotyping were performed by Reverse Line Blot analysis. Bioinformatics analysis revealed GGTLA4, FKBP6, ZNF516, SAP130, and INTS1 to be differentially methylated in cancer and normal tissues in the Discovery cohort. In the Validation cohort FKBP6 promoter methylation had 73% sensitivity and 80% specificity (AUC = 0.80). ZNF516 promoter methylation was the best biomarker, with both sensitivity and specificity of 90% (AUC = 0.92), results subsequently corroborated in a Prevalence cohort. Together, ZNF516 and FKBP6 exhibited a sensitivity of 84% and specificity of 81%, when considering both cohorts. Our genome wide DNA methylation assessment approach (MeDIP-chip) successfully identified novel biomarkers that differentiate between cervical cancer and normal samples, after adjusting for age and HPV status. These biomarkers need to be further explored in case-control and prospective cohorts to validate them as cervical cancer biomarkers.

Decreased SMAD4 expression is associated with induction of epithelial-to-mesenchymal transition and cetuximab resistance in head and neck squamous cell carcinoma

Epidermal growth factor receptor (EGFR) is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC) and cetuximab, a monoclonal antibody targeting this receptor, is widely used to treat these patients. In the following investigation, we examined the role of SMAD4 down-regulation in mediating epithelial-to-mesenchymal transition (EMT) and cetuximab resistance in HNSCC. We determined that SMAD4 downregulation was significantly associated with increased cell motility, increased expression of vimentin, and cetuximab resistance in HNSCC cell lines. In the HNSCC genomic dataset obtained from The Cancer Genome Atlas, SMAD4 was altered in 20/279 (7%) of HNSCC via homozygous deletion, and nonsense, missense, and silent mutations. When SMAD4 expression was compared with respect to human papillomavirus (HPV) status, HPV-positive tumors had higher expression compared to HPV-negative tumors. Furthermore, higher SMAD4 expression also correlated with higher CDKN2A (p16) expression. Our data suggest that SMAD4 down-regulation plays an important role in the induction of EMT and cetuximab resistance. Patients with higher SMAD4 expression may benefit from cetuximab use in the clinic.

SMAD4 Loss Is Associated with Cetuximab Resistance and Induction of MAPK/JNK Activation in Head and Neck Cancer Cells

Purpose: We previously demonstrated an association between decreased SMAD4 expression and cetuximab resistance in head and neck squamous cell carcinoma (HNSCC). The purpose of this study was to further elucidate the clinical relevance of SMAD4 loss in HNSCC. Experimental Design: SMAD4 expression was assessed by IHC in 130 newly diagnosed and 43 patients with recurrent HNSCC. Correlative statistical analysis with clinicopathologic data was also performed. OncoFinder, a bioinformatics tool, was used to analyze molecular signaling in TCGA tumors with low or high SMAD4 mRNA levels. The role of SMAD4 was investigated by shRNA knockdown and gene reconstitution of HPV-negative HNSCC cell lines in vitro and in vivo. Results: Our analysis revealed that SMAD4 loss was associated with an aggressive, HPV-negative, cetuximab-resistant phenotype. We found a signature of prosurvival and antiapoptotic pathways that were commonly dysregulated in SMAD4-low cases derived from TCGA-HNSCC dataset and an independent oral cavity squamous cell carcinoma (OSCC) cohort obtained from GEO. We show that SMAD4 depletion in an HNSCC cell line induces cetuximab resistance and results in worse survival in an orthotopic mouse model in vivo. We implicate JNK and MAPK activation as mediators of cetuximab resistance and provide the foundation for the concomitant EGFR and JNK/MAPK inhibition as a potential strategy for overcoming cetuximab resistance in HNSCCs with SMAD4 loss. Conclusions: Our study demonstrates that loss of SMAD4 expression is a signature characterizing the cetuximab-resistant phenotype and suggests that SMAD4 expression may be a determinant of sensitivity/resistance to EGFR/MAPK or EGFR/JNK inhibition in HPV-negative HNSCC tumors. Clin Cancer Res; 23(17); 5162–75. ©2017 AACR.

Genome-Wide and Gene-Specific Epigenomic Platforms for Hepatocellular Carcinoma Biomarker Development Trials

The majority of the epigenomic reports in hepatocellular carcinoma have focused on identifying novel differentially methylated drivers or passengers of the oncogenic process. Few reports have considered the technologies in place for clinical translation of newly identified biomarkers. The aim of this study was to identify epigenomic technologies that need only a small number of samples to discriminate HCC from non-HCC tissue, a basic requirement for biomarker development trials. To assess that potential, we used quantitative Methylation Specific PCR, oligonucleotide tiling arrays, and Methylation BeadChip assays. Concurrent global DNA hypomethylation, gene-specific hypermethylation, and chromatin alterations were observed as a hallmark of HCC. A global loss of promoter methylation was observed in HCC with the Illumina BeadChip assays and the Nimblegen oligonucleotide arrays. HCC samples had lower median methylation peak scores and a reduced number of significant promoter-wide methylated probes. Promoter hypermethylation of RASSF1A, SSBP2, and B4GALT1 quantified by qMSP had a sensitivity ranging from 38% to 52%, a specificity of 100%, and an AUC from 0.58 to 0.75. A panel combining these genes with HCC risk factors had a sensitivity of 87%, a specificity of 100%, and an AUC of 0.91.

Clinical and public health research using methylated DNA Immunoprecipitation (MeDIP): A comparison of commercially available kits to examine differential DNA methylation across the genome

The methylated DNA immunoprecipitation method (MeDIP) is a genome-wide, high-resolution approach that detects DNA methylation with oligonucleotide tiling arrays or high throughput sequencing platforms. A simplified high-throughput MeDIP assay will enable translational research studies in clinics and populations, which will greatly enhance our understanding of the human methylome. We compared three commercial kits, MagMeDIP Kit TM (Diagenode), Methylated-DNA IP Kit (Zymo Research) and Methylamp™ Methylated DNA Capture Kit (Epigentek), in order to identify which one has better reliability and sensitivity for genomic DNA enrichment. Each kit was used to enrich two samples, one from fresh tissue and one from a cell line, with two different DNA amounts. The enrichment efficiency of each kit was evaluated by agarose gel band intensity after Nco I digestion and by reaction yield of methylated DNA. A successful enrichment is expected to have a 1:4 to 10:1 conversion ratio and a yield of 80% or higher. We also evaluated the hybridization efficiency to genome-wide methylation arrays in a separate cohort of tissue samples. We observed that the MagMeDIP kit had the highest yield for the two DNA amounts and for both the tissue and cell line samples, as well as for the positive control. In addition, the DNA was successfully enriched from a 1:4 to 10:1 ratio. Therefore, the MagMeDIP kit is a useful research tool that will enable clinical and public health genome-wide DNA methylation studies.

Early detection of gastric cancer using global, genome-wide and IRF4, ELMO1, CLIP4 and MSC DNA methylation in endoscopic biopsies

Clinically useful molecular tools to triage gastric cancer patients are not currently available. We aimed to develop a molecular tool to predict gastric cancer risk in endoscopy-driven biopsies obtained from high-risk gastric cancer clinics in low resource settings. We discovered and validated a DNA methylation biomarker panel in endoscopic samples obtained from 362 patients seen between 2004 and 2009 in three high-risk gastric cancer clinics in Lima, Perú, and validated it in 306 samples from the Cancer Genome Atlas project (“TCGA”). Global, epigenome wide and gene-specific DNA methylation analyses were used in a Phase I Biomarker Development Trial to identify a continuous biomarker panel that combines a Global DNA Methylation Index (GDMI) and promoter DNA methylation levels of IRF4, ELMO1, CLIP4 and MSC. We observed an inverse association between the GDMI and histological progression to gastric cancer, when comparing gastritis patients without metaplasia (mean = 5.74, 95% CI, 4.97−6.50), gastritis patients with metaplasia (mean = 4.81, 95% CI, 3.77−5.84), and gastric cancer cases (mean = 3.38, 95% CI, 2.82−3.94), respectively (p < 0.0001). Promoter methylation of IRF4 (p < 0.0001), ELMO1 (p < 0.0001), CLIP4 (p < 0.0001), and MSC (p < 0.0001), is also associated with increasing severity from gastritis with no metaplasia to gastritis with metaplasia and gastric cancer. Our findings suggest that IRF4, ELMO1, CLIP4 and MSC promoter methylation coupled with a GDMI>4 are useful molecular tools for gastric cancer risk stratification in endoscopic biopsies.

CoGAPS matrix factorization algorithm identifies transcriptional changes in AP-2alpha target genes in feedback from therapeutic inhibition of the EGFR network

Patients with oncogene driven tumors are treated with targeted therapeutics including EGFR inhibitors. Genomic data from The Cancer Genome Atlas (TCGA) demonstrates molecular alterations to EGFR, MAPK, and PI3K pathways in previously untreated tumors. Therefore, this study uses bioinformatics algorithms to delineate interactions resulting from EGFR inhibitor use in cancer cells with these genetic alterations. We modify the HaCaT keratinocyte cell line model to simulate cancer cells with constitutive activation of EGFR, HRAS, and PI3K in a controlled genetic background. We then measure gene expression after treating modified HaCaT cells with gefitinib, afatinib, and cetuximab. The CoGAPS algorithm distinguishes a gene expression signature associated with the anticipated silencing of the EGFR network. It also infers a feedback signature with EGFR gene expression itself increasing in cells that are responsive to EGFR inhibitors. This feedback signature has increased expression of several growth factor receptors regulated by the AP-2 family of transcription factors. The gene expression signatures for AP-2alpha are further correlated with sensitivity to cetuximab treatment in HNSCC cell lines and changes in EGFR expression in HNSCC tumors with low CDKN2A gene expression. In addition, the AP-2alpha gene expression signatures are also associated with inhibition of MEK, PI3K, and mTOR pathways in the Library of Integrated Network-Based Cellular Signatures (LINCS) data. These results suggest that AP-2 transcription factors are activated as feedback from EGFR network inhibition and may mediate EGFR inhibitor resistance.

Abstract 1889: Smad4 inactivation correlates with EMT and cetuximab resistance in head and neck squamous cell carcinoma

Introduction: Epidermal growth factor receptor (EGFR) is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC) and cetuximab, a monoclonal antibody targeting EGFR, is widely used to treat patients with HNSCC. However, the majority of patients do not respond or develop resistance to cetuximab. Recent studies have shown that epithelial-to-mesenchymal transition (EMT) is associated with EGFR inhibitor resistance in diverse tumors including HNSCC. To further understand the role of EMT in cetuximab resistance, we examined the HNSCC cell lines for EMT- related genes and proteins and correlated their expression with cetuximab response in vitro. Methods: Responses to cetuximab in the panel of 25 HNSCC cell lines and a spontaneously immortalized keratinocyte (HaCaT) were determined by matrigel colony formation assay. Global gene and microRNA expression of the cell lines were determined using Affymetrix and Taqman microRNA arrays. Differential expression of EMT-related genes was confirmed by qRT-PCR and western blots. Collagen migration assay was performed to assess migration and stable knockdowns of Smad4 and EGFR were achieved using lentiviral shRNAs. The ligand levels in cell cultures were quantified by enzyme-linked immunosorbent assays (ELISA). Results: Both the array and qRT-PCR data confirmed that the EMT-related genes were generally correlated with cetuximab resistance. In two pairs of isogenic cell lines with acquired resistance to cetuximab, SCC1/1Cc8 and SCC25/CTX-10 and CTX-11, the resistant clones show lower E-cadherin, higher vimentin expression and increased migration rate compared to parental cells. We further examined the TGF-β pathway known to induce EMT in HNSCC. Smad4 levels were lower in the resistant clones. Knockdown of Smad4 rendered resistance in the cetuximab sensitive parent cells by activating TGF-β and c-Met pathways. In addition, knockdown of Smad4 resulted in increased p-AKT levels and EMT cellular phenotypes in the SCC1. To further examine the regulation of Smad4 by microRNAs, we determined differentially expressed microRNAs between cetuximab sensitive and resistant isogenic cell lines. We found that miRNA-146a was upregulated in the resistant clones, and Smad4 and EGFR expression were regulated by miRNA-146a mimic and inhibitor. TGFβR1 inhibitor reversed acquired and intrinsic resistance to cetuximab in vitro. Conclusion: We confirmed that EMT is associated with cetuximab resistance and TGF-β and c-MET signaling pathways may play an important role in induction of EMT in a subset of HNSCC. Our results suggest a combination strategy to improve the therapeutic index of cetuximab therapy in HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1889. doi:1538-7445.AM2012-1889

Abstract 3100: MicroRNA profiling reveals miR-205 upregulation is associated with head and neck squamous cell carcinoma and modulates E2F1 signaling.

Introduction: Based on our previous screen of miRNAs in head and neck squamous cell carcinoma (HNSCC), we sought to functionally evaluate candidate transcripts that could modify Rb/E2F signaling. Thus, we chose to investigate miR-205 as a putative HNSCC oncogene and regulator of E2F1 protein expression. Methods: miRNAs were isolated from 29 newly diagnosed HNSCC tumors (HPV-positive: 14, HPV-negative: 15) and 4 normal mucosa samples using the PureLink RNA Isolation Kit (Invitrogen). miRNA was analyzed using the ABI Megaplex protocol without pre-amplification (Applied Biosystems). Reactions were run using the miRNA Reverse Transcription Kit and Megaplex RT Human Pool A primers, then analyzed by TaqMan Low-Density Array (TLDA) cards. miRNA data was normalized to MammU6 expression, with undetectable transcripts assigned a C t value of 40. Empirical Bayes moderated t-statistics were used to assess miRNA differential expression and the Benjamini- Hotchberg correction was applied to these p-values to account for multiple hypothesis testing. miR-205 expression was then transiently modified (Dharmacon) in an HPV-positive and -negative HNSCC cell line. These cells were characterized by Western blot and assayed for changes in proliferation utilizing two- and three-dimensional growth assays. Results: HNSCC miRNA expression was characterized by a general upregulation of individual transcripts and miRNA families compared to normal mucosa. This difference in expression was independent of HPV-status, with only two miRNAs demonstrating differential expression between HPV-positive and -negative tumors: miR-449a and miR-129-3p. miR-205, a transcript upregulated in both subtypes, was able to modulate proliferation and E2F1 expression levels in 93VU147T (HPV+), but not UM-SCC-15 (HPV-). Further Western blot analysis concluded modulations in Rb-phosphorylation status and apoptotic signaling factors may explain these E2F1-mediated effects. Conclusions: While significant differences are evident in the miRNA profiles of HNSCC compared to normal mucosa, a striking degree of similarity exists between HPV-positive and -negative miRNA deregulation. A functional evaluation of miR-205 determined this transcript is capable of modulating HNSCC growth in the genetic context of p16 expression and proapoptotic signaling. Citation Format: Jason D. Howard, Haixia Cheng, Elena Ratner, Elana J. Fertig, Jimena Perez, Harry Quon, Michael Considine, Michael Ochs, Joanne Weidhaas, Christine H. Chung. MicroRNA profiling reveals miR-205 upregulation is associated with head and neck squamous cell carcinoma and modulates E2F1 signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3100. doi:10.1158/1538-7445.AM2013-3100