Triple-negative breast cancer (TNBC) is a subtype of breast cancer with poor outcome and lacks of approved targeted therapy. Overexpression of epidermal growth factor receptor (EGFR) is found in more than 50% TNBC and is suggested as a driving force in progression of TNBC; however, targeting EGFR using antibodies to prevent its dimerization and activation shows no significant benefits for TNBC patients. Here we report that EGFR monomer may activate signal transducer activator of transcription-3 (STAT3) in the absence of transmembrane protein TMEM25, whose expression is frequently decreased in human TNBC. Deficiency of TMEM25 allows EGFR monomer to phosphorylate STAT3 independent of ligand binding, and thus enhances basal STAT3 activation to promote TNBC progression in female mice. Moreover, supplying TMEM25 by adeno-associated virus strongly suppresses STAT3 activation and TNBC progression. Hence, our study reveals a role of monomeric-EGFR/STAT3 signaling pathway in TNBC progression and points out a potential targeted therapy for TNBC.

Background:Growing evidence supports the modulatory role of human gut microbiome on neoadjuvant chemotherapy (NAC) efficacy. However, the relationships among the gut microbiome, tumor-infiltrating lymphocytes (TILs), and NAC response for breast cancer (BC) patients remain unclear. We thus proposed this preliminary study to investigate the relationship between gut microbiome and BC patients' responses to NAC treatment as well as underlying mechanisms.Methods:Prior to receiving NAC, the fecal metagenome collected from 23 patients with invasive BC was analyzed. Patients were subsequently assigned to the NAC non-effectual group and the NAC effectual group based on their response to NAC. The peripheral T lymphocyte subset counts were examined by flow cytometry methods. CellMinor analysis was employed to explore the relationship between CD4 mRNA expression and the reaction of tumor cells to NAC drugs.Results:The gut microbiomes of the NAC non-effectual group showed characteristics of low diversity with low abundances, distinct metagenomic composition with decreased butyrate-producing and indolepropionic acid-producing bacteria, and increased potential pathobionts compared with the NAC effectual group. The combination of Coprococcus, Dorea, and uncultured Ruminococcus sp. serves as signature bacteria for distinguishing NAC non-effectual group patients from the NAC effectual group. The absolute numbers of CD4+ and CD8+ TIL infiltration in tumors in the NAC non-effectual group were significantly lower than those in the effectual group. Similar findings were reported for the CD4+ T lymphocytes in the peripheral blood (p's < 0.05). NAC effectual-related signature bacteria were proportional to these patients' CD4+ T lymphocyte counts in peripheral blood and tumors (p's < 0.05). CellMinor analysis showed that the CD4 mRNA expression level dramatically climbed with increased sensitivity of tumor cells to NAC drugs such as cyclophosphamide, cisplatin, and carboplatin (p's < 0.05).Conclusions:The composition of the gut microbial community differs between BC patients for whom NAC is effective to those that are treatment resistant. The modulation of the gut microbiota on host CD4+ T lymphocytes may be one critical mechanism underlying chemosensitivity and NAC pathologic response. Taken together, gut microbiota may serve as a potential biomarker for NAC response, which sheds light on novel intervention targets in the treatment of NAC non-effectual BC patients.

The early in vivo diagnosis of infectious disease foci is largely hindered by invasion and concealment of pathogens in host cells, making it difficult for conventional probes to detect and analyze intracellular pathogens. Taking advantage of the excessively produced reactive oxygen species (ROS) within host cells, herein we report the design of thiol-hemiketal blocked N-azidoacetyl galactosamine (Ac3GalNAzSP), an azido unnatural sugar bearing an unprecedent designed ROS-responsive moiety for targeted labelling of infected host cells. Ac3GalNAzSP showed great stability under physiological conditions, specifically released active unnatural sugar in host cells overproducing ROS, metabolically labeled infected host cells with azido groups, and enabled targeting in vivo infection sites by subsequent Click Chemistry reactions, substantiating an unprecedented approach for targeting infected host cells. This technique could be a powerful tool for early in vivo diagnosis and targeted treatment of infectious disease.

Human growth factor receptor-bound protein-7 (GRB7) is a pivotal mediator involved in receptor tyrosine kinase signaling and governing diverse cellular processes. Aberrant upregulation of GRB7 is frequently associated with the progression of human cancers. However, the molecular mechanisms leading to the upregulation of GRB7 remain largely unknown. Here, we propose that the epigenetic modification of GRB7 at the post-transcriptional level may be a crucial factor leading to GRB7 upregulation in ovarian cancers. Methods: The upstream miRNA regulators were predicted by in silico analysis. Expression of GRB7 was examined by qPCR, immunoblotting and immunohistochemical analyses, while miR-193a-3p levels were evaluated by qPCR and in situ hybridization in ovarian cancer cell lines and clinical tissue arrays. MS-PCR and pyrosequencing analyses were used to assess the methylation status of miR-193a-3p. Stable overexpression or gene knockdown and Tet-on inducible approaches, in combination with in vitro and in vivo tumorigenic assays, were employed to investigate the functions of GRB7 and miR-193a-3p in ovarian cancer cells. Results: Both miR-193a-3p and its isoform, miR-193b-3p, directly targeted the 3' UTR of GRB7. However, only miR-193a-3p showed a significantly inverse correlation with GRB7-upregulated ovarian cancers. Epigenetic studies revealed that methylation-mediated silencing of miR-193a-3p led to a stepwise decrease in miR-193a-3p expression from low to high-grade ovarian cancers. Intriguingly, miR-193a-3p not only modulated GRB7 but also ERBB4, SOS2 and KRAS in the MAPK/ERK signaling pathway to enhance the oncogenic properties of ovarian cancer cells in vitro and in vivo. Conclusion: These findings suggest that epigenetic silencing of miR-193a-3p by DNA hypermethylation is a dynamic process in ovarian cancer progression, and miR-193a-3p may be explored as a promising miRNA replacement therapy in this disease.

Abstract The human growth factor receptor-bound protein-7 (GRB7) is a pivotal mediator involved in receptor tyrosine kinase signaling and governing diverse cellular processes. We and others have reported that GRB7 is frequently upregulated and associated with the progression of human cancers. Clinicopathological analysis has shown that the overexpressed GRB7 is correlated with high grade and metastatic ovarian cancers. However, the molecular mechanisms leading to the upregulation of GRB7 remain largely unknown. In this study, we propose that the epigenetic modification of GRB7 at the post-transcriptional level may be a crucial factor leading to GRB7 upregulation in ovarian cancers. We found that the protein level of GRB7 was much higher than its mRNA levels in a subset of ovarian cancer cell lines, indicating aberrant post-transcriptional alterations in GRB7. Using an in silico study, we identified miR-193a-3p and its isoform, miR-193b-3p, by specifically targeting the conserved site (GGCCAGT) at position 332-338 in the 3’ UTR (length: 387) of the human GRB7 gene. QPCR and western blot analyses revealed that only the expression levels of miR-193a-3p but not miR-193b-3p were inversely correlated in ovarian cancer cell lines accompanied with high levels of GRB7 protein levels. Further biochemical analyses using luciferase reporter assays in combination with mutational analyses identified miR-193a-3p specifically targeting GRB7 3’UTR, confirming miR-193a-3p, instead of its isoform miR-193b-3p, has a more important role in GRB7 post-transcriptional regulation in ovarian cancer cells. Constitutive expression or knockdown of miR-193a-3p could significantly alter GRB7 expression, as well as oncogenic capacities of ovarian cancer cells such as cell growth, cell migration and cell invasion. Importantly, we observed that there was a significant stepwise decrease of miR-193a-3p expression from low to high grade ovarian cancers, and was inversely correlated with GRB7 expressions. Intriguingly, treatment with the DNA methyl transferase inhibitor (5’-Aza-dc) could restore the expression of miR-193a-3p, suggesting the epigenetic inhibition of miR-193a-3p is clinically relevant. Importantly, such epigenetic alteration may be the clue leading to overexpression of GRB7 in ovarian cancer cells. Taken together, our findings suggest that epigenetically silencing of miR-193a-3p upregulates GRB7 and contribute to ovarian cancer aggressiveness in tumor progression. Citation Format: Kangmei Chen, Hextan YS Ngan, David W. Chan. Methylation-associated silencing of miR-193a-3p promotes ovarian cancer aggressiveness via targeting GRB7 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-324. doi:10.1158/1538-7445.AM2017-LB-324

Abstract The unique structure and function of normal tissues is known to be regulated by epigenetic mechanisms. Understanding how normal cells in their respective tumor milieus might affect their susceptibility to become not only malignant but acquire breast cancer (BC) subtype-specific phenotypes, may determine tumor clinical behavior outcomes. The goal was to compare genome wide methylation profiles of non-coding miRNAs of breast cancer tissue and normal breast epithelium from estrogen receptor (ER) negative (-) and ER positive (+) tumors, and assess their miRNA methylomes in the context of tumor ER phenotypes as either ER- or ER+. BC tissue from 79 patients (39 ER- and 40 ER+) and normal tissue from 39 of these patients (19 ER- and 20-ER+) were assayed using the Illumina 450K bead array. A sub analysis focused on 2249 miRNA CpGs assigned to 615 unique miRNAs. M-values were computed as a logit function [(log (beta/ (1-beta))] of the methylation beta values. T-tests were used to compare the means of the M-values for the ER+ and ER- groups. The t-test p-values were used to generate adaptive FDR (aFDR) levels and aFDRs of 0.05 or lower were considered to be statistically significant (Tier 1). Tier 1 CpGs were subsequently filtered to select only those with a mean beta ratio between ER+ and ER- of under 0.5 or over 2.0 (Tier 2). The Tier 2 CpGs were further filtered to select only those with a mean beta difference of 0.2 or more (Tier 3). In the tumor cohort, 1224/2249 (54%) CpGs were differentially methylated between ER- and ER+ BC at Tier 1. Of the 1224, 963 (78.7%) were hypermethylated, and 1035 (84.6%) were in promoter regions. The 1224 CpGs at Tier 1, the 24 at Tier 2, and 2 CpGs at Tier 3 were associated with 379, 22 and 2 genes respectively. When the same analysis was performed on normal tissue only (19 ER- and 20-ER+), 76 of the 2249 CpGs had significant aFDR values and none of those met the Tier 2 or Tier 3 criteria. Seventy-one of the 76 (93.4%) were hypermethylated, and 65 (85.5%) were in promoter regions. The 76 significant Tier 1 (aFDR) differentially methylated CpGs were associated with 48 genes of which 43 were common to tumor Tier 1 differentially methylated miRNA genes, 10 were common to tumor Tier 2 genes, and 5 were restricted to normal tissue only. Normal epithelial tissues demonstrated similar differential methylation directionality as their respective tumor counterparts, favoring promoter region localization. Accordingly, the recognition of normal breast tissue-specific epigenetic propensities that align with their tumor phenotypes, suggest the possibility of progression markers specific for ER status as well as markers not associated with progression. This provides insights into our view of possible links between epigenetic programming, progression continuums, and how hormonal receptor subtypes may be determined. Support: Komen Foundation: KG110218 Citation Format: Kang Mei Chen, Josena K. Stephen, Indrani Datta, Dhananjay Chitale, George Divine, Maria J. Worsham. MicroRNA methylomes of normal breast tissue from ER negative and ER positive breast cancer identify progression markers specific for estrogen receptor status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4434. doi:10.1158/1538-7445.AM2017-4434

Abstract Many recent studies examining aberrant DNA methylation in thyroid cancer are restricted to either candidate genes or genome-wide methylation in specific thyroid tumor subtypes. The goal of this study was to identify differentially methylated genes globally and their association with molecular pathways and signaling networks. Common mutations in papillary thyroid cancer (PTC), follicular thyroid cancer (FTC), follicular adenoma (FA) and normal thyroid were also examined. Genome-wide methylation profiling using the Infinium HumanMethylation450FFPE (formalin fixed paraffin embedded) BeadChip Array was performed on 24 thyroid cases (8 PTC, 8 FTC, 4 FA and 4 normal thyroid). Ingenuity Pathway Analysis (IPA) was utilized to assess the roles of significantly differentially methylated genes in biological functions, signaling/metabolic pathways, and networks. Common mutations in 4 genes (BRAF, NRAS, HRAS, and KRAS) were assessed using TaqMan Mutation Detection assay. Twelve genes were significantly differentially methylated among 4 comparison groups: cancer vs normal, cancer vs adenoma, PTC vs normal, and PTC follicular variant (PTC-FV) vs PTC-Classic. CTU1 and HLA-DPB1 were significantly hypermethylated and AARS2, TMSB10, RNF216L, KIF15, KIAA1143, and SLC2A13 were significantly hypomethylated between cancer and normal. Significant differential hypermethylation was noted for PNPLA7 and NPC1L1 in cancer vs adenoma and SNX6 in PTC-FV vs PTC-Classic. NT5C1B was hypermethylated and AARS2 was hypomethylated in PTC vs normal. IPA identified 2 gene networks, involving 11/12 genes, characterized by 1) Cellular development, Cellular growth and proliferation, Connective tissue development and function and 2) Drug metabolism, Cell-mediated immune response, Cellular development. NT5C1B was involved in all 4 highly ranked canonical nucleotide degradation pathways. Several significant bio-functions involved NPC1L1. Mutations of NRAS codon 61 were identified in 1 sample each of FTC-Classic, PTC-FV and FA and BRAF V600E in one PTC-Classic sample. Differential methylation of AARS2, CTU1, HLA-DPB1, SLC2A13, PNPLA7, NPC1L1, NT5C1B and SNX6 suggest potential markers for discriminating thyroid cancers from adenomas and normal. NT5C1B was noted in highly ranked canonical pathways, suggesting a role in nucleotide degradation. Pathway analysis of differentially methylated genes support important biological processes in thyroid cancer pathogenesis. Supported by JFCI/CRAG A20038 Citation Format: Josena K. Stephen, Kang Mei Chen, Jason Merritt, Indrani Datta, Dhananjay Chitale, George Divine, Maria J. Worsham. Methylome differences in differentiated thyroid cancers and benign adenomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3361. doi:10.1158/1538-7445.AM2017-3361

Objectives/Hypothesis The human papillomavirus (HPV) is known to infect the tissues of the oropharynx as demonstrated in HPV‐positive oropharyngeal squamous cell carcinoma (OPSCC). HPV has also been shown to induce benign lymphoid hypertrophy. We sought to investigate an association between obstructive sleep apnea (OSA) and the presence of HPV in palatine and lingual tonsillar oropharyngeal tissue. Study Design Case series with chart review. Methods This retrospective laboratory‐based study of oropharyngeal tissue from patients with OSA included patients >18 years old who underwent surgical treatment for OSA at a single institution between January 2012 and May 2014. Surgical specimens of adequate size were analyzed for HPV6, 11, and 16 using real‐time quantitative polymerase chain reaction from DNA extracted from formalin‐fixed paraffin‐embedded tissue blocks. Student t test, Pearson χ 2 test, and linear logistic regression were used to assess comparisons of body mass index (BMI), apnea‐hypopnea index (AHI), age, and gender between HPV‐positive and HPV‐negative groups. Results Of 99 cases included in the study, six were positive for HPV: two with HPV16 and four with HPV6. BMI, AHI, age, and gender showed no significant differences between the HPV‐positive and HPV‐negative groups. Logistic regression to predict HPV positivity accounting for each variable and multivariate analysis were not statistically significant. Conclusions Our study did not show HPV to have a statistically significant association with OSA. None of the covariates analyzed (BMI, AHI, gender, age) predicted HPV positivity in surgically resected oropharyngeal tissue from OSA patients. Level of Evidence 4 Laryngoscope , 127:1231–1234, 2017

The majority of published studies investigating driver genes have focused primarily on genomic mutations which have led to novel study designs (basket trials) where patients with a rare mutation, regardless of tumor histology, are matched to a drug expected to work through the mutated pathway. This dominant focus on genomic mutations has overshadowed consideration of inclusion of epigenetic information. Epigenetic silencing of driver genes leads to various genomic alterations, including mismatch repair deficiency, altered DNA repair and loss of chromosomal stability. In human papilloma virus (HPV) positive head and neck squamous cell carcinoma (HNSCC), recent studies are beginning to establish a mechanistic role for DNA methylation with potential to impact improved survival outcomes. The purpose of this study was to illustrate network integration of epigenomic data in head and neck cancer to prioritize elements that drive biological states. Causal Networks are small hierarchical networks of regulators whose activity can be modulated by the expression of downstream target genes to enhance understanding of the effect of upstream master regulators on disease or function. To further establish the driver potential of 11 genes: C14orf162/ CCDC177, CDH8, CRMP1, ELMO1, HTR1E, MEI1, MSX2, PCDH10, PCDHB11, PITX2, SYN2 previously reported as significantly differentially methylated between HPV positive and HPV negative HNSCC tumor samples, and subsequently validated by our group in two independent sample sets, their master regulatory networks were identified utilizing Causal Network Analysis (CNA) software from Ingenuity Pathway Analysis. To reflect expected gene expression direction implied by methylation changes, the inverse of the methylation ratio from HPV positive vs. HPV negative HNSCC was used for CNA. CNA identified 23 top hierarchical networks (significant z score of absolute 2) associated with HNSCC and their corresponding master regulatory molecules characterized as transcription regulators and kinase inhibitors among others. Of the 11 target genes, 7 had representation in multiple networks; CRMP1 in 21/23, MSX2 and SYN2 in 20/23, CDH8 and PITX2 in 17/23, PCDH10 in 10/23, and ELMO1 in 5/23 networks. Of the 23 networks, 12 indicated activation and 11 inhibition by at least 4 or more of the 7 target genes. Master regulators were within 2 or 3 hops (intermediate regulators) to the target genes. CNA raised the profile of CDH8, CRMP1, ELMO1, MSX2, PCDH10, SYN2, and PITX2 (7/11 target genes) for further consideration as epigenetic drivers of HPV-associated HNSCC. Support: Komen Foundation: KG110218 Citation Format: Maria J. Worsham, Kang Mei Chen, Indrani Datta, Josena K. Stephen, Dhananjay Chitale, Tamer Ghanem, Lamont Jones, Laura Garcia-Rodriquez, George Divine. Network integration of epigenomic data in HPV-associated head and neck cancer [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr A26.

Background: Cancer metastasis is determined by the formation of the metastatic niche and the ability of cancer cells to adapt to microenvironmental stresses. Anoikis resistance is a fundamental feature of metastatic cancer cell survival during metastatic cancer progression. However, the mechanisms underlying anoikis resistance in ovarian cancer are still unclear.Methods: Expressions of miRNA-141 and its downstream targets were evaluated by qPCR, Western blotting, Immunohistochemical (IHC) and in situ hybridization (ISH) assays. The luciferase assays were used to prove KLF12 as the downstream target of miR-141. The cDNA microarray and apoptotic protein arrays were used to identify the targets of miR-141 and KLF12. The competition of KLF12 and Sp1 on survivin promoter was examined by ChIP assay. IHC analysis on ovarian cancer tissue array was used to evaluate the expressions of KLF12 and miR-141 and to show the clinical relevance. The functional studies were performed by in vitro and in vivo tumorigenic assays.Results: Enforced expression of miR-141 promotes, while knockdown of miR-141 expression inhibits, cell proliferation, anchorage-independent capacity, anoikis resistance, tumor growth and peritoneal metastases of ovarian cancer cells. Bioinformatics and functional analysis identified that Kruppel-related zinc finger protein AP-2rep (KLF12) is directly targeted by miR-141. Consistent with this finding, knockdown of KLF12 phenocopied the effects of miR-141 overexpression in ovarian cancer cells. In contrast, restoration of KLF12 in miR-141-expressing cells significantly attenuated anoikis resistance in ovarian cancer cells via interfering with Sp1-mediated survivin transcription, which inhibits the intrinsic apoptotic pathway and is crucial for ovarian cancer cell survival, anoikis resistance and peritoneal metastases. Immunohistochemical (IHC) and in situ hybridization (ISH) assays confirmed that miRNA-141 expression is inversely correlated with KLF12 expression and significantly associated with advanced ovarian cancers accompanied with distal metastases, underscoring the clinical relevance of our findings.Conclusions: Our data identify a novel signaling axis of miR-141/KLF12/Sp1/survivin in enhancing anoikis resistance and likely serves as a potential therapeutic target for metastatic ovarian cancer.

The majority of published studies investigating driver genes have focused primarily on genomic mutations which have led to novel study designs (basket trials) where patients with a rare mutation, regardless of tumor histology, are matched to a drug expected to work through the mutated pathway. This dominant focus on genomic mutations has overshadowed consideration of inclusion of epigenetic information. Epigenetic silencing of driver genes leads to various genomic alterations, including mismatch repair deficiency, altered DNA repair and loss of chromosomal stability. In human papilloma virus (HPV) positive head and neck squamous cell carcinoma (HNSCC), recent studies are beginning to establish a mechanistic role for DNA methylation with potential to impact improved survival outcomes. The purpose of this study was to illustrate network integration of epigenomic data in head and neck cancer to prioritize elements that drive biological states. Causal Networks are small hierarchical networks of regulators whose activity can be modulated by the expression of downstream target genes to enhance understanding of the effect of upstream master regulators on disease or function. To further establish the driver potential of 11 genes: C14orf162/ CCDC177, CDH8, CRMP1, ELMO1, HTR1E, MEI1, MSX2, PCDH10, PCDHB11, PITX2, SYN2 previously reported as significantly differentially methylated between HPV positive and HPV negative HNSCC tumor samples, and subsequently validated by our group in two independent sample sets, their master regulatory networks were identified utilizing Causal Network Analysis (CNA) software from Ingenuity Pathway Analysis. To reflect expected gene expression direction implied by methylation changes, the inverse of the methylation ratio from HPV positive vs. HPV negative HNSCC was used for CNA. CNA identified 23 top hierarchical networks (significant z score of absolute 2) associated with HNSCC and their corresponding master regulatory molecules characterized as transcription regulators and kinase inhibitors among others. Of the 11 target genes, 7 had representation in multiple networks; CRMP1 in 21/23, MSX2 and SYN2 in 20/23, CDH8 and PITX2 in 17/23, PCDH10 in 10/23, and ELMO1 in 5/23 networks. Of the 23 networks, 12 indicated activation and 11 inhibition by at least 4 or more of the 7 target genes. Master regulators were within 2 or 3 hops (intermediate regulators) to the target genes. CNA raised the profile of CDH8, CRMP1, ELMO1, MSX2, PCDH10, SYN2, and PITX2 (7/11 target genes) for further consideration as epigenetic drivers of HPV-associated HNSCC. Support: Komen Foundation: KG110218 Citation Format: Maria J. Worsham, Kang Mei Chen, Indrani Datta, Josena K. Stephen, Dhananjay Chitale, Tamer Ghanem, Lamont Jones, Laura Garcia-Rodriquez, George Divine. Network integration of epigenomic data in HPV-associated head and neck cancer [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr A26.

Keloids are benign fibroproliferative tumors of the skin which commonly occur after injury mainly in darker skinned patients. Medical treatment is fraught with high recurrence rates mainly because of an incomplete understanding of the biological mechanisms that lead to keloids. The purpose of this project was to examine keloid pathogenesis from the epigenome perspective of DNA methylation. Genome-wide profiling used the Infinium HumanMethylation450 BeadChip to interrogate DNA from 6 fresh keloid and 6 normal skin samples from 12 anonymous donors. A 3-tiered approach was used to call out genes most differentially methylated between keloid and normal. When compared to normal, of the 685 differentially methylated CpGs at Tier 3, 510 were hypomethylated and 175 were hypermethylated with 190 CpGs in promoter and 495 in nonpromoter regions. The 190 promoter region CpGs corresponded to 152 genes: 96 (63%) were hypomethylated and 56 (37%) hypermethylated. This exploratory genome-wide scan of the keloid methylome highlights a predominance of hypomethylated genomic landscapes, favoring nonpromoter regions. DNA methylation, as an additional mechanism for gene regulation in keloid pathogenesis, holds potential for novel treatments that reverse deleterious epigenetic changes. As an alternative mechanism for regulating genes, epigenetics may explain why gene mutations alone do not provide definitive mechanisms for keloid formation.

Objectives/Hypothesis: To generate novel insights and hypotheses in keloid development from potential master regulators.Study Design: Prospective cohort.Methods: Six fresh keloid and six normal skin samples from 12 anonymous donors were used in a prospective cohort study. Genome-wide profiling was done previously on the cohort using the Infinium HumanMethylation450 BeadChip (Illumina, San Diego, CA). The 190 statistically significant CpG islands between keloid and normal tissue mapped to 152 genes (P<.05). The top 10 statistically significant genes (VAMP5, ACTR3C, GALNT3, KCNAB2, LRRC61, SCML4, SYNGR1, TNS1, PLEKHG5, PPP1R13-alpha, false discovery rate <.015) were uploaded into the Ingenuity Pathway Analysis software's Causal Network Analysis (QIAGEN, Redwood City, CA). To reflect expected gene expression direction in the context of methylation changes, the inverse of the methylation ratio from keloid versus normal tissue was used for the analysis. Causal Network Analysis identified disease-specific master regulator molecules based on downstream differentially expressed keloid-specific genes and expected directionality of expression (hypermethylated vs. hypomethylated).Results: Causal Network Analysis software identified four hierarchical networks that included four master regulators (pyroxamide, tributyrin, PRKG2, and PENK) and 19 intermediate regulators.Conclusions: Causal Network Analysis of differentiated methylated gene data of keloid versus normal skin demonstrated four causal networks with four master regulators. These hierarchical networks suggest potential driver roles for their downstream keloid gene targets in the pathogenesis of the keloid phenotype, likely triggered due to perturbation/injury to normal tissue.

Background: Micro RNAs (miRNA) are endogenous, small non-coding RNAs that control gene expression by directing their target mRNAs for degradation and/or posttranscriptional repression. Compared to mRNA signatures, miRNAs have better and stronger biomarker properties with 20 times more power in biomarker studies as compared to mRNAs (when comparing 20,000 mRNAs to ∼1,000 miRNAs). Emerging evidence now supports the idea that DNA methylation is crucially involved in the dysregulation of miRNAs in cancer, representing a novel class of potential biomarkers for diagnosis, prediction of treatment, or prognosis. ER-negative breast cancer (BC) is an aggressive histological subtype with limited treatment options and very poor prognosis. Our long term objective is to derive a diagnostic, prognostic, and predictive ER-negative specific miRNA panel for detection of early cancer, recurrence/metastasis, and as potential therapeutic targets for better management of ER-negative BC. Methods: The initial discovery step profiled 39 primary ER negative and 40 ER positive BC cases using the Illumina Infinium HumanMethylation450 BeadChip followed by a subanalysis focusing on 2249 miRNA CpGs assigned to 615 unique miRNAs. T-tests were used to compare the means of the M-values for the ER-positive and ER-negative groups. The t-test p-values were used to generate adaptive FDR (aFDR) levels and aFDRs of 0.05 or lower were considered to be statistically significant (Tier 1). Tier 1 CpGs were subsequently filtered to select only those with a mean beta ratio between ER positive and ER negative of under 0.5 or over 2.0 (Tier 2). The Tier 2 CpGs were further filtered to select only those with a mean beta difference of 0.2 or more (Tier 3). Because miRNAs perform their important functions via their targets, the targets of miRNAs were assessed for functional enrichment analysis in IPA for biologic involvement. Results: Over half of the miRNA CpGs (1224/2249, 54%) were differentially methylated between ER negative and ER positive BC with significant aFDR levels. The 1224 CpGs at Tier 1 were associated with 379 miRNAs; the 24 and 2 CpGs for Tiers 2 and 3 with 22 and 2 miRNAs, respectively. The 22 miRNA genes were assigned to 4621 targets using online databases that predict miRNA targets. The degree of confidence that a target gene is associated with a miRNA is characterized in these databases as either observed, or just as high (predicted). Of these 4621 targets, 87 were designated as experimentally observed and were examined in IPA. Top pathways and networks designated by miRNA targets included the cell cycle G1/S checkpoint regulation canonical pathway, and the cell-to-cell interaction/cancer networks among others. MiRNA targets in top pathways and networks were circled back to their respective miRNAs revealing cooperatively mediated pathway dysregulation of ER negative BC. Conclusion: Aberrantly methylated miRNAs showed perturbation of biologically significant pathways and networks, suggesting that miRNAs mediate pathway dysregulation in a coordinated manner, strengthening the case for utility of miRNAs as viable biomarkers in ER negative BC. Support: Komen Foundation: KG110218. Citation Format: Worsham MJ, Chen KM, Datta I, Stephen JK, Chitale D, Divine G. Epigenetically altered microRNA mediated pathway dysregulation in ER negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-09-10.

Abstract The majority of published studies investigating driver genes have focused primarily on genomic mutations which have led to novel study designs (basket trials) where patients with a rare mutation, regardless of tumor histology, are matched to a drug expected to work through the mutated pathway. This dominant focus on genomic mutations has yet to configure in epigenetics. There has been relatively little advancement in changing the management of women with ER negative BC, mainly due to a dearth of actionable therapeutic targets. Our drill-down approach to identified an ER negative-specific 16 gene methylation signature (AHNAK, ALPL, ANXA2R, CCND1, CIRBP, CPQ, DST, EGFR, ESR1, GPRC5B, HERC5, IL22RA2, MITF, OBSL1, POU3F3, RB1CC1) starting from a discovery approach (Illumina Infinium HumanMethylation450 BeadChip, 40 ER negative vs. 40 ER positive BC) followed by expression verification, significant rankings in biological pathways (Ingenuity Pathway Analysis), and confirmation by targeted sequencing using Illumina MiSeq. Causal Networks are small hierarchical networks of regulators that control the expression/methylation of dataset targets. They can enhance understanding of the effect of master regulators on disease or function. The objective of this study was to identify regulatory networks utilizing IPA's Causal Network Analysis (CNA) in order to illuminate possible causes and mechanisms underlying the biological activities of the 16 candidate gene signature differentiating ER negative from ER positive BC. To reflect expected gene expression direction implied by methylation changes for the 16 candidate genes, the inverse of the methylation ratio from ER negative vs. ER positive tissue was used for CNA. CNA software identified 4 hierarchical networks and their corresponding master regulatory molecules, diethylstilbestrol, MSH2, 15-ketoprotaglandin E2, and transcription regulator SP1. Diethylstilbestrol and SP1 had direct regulatory influence (depth level 1) to the candidate molecules ALPL, CCND1, EGFR, ESR1 and CCND1, CIRBP, EGFR, ESR1, respectively. CNA raised the profile of ALPL, CCND1, CIRBP, EGFR, ESR1 (5/16 candidate genes) for further consideration as potential epigenetic drivers of ER negative BC. Support: Komen Foundation: KG110218 Citation Format: Maria J. Worsham, Kang Mei Chen, Indrani Datta, Josena K. Stephen, Dhananjay Chitale, George Divine. Differentially methylation between ER negative and ER positive breast cancer identifies master regulators to expose potential epigenetic drivers of aggressive disease. [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 4484.

In recent years, studies have suggested that promoter methylation in human papilloma virus (HPV) positive head and neck squamous cell carcinoma (HNSCC) has a mechanistic role and has the potential to improve patient survival. The present study aimed to replicate key molecular findings from previous analyses of the methylomes of HPV positive and HPV negative HNSCC in an independent cohort, to assess the reliability of differentially methylated markers in HPV-associated tumors. HPV was measured using real-time quantitative PCR and the biological significance of methylation differences was assessed by Ingenuity Pathway Analysis (IPA). Using an identical experimental design of a 450K methylation platform, 7 of the 11 genes were detected to be significantly differentially methylated and all 11 genes were either hypo- or hypermethylated, which was in agreement with the results of a previous study. IPA's enriched networks analysis identified one network with msh homeobox 2 (MSX2) as a central node. Locally dense interactions between genes in networks tend to reflect significant biology; therefore MSX2 was selected as an important gene. Sequestration in the top four canonical pathways was noted for 5-hydroxytryptamine receptor 1E (serotonin signaling), collapsin response mediator protein 1 (semaphorin signaling) and paired like homeodomain 2 (bone morphogenic protein and transforming growth factor-β signaling). Placement of 9 of the 11 genes in highly ranked pathways and bionetworks identified key biological processes to further emphasize differences between HNSCC HPV positive and negative pathogenesis.

Abstract There has been relatively little advancement in changing the management of women with ER negative (ER-) breast cancer (BC). Our objective is to examine the contribution of aberrantly methylated genes to the pathogenesis of ER- BC, determine their utility in refining classification of ER- subtypes [basal-like (BL) and Her2-enriched], in differentiating ER- BC in African American (AA) and Caucasian American (CA) women, and as new prognostic and therapeutic targets for better management of ER- BC. The methylation status of 40 ER- and 40 ER positive (ER+) breast cancer samples was assessed using the Illumina 450K. A 3-tiered approach was used to call out genes most differentially methylated between ER- and ER+. The top 70 genes were further downsized using the TaqMan low-density array expression assays, Ingenuity Pathway Analysis (IPA) to assess their biologic significance, followed by targeted sequencing (Illumina Miseq). Further assessment of the genes in the 450K TCGA data sets (UCSC Brower 2015) was used for final downsizing. Of the 70 highly ranked differentially methylated genes, 56 were hypermethylated and 14 hypomethylated, with positive correlation with gene expression for the majority of genes. Expression verification and significant rankings in canonical pathways and bionetworks resulted in further downsizing to 41 genes upon confirmation by targeted sequencing. Further filtering of the 41 genes with 450K TCGA data sets provided additional refinement to a 10 gene candidate panel currently being vetted in a retrospective cohort of over 1000 ER negative BC (with 40% AA). Our iterative approach to identify an ER negative-specific gene signature has the potential to offer predictive and prognostic markers of improved classification for ER- BC in AA and CA women. Confirmation and validation of these methylation profiles in independent cohorts of BL BC defined by cDNA microarrays should further stratify poor prognosis breast cancers subgroups and serve as targets for epigenetic-modifying agents individualized to AA and CA women with ER- BC. Human therapeutic intervention trials to reverse deleterious epigenetic changes have shown promise in halting tumor growth by reactivation of the tumor suppressor genes or by reversing DNA hypomethylation. Support: Komen Foundation: KG110218 Citation Format: Maria J. Worsham, Dhananjay Chitale, Kang Mei Chen, Indrani Datta, Josena K. Stephen, George Divine. A drill-down approach to identifying an ER negative-specific gene signature. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4765. doi:10.1158/1538-7445.AM2015-4765

Currently available markers routinely used in clinical practice are of limited value to patients with estrogen receptor-negative (ER−) breast cancer [basal-like and HER2neu-positive (HER+)], an aggressive subtype. Our aim was to uncover molecular pathways and signaling networks exposed by differentially methylated genes informative of the biology of ER− breast cancer (BC) subtypes versus ER-positive (ER+). Whole-genome methylation array analysis was carried out using the Illumina Infinium HumanMethylation27 BeadChip on 14 primary BC: five ER+, four triple-negative (TNBC), and five ER−HER2+. Degree of methylation was calculated as a β-value (ranging from 0 to 1), and M-values [log (β/(1 − β)] were used for significance tests. To identify methylated genes associated with ER− subtypes (TNBC and ER−HER2+) and distinct from ER+, a weighted algorithm, developed to increase statistical rigor, called out genes in which methylation changed dramatically between ER+ and ER− subtypes. Differentially methylated gene lists examined using Ingenuity Pathway Analysis called out canonical pathways and networks with clues to biological distinctiveness as well as relatedness between ER− subtypes as compared to ER+ BC. The study highlights the interplay of ER− subtype-specific genes and their signaling pathways as potential putative fingerprints in refining classification of BC subtypes and as potential biological markers designed to hit multiple targets.

IMPORTANCE Human papillomavirus (HPV) is a known causative agent for oropharyngeal squamous cell carcinoma (OPSCC). Whereas it is becoming more firmly established that HPV-positive head and neck squamous cell carcinoma is associated with better survival outcomes, believed to be because of better response to chemoradiation therapy, the specific mechanisms for these improved survival outcomes remain underexplored.OBJECTIVE To examine the relationship between HPV status and promoter methylation in an OPSCC cohort.DESIGN, SETTING, AND PARTICIPANTS Real-time quantitative polymerase chain reaction was used to examine oncogenic HPV type 16 in a retrospective cohort of 121 patients with primary OPSCC. Aberrant promoter methylation of IGSF4, DAPK1, and ESR1 genes, known to be methylated in head and neck squamous cell carcinoma, including OPSCC, was examined by means of quantitativemethylation-specific polymerase chain reaction.INTERVENTIONS Patients received standard therapy. MAINOUTCOMES AND MEASURES Univariate associations between HPV and methylation were analyzed using Fisher exact tests followed by multivariable logistic regression. Cox proportional-hazards regression was used to model the risk of death given age, race, sex, HPV status, methylation, stage, smoking, and treatment.RESULTS In univariate logistic regression analyses, HPV-positive status was significantly associated with Caucasian race (P = .02), treatment (radiotherapy only, P = .01; chemoradiotherapy, P = .007), and IGSF4 methylation (P = .005). The final multivariate logistic model, after controlling for patient characteristics (sex, age, smoking, race, and treatment) with backward variable selection among genes, retained IGSF4 methylation (OR, 4.5 [95% CI, 1.6-12.8]; P = .005), Caucasian race (OR, 2.9 [95% CI, 1.0-8.3]; P = .053), treatment (radiotherapy only vs neither: OR, 11.62 [95% CI, 2.02-66.82]; P = .02; chemoradiotherapy vs neither: OR, 11.15 [95% CI, 1.92-64.65]; P = .01), male sex (OR, 4.7 [95% CI, 1.3-17.0]; P = .02), and younger age (OR, 0.9 [95% CI, 0.90-1.0]; P = .008) as independent predictors of HPV-positive status. Cox regression modeling indicated HPV-negative status, age, male sex, smoking, and radiation treatment as independent predictors of mortality.CONCLUSIONS AND RELEVANCE Methylation of IGSF4 is an independent predictor of HPV-positive status. DNA methylation in conjunction with HPV infection appears to play a role in OPSCC.