Kristi L. Bennett

Germline epigenetic regulation of KILLIN in Cowden and Cowden-like syndrome.

CONTEXT Germline loss-of-function phosphatase and tensin homolog gene (PTEN) mutations cause 80% of Cowden syndrome, a rare autosomal-dominant disorder (1 in 200,000 live births), characterized by high risks of breast, thyroid, and other cancers. A large heterogeneous group of individuals with Cowden-like syndrome, who have various combinations of Cowden syndrome features but who do not meet Cowden syndrome diagnostic criteria, have PTEN mutations less than 10% of the time, making molecular diagnosis, prediction, genetic counseling, and risk management challenging. Other mechanisms of loss of function such as hypermethylation, which should result in underexpression of PTEN or of KILLIN, a novel tumor suppressor transcribed in the opposite direction, may account for the remainder of Cowden syndrome and Cowden-like syndrome. OBJECTIVE To determine whether germline methylation is found in Cowden syndrome or Cowden-like syndrome in individuals lacking germline PTEN mutations. DESIGN, SETTING, AND PARTICIPANTS Nucleic acids from prospective nested series of 123 patients with Cowden syndrome or Cowden-like syndrome and 50 unaffected individuals without PTEN variants were analyzed for germline methylation and expression of PTEN and KILLIN at the Cleveland Clinic, August 2008-June 2010. Prevalence of component cancers between groups was compared using the Fisher exact test. MAIN OUTCOME MEASURES Frequency of germline methylation in PTEN mutation-negative Cowden syndrome and Cowden syndrome-like individuals. Prevalence of component cancers in methylation-positive and PTEN mutation-positive individuals. RESULTS Of 123 patients with Cowden syndrome or Cowden-like syndrome, 45 (37%; 95% confidence interval [CI], 29%-45%) showed hypermethylation upstream of PTEN but no transcriptional repression. The germline methylation was found to transcriptionally down-regulate KILLIN by 250-fold (95% CI, 45-14 286; P = .007) and exclusively disrupted TP53 activation of KILLIN by 30% (95% CI, 7%-45%; P = .008). Demethylation treatment increased only KILLIN expression 4.88-fold (95% CI, 1.4-18.1; P = .05). Individuals with KILLIN -promoter methylation had a 3-fold increased prevalence of breast cancer (35/42 vs 24/64; P < .0001) and a greater than 2-fold increase of kidney cancer (4/45 vs 6/155; P = .004) over individuals with germline PTEN mutations. CONCLUSIONS Germline KILLIN methylation is common among patients with Cowden syndrome or Cowden-like syndrome and is associated with increased risks of breast and renal cancer over PTEN mutation-positive individuals. These observations need to be replicated.

TWIST2 Demonstrates Differential Methylation in Immunoglobulin Variable Heavy Chain Mutated and Unmutated Chronic Lymphocytic Leukemia

PURPOSE Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease for which natural history can be predicted based on the presence or absence of immunoglobulin (Ig) variable heavy chain (V(H)) gene mutations. Herein we report selective epigenetic silencing of the transcription factor TWIST2 (DERMO1) in Ig V(H) mutated CLL and describe a semiquantitative assay to study promoter methylation of this gene in primary tumor cells. MATERIALS AND METHODS TWIST2 promoter methylation was identified by restriction landmark genome scanning. Southern blot (SB), bisulfite sequencing, and combined bisulfite restriction analysis (COBRA), and quantitative SB-COBRA was performed to study methylation of the TWIST2 promoter. Reverse transcription polymerase chain reaction assays were used to study TWIST2 expression in CLL cells. RESULTS Following identification and confirmation of TWIST2 methylation in CLL patients, we demonstrated that expression of this transcription factor is related to the degree of promoter methylation. Expression of TWIST2 in a CLL cell line in which the promoter is methylated was increased following decitabine treatment. We next studied 53 patients by COBRA and demonstrated that 72% of patient samples with mutated Ig V(H) show TWIST2 methylation, while only 16% of patient samples with unmutated Ig V(H) were methylated (P < .001). In a subset of patients, methylation of TWIST2 correlated with mRNA expression. CONCLUSION TWIST2 is differentially methylated in CLL cells relative to Ig V(H) mutational status and can be quantitatively monitored by SB-COBRA. Based on the known role of TWIST2 in silencing p53 function in other malignancies, future studies should focus on the role of TWIST2 in CLL and related lymphoproliferative diseases.

HPV status-independent association of alcohol and tobacco exposure or prior radiation therapy with promoter methylation of FUSSEL18, EBF3, IRX1, and SEPT9, but not SLC5A8, in head and neck squamous cell carcinomas.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy with more than half a million people being diagnosed with the disease annually. Within the last 2 decades, the human papillomavirus (HPV) has been found to be associated with this malignancy. More recently, HPV‐infected HNSCC has been found to exhibit higher levels of global DNA methylation. In a recent study, we identified five tumor suppressive genes (IRX1, EBF3, SLC5A8, SEPT9, and FUSSEL18) as frequently methylated in HNSCC biopsies using a global methylation analysis via restriction landmark genomic scanning. In this study, we verify these genes as valid methylation markers in two separate sets of HNSCC specimens. By using the available clinical information linked to the patient specimens, we found a strong association between promoter methylation of FUSSEL18, IRX1, and EBF3 and prior radiation therapy (P < 0.0001) irrespective of HPV status. Also, promoter methylation of FUSSEL18 and SEPTIN9 was found to correlate significantly with exposure to alcohol and tobacco (P = 0.021). Importantly, in this study, we preliminarily show a trend between HPV16 positivity and specific target gene hypermethylation of IRX1, EBF3, SLC5A8, and SEPT9. If replicated in a larger study, the HPV status may be a patient selection biomarker when determining the most efficacious treatment modality for these different subsets of patients (e.g., inclusion or exclusion of epigenetic therapies). Equally notable and independent of HPV status, hypermethylation of the promoters of a subset of these genes in recurrences especially in the setting of prior radiation or in the setting of alcohol and tobacco use might help guide adjunctive inclusion or exclusion or epigenetic therapy.

Microbiomic subprofiles and MDR1 promoter methylation in head and neck squamous cell carcinoma

Clinical observations and epidemiologic studies suggest that the incidence of head and neck squamous cell carcinoma (HNSCC) correlates with dental hygiene, implying a role for bacteria-induced inflammation in its pathogenesis. Here we begin to explore the pilot hypothesis that specific microbial populations may contribute to HNSCC pathogenesis via epigenetic modifications in inflammatory- and HNSCC-associated genes. Microbiomic profiling by 16S rRNA sequencing of matched tumor and adjacent normal tissue specimens in 42 individuals with HNSCC demonstrate a significant association of specific bacterial subpopulations with HNSCC over normal tissue (P < 0.01). Furthermore, microbial populations can separate tumors by tobacco status (P < 0.008), but not by alcohol status (P = 0.41). If our subhypothesis regarding a mechanistic link from microorganism to carcinogenesis via inflammation and consequent aberrant DNA methylation is correct, then we should see hypermethylation of relevant genes associate with specific microbiomic profiles. Methylation analysis in four genes (MDR1, IL8, RARB, TGFBR2) previously linked to HNSCC or inflammation shows significantly increased methylation in tumor samples compared with normal oral mucosa. Of these, MDR1 promoter methylation associates with specific microbiomic profiles in tumor over normal mucosa. Additionally, we report that MDR1 methylation correlates with regional nodal metastases in the context of two specific bacterial subpopulations, Enterobacteriaceae and Tenericutes (P < 0.001 for each). These associations may lead to a different, and potentially more comprehensive, perspective on the pathogenesis of HNSCC, and support further exploration of mechanistic linkage and, if so, novel therapeutic strategies such as demethylating agents and probiotic adjuncts, particularly for patients with advanced or refractory disease.

Germline and somatic DNA methylation and epigenetic regulation of KILLIN in renal cell carcinoma

We recently identified germline methylation of KILLIN, a novel p53‐regulated tumor suppressor proximal to PTEN, in >1/3 Cowden or Cowden syndrome‐like (CS/CSL) individuals who are PTEN mutation negative. Individuals with germline KILLIN methylation had increased risks of renal cell carcinoma (RCC) over those with PTEN mutations. Therefore, we tested the hypothesis that KILLIN may be a RCC susceptibility gene, silenced by germline methylation. We found germline hypermethylation by combined bisulfite restriction analysis in at least one of the four CpG‐rich regions in 23/41 (56%) RCC patients compared to 0/50 controls (P < 0.0001). Of the 23, 11 (48%) demonstrated methylation in the −598 to −890 bp region in respect to the KILLIN transcription start site. Furthermore, 19 of 20 advanced RCC showed somatic hypermethylation upstream of KILLIN, with the majority hypermethylated at more than one CpG island (13/19 vs. 3/23 with germline methylation, P < 0.0001). qRT‐PCR revealed that methylation significantly downregulates KILLIN expression (P = 0.05), and demethylation treatment by 5‐aza‐2′deoxycytidine significantly increased KILLIN expression in all RCC cell lines while only increasing PTEN expression in one line. Furthermore, targeted in vitro methylation revealed a significant decrease in KILLIN promoter activity only. These data reveal differential epigenetic regulation by DNA promoter methylation of this bidirectional promoter. In summary, we have identified KILLIN as a potential novel cancer predisposition gene for nonsyndromic clear‐cell RCC, and the epigenetic mechanism of KILLIN inactivation in both the germline and somatic setting suggests the potential for treatment with demethylating agents.

Disruption of Transforming Growth Factor-β Signaling by Five Frequently Methylated Genes Leads to Head and Neck Squamous Cell Carcinoma Pathogenesis

Head and neck squamous cell carcinoma (HNSCC) is an aggressive cancer with low survival rates in advanced stages. To facilitate timely diagnosis and improve outcome, early detection markers (e.g., DNA methylation) are crucial for timely cancer diagnosis. In a recent publication, an epigenome-wide screen revealed a set of genes that are commonly methylated and downregulated in head and neck cancers (SEPT9, SLC5A8, FUSSEL18, EBF3, and IRX1). Interestingly, these candidates are potentially involved in the transforming growth factor-beta (TGF-beta) signaling pathway, which is often disrupted in HNSCC. Therefore, we sought to determine coordinated epigenetic silencing of these candidate genes in HNSCC as potential key disruptors of TGF-beta signaling, which could ultimately result in HNSCC progression. Through immunoprecipitation studies, all five of the investigated candidate genes were found to interact with components of the TGF-beta pathway. Overexpression of SLC5A8, EBF3, and IRX1 resulted in decreased mitotic activity and increased apoptosis. In addition, EBF3 was found to increase p21 promoter activity, and SMAD2 significantly increased IRX1 promoter activity. These findings are significant because they reveal a set of genes that interact with components of the TGF-beta pathway, and their silencing via methylation in HNSCC results in coordinated decrease in apoptosis, increased proliferation, and decreased differentiation.

Activator protein 2 alpha (AP2α) suppresses 42 kDa C/CAAT enhancer binding protein α (p42 cEBPα) in head and neck squamous cell carcinoma

The tumor suppressor C/CAAT enhancer binding protein alpha (C/EBPα) is a transcription factor involved in cell cycle control and cellular differentiation. A recent study showed that C/EBPα is frequently downregulated in head and neck squamous cell carcinoma (HNSCC) by DNA methylation in an upstream regulatory region. Here, we investigated how DNA methylation in the upstream regulatory region disrupts the transcriptional regulation of C/EBPα in HNSCC. The results reveal that aberrant methylation correlates with methyl binding domain protein binding and repressive histone modifications. This methylated region contains previously uninvestigated AP2α binding sites. AP2α suppresses C/EBPα promoter activity and protein expression. Interestingly, silencing AP2α by shRNA increases the antiproliferative isoform of C/EBPα (p42C/EBPα). Furthermore, growth analysis revealed that these 2 isoforms yield very different proliferative properties in HNSCC.

Abstract 4087: Microbiomic subprofiles and MDR1 promoter methylation in head and neck squamous cell carcinoma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Clinical observations and epidemiologic studies suggest that the incidence of head and neck squamous cell carcinoma (HNSCC) correlates with dental hygiene, implying a role for bacteria-induced inflammation in its pathogenesis. Differences in microbiota in HNSCC compared to matched normal oral mucosa, and whether these differences are accompanied by epigenetic modifications in selected HNSCC-associated genes and clinico-pathologic features has not yet been investigated. Standard microbiologic culturing techniques miss many fastidious organisms. Methods: Human genomic DNA and associated microbial DNA were isolated from 42 prospectively accrued consecutive HNSCC tumors and paired normal oral mucosae. Microbiomic profiling was carried out by serially characterizing bacteria-specific 16S rRNA and taxa classified by the presence of specific polymorphisms in the 16S rDNA genes. Promoter methylation and microbial populations of each sample was determined. Microbiomic profiles were compared between HNSCC and matched normal mucosa, to specific promoter methylation in four genes (MDR1, IL8, RARB, TGFBR2) previously linked to HNSCC or inflammation, and to clinico-pathologic features. Results: Specific microbiomic profiles significantly associate with HNSCC over normal mucosae. While methylation of both MDR1 (p<10-9) and IL8 (p<0.02) genes significantly associate with HNSCC, only MDR1 methylation associates with specific microbiomic profiles in HNSCC over normal mucosae. Furthermore, MDR1 methylation correlates with regional nodal metastases in the context of two specific bacterial taxa, Enterobacteriaceae and Tenericutes. Additionally, microbial populations can separate tumors by tobacco status (p<0.005), but not by alcohol status (p=0.37). Conclusions: Specific microbial populations may contribute to HNSCC pathogenesis, possibly by triggering inflammation and consequent aberrant DNA methylation. These associations could lead to better understanding of the pathogenesis and treatment of HNSCC, suggesting novel roles for demethylating agents and probiotic adjuncts particularly for patients with advanced or refractory disease. 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 4087. doi:1538-7445.AM2012-4087

Abstract 4911: HPV-independent methylation of specific TGF-B pathway-relevant genes in head and neck squamous cell carcinomas associated with prior radiation therapy and exposure to alcohol and tobacco

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy with more than half a million people being diagnosed with the disease annually. Therefore, it is critical to identify early detection markers, such as DNA methylation, for more timely diagnosis. In a recent study, we identified five tumor suppressive genes (IRX1, EBF3, SLC5A8, SEPT9, and FUSSEL18) as frequently methylated in HNSCC biopsies using a global methylation analysis via restriction landmark genomic scanning (RLGS). Interestingly, we found these candidates are all potentially involved in the TGF-B signaling pathway, which is often disrupted in HNSCC. Therefore, we sought to determine coordinated epigenetic silencing of these candidate genes in HNSCC as potential key disruptors of TGF-B signaling, which could ultimately result in HNSCC progression. Through immunoprecipitation studies, all five of the investigated candidate genes were found to interact with components of the TGF-B pathway. Also, overexpression of SLC5A8, EBF3, and IRX1 resulted in decreased mitotic activity and increased apoptosis. These findings are significant because they reveal a set of genes that interact with components of the TGF-B pathway, and their silencing via methylation in HNSCC results in coordinated decrease in apoptosis, increased proliferation, and decreased differentiation. We went on to define the methylation of these genes in relationship to HPV16-positivity, radiation therapy, and alcohol and tobacco exposure. By utilizing the available clinical information linked to the patient specimens, we found a strong association between promoter methylation of FUSSEL18, IRX1, and EBF3 and prior radiation therapy (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4911.