HPP1 Ectodomain Shedding is Mediated by ADAM17 and is Necessary for Tumor Suppression in Colon Cancer.

BACKGROUND: Hyperplastic polyposis protein 1 (HPP1) encodes a tumor-suppressive transmembrane cleavable epidermal growth factor-like ligand. It is unclear as to whether cleavage and shedding of HPP1 are essential steps in achieving its tumor suppressive properties. ADAM proteins are key players in cellular ectodomain shedding processes with ADAM17 being well characterized and representing the most likely sheddase for HPP1. In this study, we explore the mechanisms and importance of ectodomain shedding in contributing to HPP1-mediated tumor suppression. METHODS: Baseline characterization of HPP1 ectodomain shedding and ADAM family member expression was performed in HCT116 colon cancer cells with forced overexpression of HPP1 and controls. Subsequent impact of attenuation of ADAM expression by short interfering RNA on HPP1 shedding was evaluated. Furthermore, we examined the functional impact of an uncleavable HPP1 mutant construct (HPP1-Δstalk) generated by site-directed mutagenesis. Cellular growth potential functions were analyzed by MTT and soft agar assays. RESULTS: Select proinflammatory cytokines enhanced HPP1 ectodomain shedding, whereas short interfering RNA-mediated knockdown of ADAM17 resulted in abrogation of HPP1 ectodomain shedding. ADAM17 knockdown concomitantly resulted in increased cell proliferation and anchorage-independent growth. HPP1-Δstalk-transfected cells exhibited significantly higher proliferation and reduced STAT1 activation relative to full-length HPP1, further suggesting a critical role for ectodomain shedding in HPP1-mediated tumor suppression. CONCLUSION: The tumor-suppressive properties of HPP1 in colorectal cancer require cleavage and shedding of its ectodomain which in turn are mediated by ADAM17. Further investigations into the regulation of HPP1 may lead to a greater understanding of epidermal growth factor-like ligand family biology and potential novel therapeutic strategies.

Epigenomic characterization of locally advanced anal cancer: a radiation therapy oncology group 98-11 specimen study.

BACKGROUND: The Radiation Therapy Oncology Group 98-11 clinical trial demonstrated the superiority of standard 5-fluorouracil/mitomycin-C over 5-fluorouracil/cisplatin in combination with radiation in the treatment of anal squamous cell cancer. Tumor size (>5 cm) and lymph node metastases are associated with disease progression. There may be key molecular differences (eg, DNA methylation changes) in tumors at high risk for progression. OBJECTIVE: The objectives of this study were to determine whether there are differences in DNA methylation at individual CpG sites and within genes among locally advanced anal cancers, with large tumor size and/or nodal involvement, compared with those that are less advanced. DESIGN: This was a case-case study among 121 patients defined as high risk (tumor size >5 cm and/or nodal involvement; n = 59) or low risk (≤5 cm, node negative; n = 62) within the mitomycin-C arm of the Radiation Therapy Oncology Group 98-11 trial. DNA methylation was measured using the Illumina HumanMethylation450 Array. SETTINGS: The study was conducted in a tertiary care cancer center in collaboration with a national clinical trials cooperative group. PATIENTS: The patients consisted of 74 women and 47 men with a median age of 54 years (range, 25-79 years). MAIN OUTCOME MEASURES: DNA methylation differences at individual CpG sites and within genes between low- and high-risk patients were compared using the Mann-Whitney test (p < 0.001). RESULTS: A total of 16 CpG loci were differentially methylated (14 increased and 2 decreased) in high- versus low-risk cases. Genes harboring differentially methylated CpG sites included known tumor suppressor genes and novel targets. LIMITATIONS: This study only included patients in the mitomycin-C arm with tumor tissue; however, this sample was representative of the trial. CONCLUSIONS: This is the first study to apply genome-wide methylation analysis to anal cancer. Biologically relevant differences in methylated targets were found to discriminate locally advanced from early anal cancer. Epigenetic events likely play a significant role in the progression of anal cancer and may serve as potential biomarkers.

Race and prevalence of human papillomavirus infection among men residing in Brazil, Mexico and the United States.

Human papillomavirus (HPV) causes anal, penile and oropharyngeal cancers in men. Genital HPV prevalence in men appears to vary by world region with men residing in Asia having among the lowest prevalence. Unfortunately, there is little information on prevalence of HPV infection in men by race. The purpose of this study was to examine HPV prevalence by race across three countries. 3,909 men ages 18-70 years enrolled in an ongoing prospective cohort study of the natural history of HPV in men (The HIM Study) were included in the analysis. Participants completed risk factor questionnaires and samples were taken from the penile epithelium and scrotum for HPV detection. HPV testing of the combined DNA extract was conducted using PCR and genotyping. Asian/Pacific Islanders had the lowest HPV prevalence of 42.2% compared to Blacks (66.2%), and Whites (71.5%). The Asian/Pacific Islander race was strongly protective in univariate analysis (prevalence ratio (PR) = 0.59; 95% confidence interval (CI): 0.48-0.74) and multivariate analysis for any HPV infection (PR = 0.65; 95% CI: 0.52-0.8). Stratified analysis by lifetime number of female partners also showed strong inverse associations with the Asian/Pacific Islander race. We consistently observed the lowest prevalence of HPV infection among Asian/Pacific Islanders with moderate inverse associations even after various adjustments for potential confounding factors. Unmeasured behavioral factors, sexual mixing with low risk women, and/or race-specific differences in the frequency of germline variations among immune regulating genes may underlie these associations. Further studies among Asian populations that incorporate measures of immuno-genetics are needed to understand this phenomenon.

Characterization of Epigenomic Alterations in HPV16+ Head and Neck Squamous Cell Carcinomas.

BACKGROUND: Epigenetic changes associated with human papillomavirus (HPV)-driven tumors have been described; however, HPV type-specific alterations are less well understood. We sought to compare HPV16-specific methylation changes with those in virus-unassociated head and neck squamous cell carcinomas (HNSCC). METHODS: Within The Cancer Genome Atlas, 59 HPV16+ HNSCC, 238 nonviral HNSCC (no detectable HPV or other viruses), and 50 normal head and neck tissues were evaluated. Significant differentially methylated regions (DMR) were selected, and key associated genes were identified. Partial least squares models were generated to predict HPV16 status in additional independent samples. RESULTS: HPV infection in HNSCC is associated with type-specific methylomic profiles. Multiple significant DMRs were identified between HPV16+, nonviral, and normal samples. The most significant differentially methylated genes, SYCP2, MSX2, HLTF, PITX2, and GRAMD4, demonstrated HPV16-associated methylation patterns with corresponding alterations in gene expression. Phylogenetically related HPV types (alpha-9 species; HPV31, HPV33, and HPV35) demonstrated a similar methylation profile to that of HPV16 but differed from those seen in other types, such as HPV18 and 45 (alpha-7). CONCLUSIONS: HNSCC linked to HPV16 and types from the same alpha species are associated with a distinct methylation profile. This HPV16-associated methylation pattern is also detected in cervical cancer and testicular germ cell tumors. We present insights into both shared and unique methylation alterations associated with HPV16+ tumors and may have implications for understanding the clinical behavior of HPV-associated HNSCC. IMPACT: HPV type-specific methylomic changes may contribute to understanding biologic mechanisms underlying differences in clinical behavior among different HPV+ and HPV- HNSCC.

Genome-wide host methylation profiling of anal and cervical carcinoma.

HPV infection results in changes in host gene methylation which, in turn, are thought to contribute to the neoplastic progression of HPV-associated cancers. The objective of this study was to identify joint and disease-specific genome-wide methylation changes in anal and cervical cancer as well as changes in high-grade pre-neoplastic lesions. Formalin-fixed paraffin-embedded (FFPE) anal tissues (n = 143; 99% HPV+) and fresh frozen cervical tissues (n = 28; 100% HPV+) underwent microdissection, DNA extraction, HPV genotyping, bisulfite modification, DNA restoration (FFPE) and analysis by the Illumina HumanMethylation450 Array. Differentially methylated regions (DMR; t test q<0.01, 3 consecutive significant CpG probes and mean Δß methylation value>0.3) were compared between normal and cancer specimens in partial least squares (PLS) models and then used to classify anal or cervical intraepithelial neoplasia-3 (AIN3/CIN3). In AC, an 84-gene PLS signature (355 significant probes) differentiated normal anal mucosa (NM; n = 9) from AC (n = 121) while a 36-gene PLS signature (173 significant probes) differentiated normal cervical epithelium (n = 10) from CC (n = 9). The CC progression signature was validated using three independent publicly available datasets (n = 424 cases). The AC and CC progression PLS signatures were interchangeable in segregating normal, AIN3/CIN3 and AC and CC and were found to include 17 common overlapping hypermethylated genes. Moreover, these signatures segregated AIN3/CIN3 lesions similarly into cancer-like and normal-like categories. Distinct methylation changes occur across the genome during the progression of AC and CC with overall similar profiles and add to the evidence suggesting that HPV-driven oncogenesis may result in similar non-random methylomic events. Our findings may lead to identification of potential epigenetic drivers of HPV-associated cancers and also, of potential markers to identify higher risk pre-cancerous lesions.

IL23 and TGF-ß diminish macrophage associated metastasis in pancreatic carcinoma.

The precise role of tumor associated macrophages remains unclear in pancreatic ductal adenocarcinoma (PDAC) while TGF-ß has an unclear role in metastases formation. In order to understand the role of IL23, an interleukin associated with macrophage polarization, we investigated IL23 in the context of TGF-ß expression in PDAC. We hypothesized that IL23 expression is associated with metastatic development and survival in PDAC. We investigated IL23 and TGF-ß protein expression on resected PDAC patient tumor sections who were divided into short-term (<12 months) survivors and long-term (>30 months) survivors. Panc-1 cells treated with IL23, TGF-ß, macrophages, or combinations thereof, were orthotopically implanted into NSG mice. Patients in the long-term survivor group had higher IL23 protein expression (P = 0.01). IL23 expression was linearly correlated with TGF-ß expression in patients in the short-term survivor group (P = 0.038). Macrophages induce a higher rate of PDAC metastasis in the mouse model (P = 0.02), which is abrogated by IL23 and TGF-ß treatment (P < 0.001). Macrophages serve a critical role in PDAC tumor growth and metastasis. TGF-ß contributes to a less tumorigenic TME through regulation of macrophages. Macrophages increases PDAC primary tumor growth and metastases formation while combined IL23 and TGF-ß pre-treatment diminishes these processes.

The interplay between histone deacetylases and c-Myc in the transcriptional suppression of HPP1 in colon cancer.

HPP1 (hyperplastic polyposis protein 1), a tumor suppressor gene, is downregulated by promoter hypermethylation in a number of tumor types including colon cancer. c-Myc is also known to play a role in the suppression of HPP1 expression via binding to a promoter region cognate E-box site. The contribution of histone deacetylation as an additional epigenetic mechanism and its potential interplay with c-Myc in the transcriptional regulation of HPP1 are unknown. We have shown that the treatment of the HPP1-non-expressing colon cancer cell lines, HCT116 and DLD-1 with HDAC inhibitors results in re-expression of HPP1. RNAi-mediated knockdown of c-Myc as well as of HDAC2 and HDAC3 in HCT116 and of HDAC1 and HDAC3 in DLD-1 also resulted in significant re-expression of HPP1. Co-immunoprecipitation (IP), chromatin IP (ChIP), and sequential ChIP experiments demonstrated binding of c-Myc to the HPP1 promoter with recruitment of and direct interaction with HDAC3. In summary, we have demonstrated that c-Myc contributes to the epigenetic regulation of HPP1 via the dominant recruitment of HDAC3. Our findings may lead to a greater biologic understanding for the application of targeted use of HDAC inhibitors for anti-cancer therapy.

Expanding epigenomics to archived FFPE tissues: an evaluation of DNA repair methodologies.

BACKGROUND: Epigenome-wide association studies are emerging in the field of cancer epidemiology with the rapid development of large-scale methylation array platforms. Until recently, these methods were only valid for DNA from flash frozen (FF) tissues. Novel techniques for repairing DNA from formalin-fixed paraffin-embedded (FFPE) tissues have emerged; however, a direct comparison of FFPE DNA repair methods before analysis on genome-wide methylation array to matched FF tissues has not been conducted. METHODS: We conducted a systematic performance comparison of two DNA repair methods (REPLI-g Ligase vs. Infinium HD Restore Kit) on FFPE-DNA compared with matched FF tissues on the Infinium 450K array. A threshold of discordant methylation between FF-FFPE pairs was set at Δß > 0.3. The correlations of ß-values from FF-FFPE pairs were compared across methods and experimental conditions. RESULTS: The Illumina Restore kit outperformed the REPLI-g ligation method with respect to reproducibility of replicates (R(2) > 0.970), highly correlated ß-values between FF-FFPE (R(2) > 0.888), and fewest discordant loci between FF-FFPE (≤0.61%). The performance of the Restore kit was validated in an independent set of 121 FFPE tissues. CONCLUSIONS: The Restore kit outperformed RELPI-g ligation in restoring FFPE-derived DNA before analysis on the Infinium 450K methylation array. Our findings provide critical guidance that may significantly enhance the breadth of diseases that can be studied by methylomic profiling. IMPACT: Epigenomic studies using FFPE tissues should now be considered among cancers that have not been fully characterized from an epigenomic standpoint. These findings promote novel epigenome-wide studies focused on cancer etiology, identification of novel biomarkers, and developing targeted therapies. See all the articles in this CEBP Focus section, "Biomarkers, Biospecimens, and New Technologies in Molecular Epidemiology."

DNA methylation profiling across the spectrum of HPV-associated anal squamous neoplasia.

BACKGROUND: Changes in host tumor genome DNA methylation patterns are among the molecular alterations associated with HPV-related carcinogenesis. However, there is little known about the epigenetic changes associated specifically with the development of anal squamous cell cancer (SCC). We sought to characterize broad methylation profiles across the spectrum of anal squamous neoplasia. METHODOLOGY/PRINCIPAL FINDINGS: Twenty-nine formalin-fixed paraffin embedded samples from 24 patients were evaluated and included adjacent histologically normal anal mucosa (NM; n = 3), SCC-in situ (SCC-IS; n = 11) and invasive SCC (n = 15). Thirteen women and 11 men with a median age of 44 years (range 26-81) were included in the study. Using the SFP(10) LiPA HPV-typing system, HPV was detected in at least one tissue from all patients with 93% (27/29) being positive for high-risk HPV types and 14 (93%) of 15 invasive SCC tissues testing positive for HPV 16. Bisulfite-modified DNA was interrogated for methylation at 1,505 CpG loci representing 807 genes using the Illumina GoldenGate Methylation Array. When comparing the progression from normal anal mucosa and SCC-IS to invasive SCC, 22 CpG loci representing 20 genes demonstrated significant differential methylation (p<0.01). The majority of differentially methylated gene targets occurred at or close to specific chromosomal locations such as previously described HPV methylation "hotspots" and viral integration sites. CONCLUSIONS: We have identified a panel of differentially methlylated CpG loci across the spectrum of HPV-associated squamous neoplasia of the anus. To our knowledge, this is the first reported application of large-scale high throughput methylation analysis for the study of anal neoplasia. Our findings support further investigations into the role of host-genome methylation in HPV-associated anal carcinogenesis with implications towards enhanced diagnosis and screening strategies.

Age-specific seroprevalence of Merkel cell polyomavirus, BK virus, and JC virus.

We produced capsids of Merkel cell polyomavirus (MCPyV) in a baculovirus expression system and developed a virus-like particle (VLP) enzyme-linked immunosorbent assay (ELISA). To determine age-specific seroprevalence, serum samples were collected from 947 individuals attending hospital outpatient clinics and ranging in age from 1 to 93 years. To evaluate the association between exposure to MCPyV and Merkel cell cancer (MCC), plasma samples were obtained from 33 MCC patients and 37 controls. MCPyV seroprevalence was 45% in children under 10 years of age, increased to 60% in the next decade of life, and peaked at 81% among those 60 to 69 years of age. Levels of MCPyV capsid antibodies were positively correlated with age (P = 0.007). Virus specificity of MCPyV seroreactivity was supported by competitive inhibition of reactivity by MCPyV VLPs and not by BK polyomavirus (BKPyV) VLPs. MCPyV seroprevalence was greater among MCC patients (91%) than controls (68%; age-adjusted P value, 0.32); the mean level of MCPyV antibodies was also greater (P = 0.04). The age-specific seroprevalence of MCPyV shares with previously known polyomaviruses, BKPyV and JC polyomavirus (JCPyV), evidence of widespread exposure in human populations beginning early in life. MCPyV age-specific seroprevalence also has unique features. Seroprevalence among children is higher than that of JCPyV but lower than that of BKPyV. Among older adults, MCPyV seroprevalence remains high, while that of BKPyV declines and that of JCPyV continues to rise. In agreement with results from other studies, we found an association between MCPyV seropositivity and MCC, and higher levels of serum MCPyV capsid antibodies in MCC patients than in controls.

Validation and field testing of library-independent microbial source tracking methods in the Gulf of Mexico.

Water quality is frequently impacted by microbial pollution from human and animal feces. Microbial source tracking (MST) can identify dominant pollution sources and improve assessment of health risk compared to indicator bacteria alone. This study aims to standardize and validate MST methods across laboratories in coastal Gulf of Mexico states. Three laboratories evaluated library-independent MST methods for human sewage detection via conventional PCR: (1) human-associated Bacteroidales, (2) human polyomaviruses (HPyVs), and (3) Methanobrevibacter smithii. All methods detected targets in human sewage seeded into buffer, freshwater or marine water (100% sensitivity). The limit of detection (LOD) for human sewage was lowest for the Bacteroidales assay (10(-5)-10(-6) dilution). LODs for HPyVs and M. smithii assays were similar to each other (10(-3)-10(-4)), but were higher than Bacteroidales. The HPyVs assay was 100% specific, showing no cross-reactivity to dog, cow, cat, bird, or wild animal feces among >300 samples from three Gulf Coast regions. The human Bacteroidales assay was 96% specific, but cross-reacted with 10% of dog and some chicken samples. The M. smithii assay was 98% specific with limited cross-reactivity with cow, dog and seagull samples. An experts' workshop concluded that all methods showed sufficient accuracy and reliability to move forward. SOPs will be distributed to collaborating laboratories for further inter-laboratory comparison, and field validation will occur in year 2.