Abstract Patients with metastatic lung cancer have a very poor prognosis indicating an urgent need to develop more effective chemotherapy. Aberrant promoter DNA methylation can result in the epigenetic silencing of tumor suppressor genes (TSGs) in lung cancer. 5-Aza-2’deoxycytidine (5-Aza-CdR, decitabine), an inhibitor of DNA methylation, is able to reactivate silent TSGs. Trimethylation of histone H3 on lysine 27 (H3K27me3) by enhancer of zeste homolog 2 (EZH2) histone methyltransferase can also silence TSGs in lung cancer. 3-Deazaneplanocin-A (DZNep), an inhibitor of EZH2, up-regulates the expression of genes silenced by H3K27me3. In this study we compared the in vitro antineoplastic activity of different inhibitors of EZH2; DZNep, U-4149 and Gsk-126, alone and in combination with 5-Aza-CdR, on the human A549 lung adenocarcinoma cells. U-4149, an analogue of DZNep, was more potent than either DZNep or Gsk-126. The reduction in colony formation was dose- and time-dependent for each EZH2 inhibitors. Combination treatment of 5-Aza-CdR with the EZH2 inhibitors showed a synergistic antineoplastic activity. 5-Aza-CdR and U-4149 was the most potent combination. The in vitro antineoplastic activity of these agents was evaluated by inhibition of growth, colony formation, induction of senescence and apoptosis. All the drug combinations induced signs of senescence and apoptosis. Analysis by gene expression by qRT-PCR showed that the combinations increased the expression of several TSGs to a greater extent that either agent alone. In conclusion, epigenetic therapy that specifically targets DNA and histone methylation has interesting potential for the treatment of lung cancer and merits further investigation.
Keywords: 5-aza-2'deoxycytidine; decitabine; 3-deazaneplanocin-A; Gsk-126; U-4149; lung cancer; antineoplastic action; epigenetics; DNA methylation; EZH2

Abstract Background and aim: Squamous cell carcinoma (SCC) is the most common cancer of the anal region, typically associated with high-risk (hr) HPV infection. Furthermore, there is evidence that Crohn's disease predisposes to adenocarcinoma in patients with perianal disease. Materials and methods: A 57-year old patient presenting with long history of Crohn's disease since the age of mid-twenties, went through several surgeries including ileocolectomy and anal fistula resection, combined with immunosuppressive therapy additionally periodically since 2008. One year before death (in 2015) a painful fistula was diagnosed with extensive high grade anal intraepithelial neoplasia (AIN-HG) and evidence of invasive growth as non-keratinizing SCC. Tissue samples from several previous and current resection specimens were re-evaluated and extensively investigated for Crohn´s type inflammation, dysplasia and HPV both by immunohistochemistry (p16/Ki67) and molecular subtyping of HPV. Results: AIN-HG and invasive anal squamous cell carcinoma turned out to be strongly positive for p16/Ki67 staining and molecular analysis disclosed a HPV-16 subtype. In contrast, HPV-analysis was negative in all available previous tissue samples including one anal fistula resected five years before (in 2009) which was lined by non-keratinized squamous epithelium without any evidence of dysplasia. Thus, the patient was diagnosed as Crohn's disease with hr-HPV infection that rapidly (< 5ys) progressed to AIN-HG and anal SCC. Finally, osseous metastases occurred and the patient died shortly after. Conclusions: This case of a patient diagnosed with SCC of the anal canal in combination with Crohn's disease as well as HPV Type 16 infection, points to the pathomechanism leading to dysplasia and finally cancer. We assume that immunosuppressive therapy in Crohn's disease may predispose to both persistent HPV infection and HPV related invasive anal carcinoma. The accelerated progression of HPV associated neoplasia in immunosuppressed patients might represent a fast-tracked process of the long-term course of precancerous or cancerous lesions in immunocompetent hosts. This might implicate, that there is a need to re-evaluate current screening guidelines for anal cancer in patients with chronic inflammatory bowel disease under immunosuppressive therapy.
Keywords: Crohn's disease; Human papillomavirus (HPV); anal squamous cell carcinoma; Inflammatory bowel disease (IBD); immunosuppression