DNA methylation reprogramming of human cancer cells by expression of a plant 5-methylcytosine DNA glycosylase

Patterns of DNA methylation, an important epigenetic modification involved in gene silencing and development, are disrupted in cancer cells. Understanding the functional significance of aberrant methylation in tumors remains challenging, due in part to the lack of suitable tools to actively modify methylation patterns. DNA demethylation caused by mammalian DNA methyltransferase inhibitors is transient and replication-dependent, whereas that induced by TET enzymes involves oxidized 5mC derivatives that perform poorly understood regulatory functions. Unlike animals, plants possess enzymes that directly excise unoxidized 5mC from DNA, allowing restoration of unmethylated C through base excision repair. Here, we show that expression of Arabidopsis 5mC DNA glycosylase DEMETER (DME) in colon cancer cells demethylates and reactivates hypermethylated silenced loci. Interestingly, DME expression causes genome-wide changes that include both DNA methylation losses and gains, and partially restores the methylation pattern observed in normal tissue. Furthermore, such methylome reprogramming is accompanied by altered cell cycle responses and increased sensibility to anti-tumor drugs, decreased ability to form colonospheres, and tumor growth impairment in vivo. Our study shows that it is possible to reprogram a human cancer DNA methylome by expression of a plant DNA demethylase.     

Calmodulin mediates Fas-induced FADD-independent survival signaling in pancreatic cancer cells via activation of Src-extracellular signal-regulated kinase (ERK)

Pancreatic cancer remains a devastating malignancy with a poor prognosis and is largely resistant to current therapies. To understand the resistance of pancreatic tumors to Fas death receptor-induced apoptosis, we investigated the molecular mechanisms of Fas-activated survival signaling in pancreatic cancer cells. We found that knockdown of the Fas-associated protein with death domain (FADD), the adaptor that mediates downstream signaling upon Fas activation, rendered Fas-sensitive MiaPaCa-2 and BxPC-3 pancreatic cells resistant to Fas-induced apoptosis. By contrast, Fas activation promoted the survival of the FADD knockdown MiaPaCa-2 and BxPC-3 cells in a concentration-dependent manner. The pharmacological inhibitor of ERK, PD98059, abrogated Fas-promoted cell survival in FADD knockdown MiaPaCa-2 and BxPC-3 cells. Furthermore, increased phosphorylation of Src was demonstrated to mediate Fas-induced ERK activation and cell survival. Immunoprecipitation of Fas in the FADD knockdown cells identified the presence of increased calmodulin, Src, and phosphorylated Src in the Fas-associated protein complex upon Fas activation. Trifluoperazine, a calmodulin antagonist, inhibited Fas-induced recruitment of calmodulin, Src, and phosphorylated Src. Consistently, trifluoperazine blocked Fas-promoted cell survival. A direct interaction of calmodulin and Src and their binding site were identified with recombinant proteins. These results support an essential role of calmodulin in mediating Fas-induced FADD-independent activation of Src-ERK signaling pathways, which promote survival signaling in pancreatic cancer cells. Understanding the molecular mechanisms responsible for the resistance of pancreatic cells to apoptosis induced by Fas-death receptor signaling may provide molecular insights into designing novel therapies to treat pancreatic tumors.     

KDM1A and KDM3A promote tumor growth by upregulating cell cycle-associated genes in pancreatic cancer

Pancreatic cancer is a highly malignant cancer of the pancreas with a very poor prognosis. Methylation of histone lysine residues is essential for regulating cancer physiology and pathophysiology, mediated by a set of methyltransferases (KMTs) and demethylases (KDMs). This study surveyed the expression of methylation regulators functioning at lysine 9 of histone 3 (H3K9) in pancreatic lesions and explored the underlying mechanisms. We analyzed KDM1A and KDM3A expression in clinical samples by immunohistochemical staining and searching the TCGA PAAD program and GEO datasets. Next, we identified the variation in tumor growth in vitro and in vivo after knockdown of KDM1A or KDM3A and explored the downstream regulators of KDM1A and KDM3A via RNA-seq, and gain- and loss-of-function assays. Eleven H3K9 methylation regulators were highly expressed in pancreatic cancer, and only KDM1A and KDM3A expression positively correlated with the clinicopathological characteristics in pancreatic cancer. High expression of KDM1A or KDM3A positively correlated with pathological grade, lymphatic metastasis, invasion, and clinical stage. Kaplan–Meier analysis indicated that a higher level of KDM1A or KDM3A led to a shorter survival period. Knockdown of KDM1A or KDM3A led to markedly impaired tumor growth in vitro and in vivo. Mechanistically, CCNA2, a cell cycle-associated gene was partially responsible for KDM1A knockdown-mediated effect and CDK6, also a cell cycle-associated gene was partially responsible for KDM3A knockdown-mediated effect on pancreatic cancer cells. Our study demonstrates that KDM1A and KDM3A are highly expressed in pancreatic cancer and are intimately correlated with clinicopathological factors and prognosis. The mechanism of action of KDM1A or KDM3A was both linked to the regulation of cell cycle-associated genes, such as CCNA2 or CDK6, respectively, by an H3K9-dependent pathway.     

Aberrant expression of N-acetylglucosaminyltransferase-IVa and IVb (GnT-IVa and b) in pancreatic cancer

The goal of this study was to identify glycosyltransferases that are specifically expressed in pancreatic cancer. To investigate the gene expression of glycosyltransferases between pancreatic cancer and normal pancreatic tissues, we performed DNA-microarray (involving about 1000 oligosaccharide-related genes) using RNA mixtures of pancreatic cancer cells and normal pancreatic tissues. Eighty-six genes were up-regulated and thirty-two were down-regulated in pancreatic cancer, compared to normal pancreatic tissue. Among these changes, it is noteworthy that the expression of GnT-IVa was decreased and the expression of GnT-IVb was increased in pancreatic cancer, compared to normal pancreatic tissues. Although GnT-IVa and -IVb are involved in the same reaction as a glycosyltransferase, their chromosomal localization is different. When 5 cases of pancreatic cancer tissues were examined using the real-time RT-PCR method, the expression of GnT-IVb was dominant in tumor tissues and the expression of GnT-IVa was dominant in the surrounding normal tissues. The expression of GnT-IVa was increased in all 3 cell lines that had been treated with 5-aza-C and butyrate. These results suggest that the down-regulation of GnT-IVa in pancreatic cancer cells is due to an epigenetic abnormality in the gene.     

Thrombomodulin is silenced in malignant mesothelioma by a poly(ADP-ribose) polymerase-1-mediated epigenetic mechanism

Malignant mesothelioma (MM) is often complicated by thromboembolic episodes, with thrombomodulin (TM) playing a critical role in the anticoagulant process. Heterogeneous expression of TM has been observed in cancer, and low or no TM expression in cancer cells is associated with poor prognosis. In this study, we analyzed TM expression in biopsies of MM patients and compared them with normal mesothelial tissue. The role of DNA methylation-associated gene silencing in TM expression was investigated. To evaluate poly(ADP-ribose) polymerase-1 (PARP1) as responsible for gene promoter epigenetic modifications, nonmalignant mesothelial cells (Met-5A) and MM cells (H28) were silenced for PARP1 and the DNA methylation/acetylation-associated TM expression evaluated. A correlation between low TM expression and high level of TM promoter methylation was found in MM biopsies. Low expression of TM was restored in MM cells by their treatment with 5-aza-2'-deoxycytidine and, to a lesser extent, with trichostatin, whereas the epigenetic agents did not affect TM expression in Met-5A cells. Silencing of PARP1 resulted in a strong down-regulation of TM expression in Met-5A cells, while restoring TM expression in H28 cells. PARP1 silencing induced TM promoter methylation in Met-5A cells and demethylation in MM cells, and this was paralleled by corresponding changes in the DNA methyltransferase activity. We propose that methylation of the TM promoter is responsible for silencing of TM expression in MM tissue, a process that is regulated by PARP1.     

Association between hypermethylation of DNA repetitive elements in white blood cell DNA and pancreatic cancer

Pancreatic cancer is a leading cause of cancer-related deaths worldwide. Methylation of DNA may influence risk or be a marker of early disease. The aim of this study was to measure the association between methylation of three DNA repetitive elements in white blood cell (WBC) DNA and pancreatic cancer.DNA from WBCs of pancreatic cancer cases (n = 559) and healthy unrelated controls (n = 603) were tested for methylation of the LINE-1, Alu and Sat2 DNA repetitive elements using MethyLight quantitative PCR assays. Odds ratios (ORs) and 95% confidence intervals (95%CI) between both continuous measures of percent of methylated sample compared to a reference (PMR) or quintiles of PMR and pancreatic cancer, adjusted for age, sex, smoking, BMI, alcohol and higher education, were estimated.The PMR for each of the three markers was higher in cases than in controls, although only LINE-1 was significantly associated with pancreatic cancer (OR per log unit = 1.37, 95%CI = 1.16–1.63). The marker methylation score for all three markers combined was significantly associated with pancreatic cancer (p-trend = 0.0006). There were no associations between measures of PMR and either presence of metastases, or timing of blood collection in relation to diagnosis, surgery, chemotherapy or death (all p > 0.1).We observed an association between methylation of LINE-1 in WBC DNA and risk of pancreatic cancer. Further studies are needed to confirm this association.     

DNA hypomethylation-mediated activation of Cancer/Testis Antigen 45 (CT45) genes is associated with disease progression and reduced survival in epithelial ovarian cancer

Epithelial ovarian cancer (EOC) is a highly lethal malignancy due to a lack of early detection approaches coupled with poor outcomes for patients with clinically advanced disease. Cancer-testis (CT) or cancer-germline genes encode antigens known to generate spontaneous anti-tumor immunity in cancer patients. CT45 genes are a recently discovered 6-member family of X-linked CT genes with oncogenic function. Here, we determined CT45 expression in EOC and fully defined its epigenetic regulation by DNA methylation. CT45 was silent and hypermethylated in normal control tissues, but a large subset of EOC samples showed increased CT45 expression in conjunction with promoter DNA hypomethylation. In contrast, copy number status did not correlate with CT45 expression in the TCGA database for EOC. CT45 promoter methylation inversely correlated with both CT45 mRNA and protein expression, the latter determined using IHC staining of an EOC TMA. CT45 expression was increased and CT45 promoter methylation was decreased in late-stage and high-grade EOC, and both measures were associated with poor survival. CT45 hypomethylation was directly associated with LINE-1 hypomethylation, and CT45 was frequently co-expressed with other CT antigen genes in EOC. Decitabine treatment induced CT45 mRNA and protein expression in EOC cells, and promoter transgene analyses indicated that DNA methylation directly represses CT45 promoter activity. These data verify CT45 expression and promoter hypomethylation as possible prognostic biomarkers, and suggest CT45 as an immunological or therapeutic target in EOC. Treatment with decitabine or other epigenetic modulators could provide a means for more effective immunological targeting of CT45.     

Complete remission of liver metastasis of pancreatic cancer under vaccination with a HLA-A2 restricted peptide derived from the universal tumor antigen survivin

Purpose: As prognosis of advanced pancreatic cancer remains gloomy, novel therapeutic modalities have to be developed. Immunotherapy, which targets tumor-associated antigens of tumor cells or tumor stroma, is currently under investigation. As survivin is expressed by neoplastic and tumor endothelial cells, but rarely by normal cells, this antigen appears as an intriguing target molecule. Methods: A 72-year old patient, suffering from pancreatic cancer refractory to gemcitabine therapy, received the survivin-based peptide vaccinations consisting of 100 μg of a modified HLA-A2 restricted survivin_96–104 epitope in Montanide®. Each visit the patient was assessed for adverse events, quality of life and immunological response. Immunemonitoring was performed by IFN-γ-ELISPOT analysis of peripheral blood lymphocytes. Clinical outcome was evaluated by repetitive computed tomography. Results: Under vaccination with survivin peptides the patient initially underwent partial remission of liver metastasis which proceeded after 6 months into a complete remission with a duration of 8 months. Immunological monitoring revealed strong vaccine-induced immune-reactivity against survivin. Unfortunately, after the patient was weaned from vaccination in state of no evidence of disease, he developed recurrent disease. Conclusion: T-cell responses against survivin-expressing cells of the tumor itself and tumor endothelium should impact tumor growth and metastasis. The presented patient with pancreatic cancer is the first example of a successful application of a survivin-based vaccination in the clinical setting. An ongoing phase I/II trial with HLA-A1, -A2 and -B35 restricted survivin peptides for patients with advanced cancer will provide further information towards this notion.     

Identification of BCP-20 (FBXO39) as a cancer/testis antigen from colon cancer patients by SEREX

Cancer/Testis (CT) antigens are considered promising target molecules for immunotherapy. To identify potential CT antigens, we performed immunoscreening of a testis cDNA library with sera from colon cancer patients by SEREX. We isolated 114 positive cDNA clones comprising 90 different antigens, designated BCP-1 through BCP-90. Quantitative real-time and conventional RT-PCR analysis showed that BCP-20, -33, and -41 antigens were expressed strongly only in a normal testis and detected in 22 cases (39%), 12 cases (21%), and 17 cases (30%), respectively, from 57 colon tumors. BCP-20 was also detected in various cancer cell lines including breast, colon, hepatoma, renal, thyroid anaplastic, ovary, sarcoma, and lung. By ELISA analysis, anti-BCP-20 antibody was detected in 3 of 50 colon cancer and 1 of 24 gastric cancer patients while healthy donors were three positive (3/50). But the BCP-20 antibody levels of patients with colon cancer showed significantly higher titers than those of healthy donors. These data suggest that the BCP-20 gene is a new CT antigen and may be useful for diagnosis and immunotherapy.     

Identification of specific reachable molecular targets in human breast cancer using a versatile ex vivo proteomic method

Targeting of tumoral tissues is one of the most promising approaches to improve both the efficacy and safety of anticancer treatments. The identification of valid targets, including proteins specifically and abundantly expressed in cancer lesions, is of utmost importance. Despite state-of-the-art technologies, the discovery of cancer-associated target proteins still faces the limitation, in human tissues, of antigen accessibility to suitable high-affinity ligands such as human mAb bound to bioactive molecules. Terminal perfusion of tumor-bearing mice or ex vivo perfusion of human cancer-bearing organs with a reactive biotin ester solution has successfully led to the identification of novel accessible biomarkers. This methodology is however restricted to perfusable organs, and excludes most of the tissues of interest to targeted therapies, e.g. primary breast cancer and metastases. Herein, we report on the development of a new chemical proteomic method that bypasses the perfusion step and thus offers the potential to identify accessible molecular targets in virtually all types of animal and human tissues. We have validated our new procedure by identifying biomarkers selectively expressed in human breast carcinoma. Overall, this powerful technology may lay the ground not only for custom-made therapies in cancer, but also for the development of therapies that need to be selectively delivered in a specific tissue.     

Inhibition of cell growth by overexpression of manganese superoxide dismutase (MnSOD) in human pancreatic carcinoma

Manganese superoxide dismutase (MnSOD) levels have been found to be low in human pancreatic cancer [Pancreas26, (2003), 23] and human pancreatic cancer cell lines [Cancer Res.63, (2003), 1297] when compared to normal human pancreas. We hypothesized that stable overexpression of pancreatic cancer cells with MnSOD cDNA would alter the malignant phenotype. MIA PaCa-2 cells were stably transfected with a pcDNA3 plasmid containing sense human MnSOD cDNA or containing no MnSOD insert by using the lipofectAMINE method. G418-resistant colonies were isolated, grown and maintained. Overexpression of MnSOD was confirmed in two selected clones with a 2-4-fold increase in MnSOD immunoreactive protein. Compared with the parental and neo control cells, the MnSOD-overexpressing clones had decreased growth rates, growth in soft agar and plating efficiency in vitro, while in vivo, the MnSOD-overexpressing clones had slower growth in nude mice. These results suggest that MnSOD may be a tumor suppressor gene in human pancreatic cancer.