Konstantinos Evangelou

Deregulated Overexpression of hCdt1 and hCdc6 Promotes Malignant Behavior

The accurate execution of DNA replication requires a strict control of the replication licensing factors hCdt1 and hCdc6. The role of these key replication molecules in carcinogenesis has not been clarified. To examine how early during cancer development deregulation of these factors occurs, we investigated their status in epithelial lesions covering progressive stages of hyperplasia, dysplasia, and full malignancy, mostly from the same patients. Abnormal accumulation of both proteins occurred early from the stage of dysplasia. A frequent cause of unregulated hCdc6 and hCdt1 expression was gene amplification, suggesting that these components can play a role per se in cancer development. Overexpression of hCdt1 and hCdc6 promoted rereplication and generated a DNA damage response, which activated the antitumor barriers of senescence and apoptosis. Generating an inducible hCdt1 cellular system, we observed that continuous stimulus by deregulated hCdt1 led to abrogation of the antitumor barriers and resulted in the selection of clones with more aggressive properties. In addition, stable expression of hCdc6 and hCdt1 in premalignant papilloma cells led to transformation of the cells that produced tumors upon injection into nude mice depicting the oncogenic potential of their deregulation.

Oncogene-induced replication stress preferentially targets common fragile sites in preneoplastic lesions. A genome-wide study

Common fragile sites (CFSs) are regions of the genome prone to breakage by replication inhibitors (extrinsic replication stress). Recently, we and others observed that oncogene-induced replication stress (RS) induces DNA damage from the earliest stages of cancer. Our aim was to perform a genome-wide analysis in precancerous and cancerous experimental models to examine whether allelic imbalance occurs within CFSs. Subsequently, CFSs sequence characteristics were assessed. We used a growth-factor-induced human skin hyperplasia and a H-ras-induced mouse hyperplastic urothelium as preneoplastic models, along with an inducible U2OS-CDT1Tet-ON cancer cell line model, all bearing established oncogene-induced RS stimuli. Human DNA was analysed with Affymetrix SNP microarrays, while mouse DNA was analysed with Nimblegen array CGH. We studied 56 aphidicolin-type CFSs and 1914 regions of control, nonfragile DNA. Our theoretical in silico analysis spanned 2.16 billion nonoverlapping bases on human chromosomes 1–22. Our results provide direct experimental evidence indicating that genomic alterations were more common within CFSs in epidermal and urothelial preneoplastic lesions as well as in cancer. CFSs were on average less flexible than nonfragile regions, contained more guanine–cytosine (GC) and Alu sequences. Importantly, regions with loss-of-heterozygosity were also less flexible and had a higher Alu percentage.

Distinct expression patterns of the transcription factor E2F-1 in relation to tumour growth parameters in common human carcinomas.

E2F‐1 is a pivotal transcription factor that integrates signals from a variety of G1/S phase regulators and modulates diverse cellular functions, such as DNA synthesis, repair, mitosis, and apoptosis. Its role in cellular proliferation and apoptosis, as depicted from experimental models and limited reports in human malignancies, remains a matter of debate. Recently, in non‐small cell lung cancer, it was observed that E2F‐1 overexpression was associated with tumour growth, implying an ‘oncogenic’ effect. To clarify further the role of E2F‐1 in carcinogenesis, the investigation was expanded in four of the most common human malignancies by examining its expression status and putative impact on tumour kinetics. These issues were addressed by immunohistochemical and molecular means in 52 breast carcinomas, 42 prostate adenocarcinomas, 58 colon adenocarcinomas, and 77 superficial bladder transitional cell carcinomas (TCCs). The following results were found: (i) in breast carcinomas, E2F‐1 expression correlated with proliferation (p < 0.001) and growth index (p = 0.001); (ii) in prostate adenocarcinomas, absence of E2F‐1 was noted, in contrast to its expression in normal and hyperplastic glands; (iii) in colon adenocarcinomas, E2F‐1 expression was inversely related to growth index (p = 0.001), being expressed in lesions with increased apoptosis (p = 0.001) and low proliferation (p < 0.001); and (iv) in superficial TCCs, E2F‐1 expression correlated with proliferation (p = 0.002). Taken together, these results suggest that E2F‐1 has a growth‐promoting effect in breast carcinomas and superficial TCC, whereas the opposite seems to be the case for colon and prostate cancer. To interpret the above findings, the status of the pRb and p53 tumour suppressor pathways, which are known to affect E2F‐1 activity, was further investigated. The results suggest that the actions of E2F‐1 are mainly dependent on the functionality of these pathways. Nevertheless, the data also imply that p53‐independent pathways may play a nodal role in the function of E2F‐1 in colon cancer. Copyright

Cdc6 expression represses E-cadherin transcription and activates adjacent replication origins

The Cdc6 replication licensing factor acts as a molecular switch at the E-cadherin locus, leading to E-cadherin transcriptional repression and local activation of replication.

Mammalian RAD52 Functions in Break-Induced Replication Repair of Collapsed DNA Replication Forks

Summary Human cancers are characterized by the presence of oncogene-induced DNA replication stress (DRS), making them dependent on repair pathways such as break-induced replication (BIR) for damaged DNA replication forks. To better understand BIR, we performed a targeted siRNA screen for genes whose depletion inhibited G1 to S phase progression when oncogenic cyclin E was overexpressed. RAD52, a gene dispensable for normal development in mice, was among the top hits. In cells in which fork collapse was induced by oncogenes or chemicals, the Rad52 protein localized to DRS foci. Depletion of Rad52 by siRNA or knockout of the gene by CRISPR/Cas9 compromised restart of collapsed forks and led to DNA damage in cells experiencing DRS. Furthermore, in cancer-prone, heterozygous APC mutant mice, homozygous deletion of the Rad52 gene suppressed tumor growth and prolonged lifespan. We therefore propose that mammalian RAD52 facilitates repair of collapsed DNA replication forks in cancer cells.

Proliferation, but Not Apoptosis, Is Associated with Distinct β-Catenin Expression Patterns in Non-Small-Cell Lung Carcinomas : Relationship with Adenomatous Polyposis Coli and G1-to S-Phase Cell-Cycle Regulators

beta-catenin (beta-cat) is a versatile component of homotypic cell adhesion and signaling. Its subcellular localization and cytoplasmic levels are tightly regulated by the adenomatous polyposis coli (APC) protein. Mutations in beta-cat (exon 3) or APC (MCR) result in beta-cat aberrant overexpression that is associated with its nuclear accumulation and improper gene activation. Data from experimental models have shown that beta-cat overexpression has a multitude of effects on cell-cycle behavior. In many of these aspects its function depends on major G(1) phase regulators. To the best of our knowledge, most of these issues have never been addressed concurrently in tumors. For this reason we investigated in a panel of 92 non-small-cell lung carcinomas, beta-cat and APC expression, and their relationship with cell-cycle kinetics (PI and AI) and ploidy status. Moreover, the above correlations were examined in relation to the main G(1)/S-phase checkpoint regulators. Four beta-cat immunohistochemical expression patterns [membranous (11.1%), membranous-cytoplasmic (54.3%), cytoplasmic (9.9%), cytoplasmic-nuclear (24.7%)] and three APC immunohistochemical expression patterns [cytoplasmic (37.7%), cytoplasmic-nuclear (58%), nuclear (4.3%)] were observed, which were further confirmed by Western blot analysis on subcellular fractions in representative samples. The frequent presence of beta-cat in the cytoplasm is an indication of aberrant expression, whereas membranous and nuclear localization were inversely related. Absence of mutations in beta-cat (exon 3) and APC (MCR) suggest that beta-cat destruction mechanisms may be functional. However, expression analysis revealed attenuated levels for APC, indicating a residual ability to degrade beta-cat. Decreased levels were associated with loss of heterozygosity at the APC region in 24% of the cases suggesting that additional silencing mechanisms may be involved. Interestingly, the 90-kd APC isoform associated with apoptosis, was found to be the predominant isoform in normal and cancerous lung tissues. The most important finding in our study, was the correlation of nuclear beta-cat immunohistochemical localization with increased proliferation, overexpression of E2F1 and MDM2, aberrant p53, and low expression of p27(KIP), providing for the first time in vivo evidence that beta-cat-associated proliferation correlates with release of E2F1 activity and loss of p53- and p27(KIP)-dependent cell-cycle checkpoints. Loss of these checkpoints is accompanied by low levels of APC, which possibly reflects a diminished ability to degrade beta-cat. Taken together our data indicate that cases with nuclear beta-cat immunohistochemical expression represent a subset of non-small-cell lung carcinomas that have gained an increased proliferation advantage in contrast to the other beta-cat immunohistochemical expression profiles.

Therapeutic Inhibition of Tyrosine Kinases in Systemic Sclerosis: A Review of Published Experience on the First 108 Patients Treated with Imatinib

OBJECTIVE Experimental and clinical evidence suggest a therapeutic role for the tyrosine kinase inhibitor imatinib in fibrosing conditions. We evaluated published data on the safety and efficacy of imatinib for patients with systemic sclerosis (SSc), a severe autoimmune disease with significant morbidity and mortality. METHODS A careful search for all original articles and abstracts on the use of imatinib in SSc published in English from 2008 through February 2012 was performed. Two additional patients from our center are also described. RESULTS Five small observational clinical trials on the use of imatinib in severe SSc have been conducted and case reports and small series of refractory to current approaches patients have been reported, adding to a total of 108 patients having received this drug to date. In most of these patients imatinib was given for skin or pulmonary fibrosis. Encouraging results were reported in 3 of 4 studies, whereas the fifth study was prematurely terminated for safety reasons. Overall, clinical results are highly variable, ranging from ineffective or toxic responses to extremely encouraging clinical improvements in some severely ill patients. These discrepancies could partly reflect imatinib-related safety issues, in particular, SSc patients or idiosyncratic resistance to imatinib, as happens in chronic myelogenous leukemia and gastrointestinal stromal tumors, the drugs approved indications. CONCLUSIONS The limited available experience suggests that imatinib could be considered as an individualized treatment approach in severe SSc and underscores the need to identify markers for selecting particular patients, who will safely respond to therapeutic inhibition of tyrosine kinases.

Robust, universal biomarker assay to detect senescent cells in biological specimens

Cellular senescence contributes to organismal development, aging, and diverse pathologies, yet available assays to detect senescent cells remain unsatisfactory. Here, we designed and synthesized a lipophilic, biotin‐linked Sudan Black B (SBB) analogue suitable for sensitive and specific, antibody‐enhanced detection of lipofuscin‐containing senescent cells in any biological material. This new hybrid histo‐/immunochemical method is easy to perform, reliable, and universally applicable to assess senescence in biomedicine, from cancer research to gerontology.

Role of functional polymorphisms of NRAMP1 gene for the development of Crohn's disease

Background: Crohns disease (CD) is characterized by chronic activation of macrophages. Natural resistance‐associated macrophage protein 1 (NRAMP1) gene exerts many pleiotropic effects on macrophage functions. Hence, NRAMP1 may be also involved in the resistance to intracellular pathogens, and this effector of the innate immunity might be involved in CD pathogenesis. Polymorphic alleles at the NRAMP1 locus have been previously associated with susceptibility both to the putative infectious agents and to autoimmune disorders. Based on these indications, in the present study we investigate its candidacy as a genetic determinant for CD in a Greek population in an association‐based study, comparing frequencies of 274 CD patients to these of 200 healthy control subjects. Methods: The 5′(GT)n promoter polymorphism and 9 either single nucleotide (SNPs) or insertion/deletion type polymorphisms were genotyped across the NRAMP1 gene. Reverse‐transcriptase polymerase chain reaction (RT‐PCR) and immunohistochemistry were performed in order to investigate the NRAMP1 mRNA levels in RNA isolated from biopsies of CD patients as well as protein expression in tissues. Results: Three NRAMP1 polymorphisms [5′(GT)n, D543N, and INT4G/C] were significantly associated with CD. Consistent with previous autoimmune disease studies, allele 3 at the functional 5′(GT)n promoter region repeat polymorphism, was significantly associated with CD when compared to healthy controls (odds ratio 1.50; 95% confidence interval [CI]: 1.16–1.95; P = 0.002). Interestingly, we observed that CD patients homozygous for allele 3 expressed higher NRAMP1 mRNA levels compared to carriers of allele 2. Furthermore, the protein levels of allele 3 carriers in tissues were also elevated compared to those of allele 2 carriers. Based on these data we can speculate that overrepresentation of allele 3 in CD patients could lead to hyperactivation of bowel‐wall macrophages that are chronically exposed to lipopolysaccharide and this could subsequently cause the autoimmune‐like phenotype characteristic of CD. Conclusions: Collectively, our data indicate that genetic polymorphisms of NRAMP1 might be associated with susceptibility to CD.

E2F-1 overexpression correlates with decreased proliferation and better prognosis in adenocarcinomas of Barrett oesophagus

Background: E2F-1 expression is positively associated with tumour growth in oesophageal squamous-cell carcinomas (OSCC), while it exhibits oncosuppressive features in colonic adenocarcinomas (AC). To date there are no data regarding E2F-1 expression and its relationship with tumour kinetics (proliferation, apoptosis) in adenocarcinomas that develop on Barrett oesophagus. Aim: As oesophageal adenocarcinomas occur almost exclusively in the metaplastic Barrett epithelium and the opposing E2F-1 behaviour seems to be cell and tissue-type dependent, we examined the manner in which E2F-1 acts in ACs of Barrett oesophagus. Methods: We estimated the immunohistochemical expression of E2F-1, Ki-67, caspase-3 and p53 immunohistochemical status in 35 Barrett oesophagus ACs. Results: E2F-1 immunopositivity correlated inversely with Ki-67, by semi-serial section and statistical analysis (p = 0.023, Spearman correlation). Semi-serial section analysis revealed a direct association between E2F-1 and caspase-3 staining. No correlation was found with p53 status. Cases with higher E2F-1 immunoexpression exhibited longer survival (p = 0.047, Cox-regression). Conclusions: E2F-1 expression was negatively related to tumour proliferation in ACs of Barrett oesophagus. Additionally, E2F-1 immunohistochemical status correlated positively with patient survival. These findings are opposite from those seen in OSCCs, suggesting that the tumour-suppressing E2F-1 behaviour in oesophageal adenocarcinomas is possibly due to the intestinal-type nature of the metaplastic Barrett mucosa.