Kostas Evangelou

Functional interplay between the DNA-damage-response kinase ATM and ARF tumour suppressor protein in human cancer

The DNA damage response (DDR) pathway and ARF function as barriers to cancer development. Although commonly regarded as operating independently of each other, some studies proposed that ARF is positively regulated by the DDR. Contrary to either scenario, we found that in human oncogene-transformed and cancer cells, ATM suppressed ARF protein levels and activity in a transcription-independent manner. Mechanistically, ATM activated protein phosphatase 1, which antagonized Nek2-dependent phosphorylation of nucleophosmin (NPM), thereby liberating ARF from NPM and rendering it susceptible to degradation by the ULF E3-ubiquitin ligase. In human clinical samples, loss of ATM expression correlated with increased ARF levels and in xenograft and tissue culture models, inhibition of ATM stimulated the tumour-suppressive effects of ARF. These results provide insights into the functional interplay between the DDR and ARF anti-cancer barriers, with implications for tumorigenesis and treatment of advanced tumours.

Downregulation of the KIP family members p27KIP1 and p57KIP2 by SKP2 and the role of methylation in p57KIP2 inactivation in nonsmall cell lung cancer

Knowing the status of molecules involved in cell cycle control in cancer is vital for therapeutic approaches aiming at their restoration. The p27KIP1 and p57KIP2 cyclin‐dependent kinase inhibitors are nodal factors controlling normal cell cycle. Their expression in normal lung raises the question whether they have a mutual exclusive or redundant role in nonsmall cell lung cancer (NSCLC). A comparative comprehensive analysis was performed in a series of 70 NSCLCs. The majority of cases showed significantly reduced expression of both members compared to normal counterparts. Low KIP protein levels correlated with increased proliferation, which seems to be histological subtype preponderant. At mechanistic level, degradation by SKP2 was demonstrated, in vivo and in vitro, by siRNA‐methodology, to be the most important downregulating mechanism of both KIPs in NSCLC. Decreased p57KIP2‐transcription complements the above procedure in lowering p57KIP2‐protein levels. Methylation was the main cause of decreased p57KIP2‐mRNA levels. Allelic loss and imprinting from LIT1 mRNA contribute also to decreased p57KIP2 transcription. In vitro recapitulation of the in vivo findings, in A549 lung cells (INK4A‐B(−/−)), suggested that inhibition of the SKP2‐degradation mechanism restores p27KIP1 and p57KIP2 expression. Double siRNA treatments demonstrated that each KIP is independently capable of restraining cell growth. An additional demethylation step is required for complete reconstitution of p57KIP2 expression in NSCLC.

Centrosome abnormalities are frequently observed in non-small-cell lung cancer and are associated with aneuploidy and cyclin E overexpression.

Centrosome abnormalities are observed in human cancers and have been associated with aneuploidy, a driving force in tumour progression. However, the exact pathways that tend to cause centrosome abnormalities have not been fully elucidated in human tumours. Using a series of 68 non‐small‐cell lung carcinomas and an array of in vitro experiments, the relationship between centrosome abnormalities, aneuploidy, and the status of key G1 to S‐phase transition cell‐cycle molecules, involved in the regulation of centrosome duplication, was investigated. Centrosome amplification and structural abnormalities were common (53%), were strongly related to aneuploidy, and, surprisingly, were even seen in adjacent hyperplastic regions, suggesting the possibility that these are early lesions in lung carcinogenesis. Cyclin E and E2F1 overexpression, but not p53 mutation, was observed to correlate with centrosome abnormalities in vivo (p = 0.029 and p = 0.015, respectively). This was further strengthened by the observation that cyclin E was specifically present in the nucleus and/or cytoplasm of the cells that contained centrosome aberrations. The cytoplasmic cyclin E signal may be attributed, in part, to the presence of truncated low‐molecular‐weight isoforms of cyclin E. In order to isolate the effect of cyclin E on the appearance of centrosome abnormalities, a U2OS tetracycline‐repressible cyclin E cell line that has a normal centrosome profile by default was used. With this system, it was confirmed in vitro that persistent cyclin E overexpression is sufficient to cause the appearance of centrosome abnormalities. Copyright

Upregulation and nuclear localization of TNF-like cytokine 1A (TL1A) and its receptors DR3 and DcR3 in psoriatic skin lesions.

Abstract:  TNF is critically involved in the pathogenesis of psoriasis. TL1A is a TNF‐like cytokine, which, after binding to death domain receptor DR3, provides costimulatory signals to lymphocytes, amplifies Th1‐ and Th17‐mediated immune responses and induces apoptotic cell death. These functions are inhibited when TL1A associates to decoy receptor DcR3. In the present study, we investigated the expression profiles for TL1A, DR3 and DcR3 in the normal skin and in psoriatic skin lesions. By use of immunohistochemistry, we were able to demonstrate constitutive cutaneous expression of DR3 and DcR3 but not of TL1A in healthy skin. On the other hand, in patients with active psoriasis, we observed abundant immunostaining for TL1A and significant upregulation of its receptors (P < 0.05 in comparison to healthy skin). TL1A, DR3 and DcR3 proteins, as well as mRNA transcripts reflecting in situ production of TL1A and DcR3, were also specifically increased in lesional as compared to non‐lesional skin from patients with psoriasis (P < 0.05). These proteins were upregulated in cell populations that are critically involved in the pathogenesis of chronic skin inflammation, such as keratinocytes, macrophages in deep dermis and cells at the perivascular/endothelial area. Finally, we provide evidence for the existence of nuclear localization of TL1A in inflammatory cells from psoriatic lesions. This was also observed in inflamed synovia from patients with rheumatoid arthritis, but not in neoplastic TL1A‐expressing cell lines. We conclude that interactions between TL1A and its two receptors may be involved in the pathogenesis of chronic skin inflammation that takes place in psoriasis.

Gene amplification is a relatively frequent event leading to ZBTB7A (Pokemon) overexpression in non-small cell lung cancer.

ZBTB7A (Pokemon) is a member of the POK family of transcriptional repressors. Its main function is the suppression of the p14ARF tumour suppressor gene. Although ZBTB7A expression has been found to be increased in various types of lymphoma, there are no reports dealing with its expression in solid tumours. Given that p14ARF inhibits MDM2, the main negative regulator of p53, we hypothesized that overexpression of ZBTB7A could lead indirectly to p53 inactivation. To this end, we examined the status of ZBTB7A and its relationship with tumour kinetics (proliferation and apoptosis) and nodal members of the p53 network in a panel of 83 non‐small cell lung carcinomas (NSCLCs). We observed, in the majority of the samples, prominent expression of ZBTB7A in the cancerous areas compared to negligible presence in the adjacent normal tissue elements. Gene amplification (two‐ to five‐fold) was found in 27.7% of the cases, denoting its significance as a mechanism driving ZBTB7A overproduction in NSCLCs. In the remaining non‐amplified group of carcinomas, analysis of the mRNA and protein expression patterns suggested that deregulation at the transcriptional and post‐translational level accounts for ZBTB7A overexpression. Proliferation was associated with ZBTB7A expression (p = 0.033) but not apoptosis. The association with proliferation was reflected in the positive correlation between ZBTB7A expression and tumour size (p = 0.018). The overexpression of ZBTB7A in both p53 mutant and p53 wild‐type cases, implies either a synergistic effect or that ZBTB7A exerts its oncogenic properties independently of the p14ARF–MDM2–p53 axis. The concomitant expression of ZBTB7A with p14ARF (p = 0.039), instead of the anticipated inverse relation, supports the latter notion. In conclusion, regardless of the pathway followed, the distinct expression of ZBTB7A in cancerous areas and the association with proliferation and tumour size pinpoints a role for this novel cell cycle regulator in the pathogenesis of lung cancer. Copyright

The DNA damage checkpoint precedes activation of ARF in response to escalating oncogenic stress during tumorigenesis

Oncogenic stimuli trigger the DNA damage response (DDR) and induction of the alternative reading frame (ARF) tumor suppressor, both of which can activate the p53 pathway and provide intrinsic barriers to tumor progression. However, the respective timeframes and signal thresholds for ARF induction and DDR activation during tumorigenesis remain elusive. Here, these issues were addressed by analyses of mouse models of urinary bladder, colon, pancreatic and skin premalignant and malignant lesions. Consistently, ARF expression occurred at a later stage of tumor progression than activation of the DDR or p16INK4A, a tumor-suppressor gene overlapping with ARF. Analogous results were obtained in several human clinical settings, including early and progressive lesions of the urinary bladder, head and neck, skin and pancreas. Mechanistic analyses of epithelial and fibroblast cell models exposed to various oncogenes showed that the delayed upregulation of ARF reflected a requirement for a higher, transcriptionally based threshold of oncogenic stress, elicited by at least two oncogenic ‘hits’, compared with lower activation threshold for DDR. We propose that relative to DDR activation, ARF provides a complementary and delayed barrier to tumor development, responding to more robust stimuli of escalating oncogenic overload.

Electron microscopy evidence that cytoplasmic localization of the p16INK4A “nuclear” cyclin-dependent kinase inhibitor (CKI) in tumor cells is specific and not an artifact. A study in non-small cell lung carcinomas

It is well established that p16INK4A protein acts as a cell cycle inhibitor in the nucleus. Therefore, cytoplasmic localization of p16 INK4A usually is disregarded by investigators as nonspecific. Three recent studies reported findings that differ from the current view concerning p16INK4A immunohistochemical localization. All three demonstrated that breast and colon cancers expressing cytoplasmic p16INK4 represent distinct biological subsets. We previously detected in a percentage of non-small cell lung carcinomas simultaneous nuclear and cytoplasmic p16INK4A staining. In view of the reports concerning breast and colon carcinomas, we conducted an ultrastructural re-evaluation of our cases to clarify the specificity of p16INK4A cytoplasmic expression. We observed p16 INK4A immunolocalization in both the nucleus and the cytoplasm of a proportion of tumor cells. Diffuse dense nuclear staining was detected in the nucleoplasm, whereas weaker granular immunoreactivity was observed in the cytoplasm near the rough endoplasmic reticulum. Negative tumor cells also were visible. In the tumor-associated stromal, cells p16INK4A immunoreactivity was detected only in the nuclei. We have demonstrated that p16INK4A cytoplasmic staining is specific and suggest that it represents a mechanism of p16INK4A inactivation similar to that observed in other tumor suppressor genes.

Hepatocyte autotaxin expression promotes liver fibrosis and cancer

Autotaxin (ATX) is a secreted lysophospholipase D that catalyzes the production of lysophosphatidic acid (LPA), a pleiotropic growth‐factor–like lysophospholipid. Increased ATX expression has been detected in various chronic inflammatory disorders and different types of cancer; however, little is known about its role and mode of action in liver fibrosis and cancer. Here, increased ATX expression was detected in chronic liver disease (CLD) patients of different etiologies, associated with shorter overall survival. In mice, different hepatotoxic stimuli linked with the development of different forms of CLDs were shown to stimulate hepatocyte ATX expression, leading to increased LPA levels, activation of hepatic stellate cells (HSCs), and amplification of profibrotic signals. Hepatocyte‐specific, conditional genetic deletion and/or transgenic overexpression of ATX established a liver profibrotic role for ATX/LPA, whereas pharmacological ATX inhibition studies suggested ATX as a possible therapeutic target in CLDs. In addition, hepatocyte ATX ablation and the consequent deregulation of lipid homeostasis was also shown to attenuate hepatocellular carcinoma (HCC) development, thus implicating ATX/LPA in the causative link of cirrhosis and HCC. Conclusion: ATX is a novel player in the pathogenesis of liver fibrosis and cancer and a promising therapeutic target. (Hepatology 2017;65:1369‐1383).

Sensitive differential detection of genetically related mycobacterial pathogens in archival material.

A polymerase chain reaction (PCR) assay targeted to the immunogenic protein MPB64 gene was used to detect members of the Mycobacterium tuberculosis complex, and an outward-primed PCR (OPPCR) designed on the IS6110 element allowed differentiation between Mycobacterium bovis and Mycobacterium tuberculosis. Additionally, the amplification of IS1110 and 16S ribosomal RNA sequences combined with a dot blotting assay were able to differentially detect Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium paratuberculosis. The validity of the experimental procedure was tested on reference material and formalin-fixed paraffin-embedded samples from patients with tuberculosis, sarcoidosis, or Crohn disease. We demonstrated mycobacterial DNA in 59 of 75 cases with histologic lesions typical of tuberculosis; we detected M tuberculosis and M paratuberculosis in 6 of 25 sarcoidosis cases and in 7 of 20 Crohn disease specimens, respectively. The proposed diagnostic procedure is directly applicable to archival material and allows differentiation of genetically related mycobacterial pathogens in more detail than other molecular methods. It provides a tool for the diagnostic study of tuberculosis, sarcoidosis, and Crohn disease.

Evaluation of claspin as a proliferation marker in human cancer and normal tissues

Claspin is a nuclear protein involved in DNA replication and the DNA damage response. Its structural and functional properties suggest that it may represent a potentially useful proliferation marker. To this end, a monoclonal antibody was generated and the expression of claspin was investigated in normal fibroblasts and various cancer cell lines, as well as in tumour and normal tissues from patients with primary epithelial carcinomas. Immunoblotting analysis confirmed the specificity of the antibody, while immunohistochemistry demonstrated its applicability in archival material. In normal cells and tissues, claspin expression was weak, whereas increased levels were observed in cancer cell lines and tumour specimens. Claspin staining correlated strongly with Ki67 staining in both normal (p < 0.001) and tumour tissues (p < 0.001). However, the labelling index (LI) of claspin was consistently lower than that of Ki67, suggesting that claspin expression may be limited to a narrower part of the cell cycle. Co‐localization assays with cyclin A and cell synchronization experiments indicated that claspin expression coincides with the S phase. Interestingly, the relative increase of the claspin LI in tumour samples compared with normal tissues was significantly higher (14‐fold) than that of the Ki67 LI (five‐fold), suggesting that claspin may be a more sensitive marker of aberrant proliferation. Copyright