Christos Kittas

Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions

DNA damage checkpoint genes, such as p53, are frequently mutated in human cancer, but the selective pressure for their inactivation remains elusive. We analysed a panel of human lung hyperplasias, all of which retained wild-type p53 genes and had no signs of gross chromosomal instability, and found signs of a DNA damage response, including histone H2AX and Chk2 phosphorylation, p53 accumulation, focal staining of p53 binding protein 1 (53BP1) and apoptosis. Progression to carcinoma was associated with p53 or 53BP1 inactivation and decreased apoptosis. A DNA damage response was also observed in dysplastic nevi and in human skin xenografts, in which hyperplasia was induced by overexpression of growth factors. Both lung and experimentally-induced skin hyperplasias showed allelic imbalance at loci that are prone to DNA double-strand break formation when DNA replication is compromised (common fragile sites). We propose that, from its earliest stages, cancer development is associated with DNA replication stress, which leads to DNA double-strand breaks, genomic instability and selective pressure for p53 mutations.

Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints.

Recent studies have indicated the existence of tumorigenesis barriers that slow or inhibit the progression of preneoplastic lesions to neoplasia. One such barrier involves DNA replication stress, which leads to activation of the DNA damage checkpoint and thereby to apoptosis or cell cycle arrest, whereas a second barrier is mediated by oncogene-induced senescence. The relationship between these two barriers, if any, has not been elucidated. Here we show that oncogene-induced senescence is associated with signs of DNA replication stress, including prematurely terminated DNA replication forks and DNA double-strand breaks. Inhibiting the DNA double-strand break response kinase ataxia telangiectasia mutated (ATM) suppressed the induction of senescence and in a mouse model led to increased tumour size and invasiveness. Analysis of human precancerous lesions further indicated that DNA damage and senescence markers cosegregate closely. Thus, senescence in human preneoplastic lesions is a manifestation of oncogene-induced DNA replication stress and, together with apoptosis, provides a barrier to malignant progression.

High risk HPV types are frequently detected in potentially malignant and malignant oral lesions, but not in normal oral mucosa.

Studies on the involvement of the human papillomavirus (HPV) in initiation and progression of oral neoplasia have generated conflicting results. The observed discrepancy is attributable mainly to the varying sensitivity of the applied methodologies and to epidemiologic factors of the examined patient groups. To evaluate the role of HPV in oral carcinogenesis, we analyzed 53 potentially neoplastic and neoplastic oral lesions consisting of 29 cases of hyperplasia, 5 cases of dysplasia, and 19 cases of squamous cell carcinomas, as well as 16 oral specimens derived from healthy individuals. A highly sensitive nested polymerase chain reaction (PCR) assay was used, along with type-specific PCR, restriction fragment length polymorphism analysis, dot blotting, and nonisotopic in situ hybridization. Nested PCR revealed the presence of HPV DNA in 48 of the 53 (91%) pathologic samples analyzed, whereas none (0%) of the normal specimens was found to be infected. Positivity for HPV was independent of histology and the smoking habits of the analyzed group of patients. At least one “high risk” type, such as HPV 16, 18, and 33, was detected by type-specific PCR in 47 (98%) infected specimens, whereas only 1 (2%) squamous cell carcinoma was solely infected by a “low risk” type (HPV 6). HPV 16 was the prevailing viral type, being present in 71% of infected cases. Single HPV 16 and HPV 18 infections were confirmed by restriction fragment length polymorphism. HPV 58 was detected by dot blotting in three hyperplastic lesions. HPV positivity and genotyping were further confirmed, and the physical status of this virus was evaluated by nonisotopic in situ hybridization. Diffuse and punctate signals, indicative of the episomal and integrative pattern of HPV infection, were observed for low- and high-risk types, respectively. Our findings are suggestive of an early involvement of high-risk HPV types in oral carcinogenesis.

Transcription factor E2F‐1 acts as a growth‐promoting factor and is associated with adverse prognosis in non‐small cell lung carcinomas

Numerous upstream stimulatory and inhibitory signals converge to the pRb/E2F pathway, which governs cell‐cycle progression, but the information concerning alterations of E2F‐1 in primary malignancies is very limited. Several in vitro studies report that E2F‐1 can act either as an oncoprotein or as a tumour suppressor protein. In view of this dichotomy in its functions and its critical role in cell cycle control, this study examined the following four aspects of E2F‐1 in a panel of 87 non‐small cell lung carcinomas (NSCLCs), previously analysed for defects in the pRb‐p53‐MDM2 network: firstly, the status of E2F‐1 at the protein, mRNA and DNA levels; secondly, its relationship with the kinetic parameters and genomic instability of the tumours; thirdly, its association with the status of its transcriptional co‐activator CBP, downstream target PCNA and main cell cycle regulatory and E2F‐1‐interacting molecules pRb, p53 and MDM2; and fourthly, its impact on clinical outcome. The protein levels of E2F‐1 and its co‐activator CBP were significantly higher in the tumour area than in the corresponding normal epithelium (p<0.001). E2F‐1 overexpression was associated with increased E2F‐1 mRNA levels in 82% of the cases examined. The latter finding, along with the low frequency of E2F‐1 gene amplification observed (9%), suggests that the main mechanism of E2F‐1 protein overexpression in NSCLCs is deregulation at the transcriptional level. Mutational analysis revealed only one sample with asomatic mutation at codon 371 (Glu→Asp) and one carrying a polymorphism at codon 393 (Gly→Ser). Carcinomas with increased E2F‐1 positivity demonstrated a significant increase in their growth indexes (r=0.402, p=0.001) and were associated with adverse prognosis (p=0.033 by Cox regression analysis). The main determinant of the positive association with growth was the parallel increase between E2F‐1 staining and proliferation (r=0.746, p<0.001), whereas apoptosis was not influenced by the status of E2F‐1. Moreover, correlation with the status of the pRb–p53–MDM2 network showed that the cases with aberrant pRb expression displayed significantly higher E2F‐1 indexes (p=0.033), while a similar association was noticed in the group of carcinomas with deregulation of the p53–MDM2 feedback loop. In conclusion, the results suggest that E2F‐1 overexpression may contribute to the development of NSCLCs by promoting proliferation and provide evidence that this role is further enhanced in a genetic background with deregulated pRb–p53–MDM2 circuitry. Copyright

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.

Alterations of the p16-pRb Pathway and the Chromosome Locus 9p21–22 in Non-Small-Cell Lung Carcinomas : Relationship with p53 and MDM2 Protein Expression

The p16-pRb and p53-MDM2 pathways represent vital cell cycle checkpoints. Recent studies provide evidence that these pathways are directly linked via MDM2-pRb interaction and p53 suppression of the RB1 gene. In the present study we investigated the alterations of this G1 phase protein network using immunohistochemical and molecular methods in a series of 68 non-small-cell lung carcinomas (NSCLCs) and correlated the findings with clinicopathological features and prognosis of the patients. Aberrant expression (Ab) of p16 and pRb was observed in 33 (49%) and 27 (40%) of the carcinomas, respectively. Analysis of the region that encodes for p16 by deletion mapping, a polymerase chain reaction (PCR)-based methylation assay and PCR single-strand conformation polymorphism (SSCP) analysis revealed that deletions and transcriptional silencing by methylation might represent the main mechanisms of CDKN2/p16ink4a inactivation in NSCLCs. The results of deletion mapping also suggest that other tumor suppressor genes may reside at the 9p21-22 region, which encodes for CDKN2/MTS1/p16ink4a, p14ARF, and MTS2/p15ink4b. In addition, microsatellite instability was observed with a frequency of 16% in the 9p21-22 chromosome area. Overexpression (P) of p53 and MDM2 proteins was found in 39 (58%) and 47 (70%) of the cases, respectively. A highly significant association was observed between p53 overexpression and p53 mutations (P = 0.006). Statistical analysis of the expression patterns of the biologically relevant molecules (p16/pRb, p53/MDM2, MDM2/pRb, and p53/pRb) showed coincident overexpression of p53 and MDM2 (P = 0.04) and that abnormal pRb was correlated with elevated levels of MDM2 (P = 0.013) and p53 (P = 0.01), respectively. We suggest that deregulated expression of these molecules may act synergistically. An important finding of the study was that multiple impairments (three and four molecules affected) of the p16/pRb/p53/MDM2 network occurred in a large proportion (43%) of the carcinomas. This finding in addition to the absence of correlation with clinical stage of the tumors suggests that multiple hits of this network may be a relatively early event in the development of a subset of NSCLCs. The relationship between the factors examined in the present study, clinicopathological features, and survival of the patients did not reveal any significant correlations with the exception of smoking, which was associated with microsatellite alterations (loss of heterozygosity and microsatellite instability) at the 9p21-22 locus (P = 0.04) and the immunophenotypes p53(P)/MDM2(P) (P = 0.04) and p16(Ab)/pRb(Ab)/p53(P)/MDM2(P) (P = 0.03), respectively. We suggest that in a subset of NSCLCs, simultaneous deregulation of the members of this network may represent one way of initiating the oncogenic procedure whereas in other NSCLC subgroups alternative pathways may play this role.

Human papilloma virus (HPV) is possibly involved in laryngeal but not in lung carcinogenesis.

Data on human papilloma virus (HPV) involvement in preneoplastic and neoplastic lesions of the larynx and lung are limited and conflicting. The presence of HPV was investigated in a series of laryngeal specimens and non-small cell lung carcinomas (NSCLCs). The laryngeal samples (154) comprised 14 cases with hyperplasia without dysplasia, 49 with dysplasia, and 91 squamous cell carcinomas (SqCCs). The NSCLCs included 31 SqCCs, 32 adenocarcinomas, and 5 undifferentiated large cell carcinomas. Furthermore, we examined, for HPV DNA sequences, 14 bronchial metaplastic squamous lesions located next to cancerous areas. We used a sensitive nested polymerase chain reaction assay (NPCR), dot blotting, and in situ hybridization. The findings were correlated with clinicopathologic features of the patients. In the laryngeal specimens, NPCR analysis showed HPV DNA in 20 (13%) of the 154 specimens. Notably, 19 of 20 HPV-positive cases were carcinomas and only one was a mild dysplastic lesion. Typing of the carcinomas showed single HPV 6, 16, 18, and 33 infection in 1 (1.1%), 12 (13.2%), 2 (2.2%), and 1 (1.1%) samples, respectively, and HPV 6/33, 16/33, and 6/18 coinfection in three carcinomas. In situ hybridization findings were in agreement with PCR results, with the exception of two cases in which HPV 18 DNA was detected only by PCR. HPV was more frequently observed in heavy smokers than in patients with low daily cigarette consumption and nonsmokers (P = .03). There was no correlation between virus infection and gender, grade, and lymph node status of the carcinomas. None of the NSCLCs or adjacent metaplastic squamous epithelium contained HPV DNA sequences. The presented data suggest a contributory role of HPV in late stages of laryngeal carcinogenesis, because all premalignant lesions were negative but one. This study does not support a potential role of HPV in the development of NSCLCs.

p53 activates ICAM-1 (CD54) expression in an NF-κB-independent manner

Intercellular adhesion molecule‐1 (ICAM‐1) is a crucial receptor in the cell–cell interaction, a process central to the reaction to all forms of injury. Its expression is upregulated in response to a variety of inflammatory/immune mediators, including cellular stresses. The NF‐κB signalling pathway is known to be important for activation of ICAM‐1 transcription. Here we demonstrate that ICAM‐1 induction represents a new cellular response to p53 activation and that NF‐κB inhibition does not prevent the effect of p53 on ICAM‐1 expression after DNA damage. Induction of ICAM‐1 is abolished after treatment with the specific p53 inhibitor pifithrin‐α and is abrogated in p53‐deficient cell lines. Furthermore, we map two functional p53‐responsive elements to the introns of the ICAM‐1 gene, and show that they confer inducibility to p53 in a fashion similar to other p53 target genes. These results support an NF‐κB‐independent role for p53 in ICAM‐1 regulation that may link p53 to ICAM‐1 function in various physiological and pathological settings.

Vascular endothelial growth factor (VEGF) is expressed by neoplastic Hodgkin-Reed-Sternberg cells in Hodgkin's disease

Vascular endothelial growth factor (VEGF) is involved in tumour angiogenesis, an important process for the growth and metastatic potential of solid tumours. Numerous studies have demonstrated up‐regulation of VEGF at both mRNA and protein level in various tumours and a correlation with advanced stage and prognosis has been demonstrated in some cases. Limited information exists about its role in lymphoid malignancies and in particular, Hodgkins disease. The present study examined the immunohistochemical expression of VEGF using the monoclonal antibody VG1 in a series of 61 cases of Hodgkins disease, including both classical Hodgkins disease and the nodular lymphocyte predominance variant, and correlated these results with microvessel density, using an anti‐CD31 monoclonal antibody. In 41 cases (70.6%) of classical Hodgkins disease and one of the three cases of nodular lymphocyte predominance Hodgkins disease, the neoplastic Reed–Sternberg and Hodgkin cells expressed VEGF. The staining observed was cytoplasmic, either diffuse or with a focal paranuclear distribution. Macrophages were always positive, while reactive lymphocytes showed occasional positivity. A variable amount of strong extracellular staining was also observed in the tissue stroma and intravascular plasma staining was prominent. There was no statistically significant relationship between VEGF expression and the subtype of Hodgkins disease or microvessel density. In vitro studies using the Reed–Sternberg cell lines L428 and KM‐H2 were also performed in both normoxia and hypoxia and VEGF protein production was assessed by flow cytometry (FACS), immunoassay of cell culture supernatant, and RT‐PCR. Analysis by FACS demonstrated a subset of cells in both cell lines reacting with VG1 and analysis of secreted VEGF (pg/ml per 1×106 cells) in cell culture supernatant confirmed the normoxic production in both cell lines and significant hypoxic induction (p<0.005). Additionally, both cell lines expressed VEGF mRNA, as demonstrated using the RT‐PCR method. In conclusion, neoplastic cells express VEGF in Hodgkins disease, as is the case in solid tumours, and this expression may be induced by hypoxia. The presence of VEGF in reactive macrophages and in the extracellular matrix might facilitate tumour progression. Copyright