Merkel cell polyomavirus small T antigen induces genome instability by E3 ubiquitin ligase targeting.

This corrects the article DOI: 10.1038/onc.2017.277.

Merkel cell polyomavirus small T antigen induces genome instability by E3 ubiquitin ligase targeting.

The formation of a bipolar mitotic spindle is an essential process for the equal segregation of duplicated DNA into two daughter cells during mitosis. As a result of deregulated cellular signaling pathways, cancer cells often suffer a loss of genome integrity that might etiologically contribute to carcinogenesis. Merkel cell polyomavirus (MCV) small T (sT) oncoprotein induces centrosome overduplication, aneuploidy, chromosome breakage and the formation of micronuclei by targeting cellular ligases through a sT domain that also inhibits MCV large T oncoprotein turnover. These results provide important insight as to how centrosome number and chromosomal stability can be affected by the E3 ligase targeting capacity of viral oncoproteins such as MCV sT, which may contribute to Merkel cell carcinogenesis.

Identification of a novel polymorphism in the fibronectin type II domain of the SEL1L gene and possible relation to the persistent hyperinsulinemic hypoglycemia of infancy.

SEL1L, a human gene located on chromosome 14q24.3-q31, is highly expressed in adult pancreas. It is proximal to D14S67 (IDDM11) a proposed type I diabetes susceptibility locus. Considering the organ specific expression of SEL1L, a fundamental role of SEL1L in pancreatic growth can be hypothesized. While screening for mutations in young diabetic patients, in children affected by persistent hyperinsulinemic hypoglycemia of infancy (PHHI), in patients with non-functional endocrine tumours and in over 100 control subjects, we identified a novel polymorphism (D162G) residing on the fourth exon of the gene. This exon encodes for the fibronectin type II domain and the nucleotide change involves a highly conserved amino acid. The D162G polymorphism induces a major change in the amino acid composition producing a possible disruptive role in collagen binding.

Detection of human papillomavirus DNA in pterygia from different geographical regions.

BACKGROUND/AIMS: The aetiology and pathogenesis of pterygia remain unclear and the involvement of human papillomavirus (HPV) is controversial. 41 pterygia from two geographic locations were evaluated for the presence of HPV DNA. METHODS: 41 pterygium biopsies (17 from Italy and 24 from Ecuador) were analysed using the L1C1 and PU-1ML primer sets by polymerase chain reaction (PCR) and DNA sequence analysis. RESULTS: 22 of the 41 pterygia (54%) were positive for HPV, including all 17 Italian cases and 5/24 (21%) Ecuadorean cases. DNA sequencing of the 22 positive cases showed that 11 were HPV type 52, four were type 54, five were candHPV90, and two of unknown genotype. CONCLUSIONS: The major differences in the frequency of HPV in geographically distant populations might suggest a possible explanation for the vast differences in the reported detection rates. Three subtypes of HPV were found in this sample of pterygia. None the less, these results suggest that HPV may have a pathogenic role in pterygium.

Review of the clinical, histological, and molecular aspects of pancreatic endocrine neoplasms.

Pancreatic endocrine neoplasms (PENs) are rare tumors, and little is known about their genetic and chromosomal alterations. Elucidation of the molecular events involved in PEN carcinogenesis has been hindered by the fact that PENs have been considered a single disease entity. The emergence of novel molecular characterization strategies has, however, made it apparent that these lesions exhibit diverse molecular fingerprints, which will facilitate the precise delineation of PEN prognosis, histopathology, and carcinogenesis.

Molecular anatomy of CCR5 engagement by physiologic and viral chemokines and HIV-1 envelope glycoproteins: differences in primary structural requirements for RANTES, MIP-1 alpha, and vMIP-II Binding.

Molecular analysis of CCR5, the cardinal coreceptor for HIV-1 infection, has implicated the N-terminal extracellular domain (N-ter) and regions vicinal to the second extracellular loop (ECL2) in this activity. It was shown that residues in the N-ter are necessary for binding of the physiologic ligands, RANTES (CCL5) and MIP-1 alpha (CCL3). vMIP-II, encoded by the Kaposi's sarcoma-associated herpesvirus, is a high affinity CCR5 antagonist, but lacks efficacy as a coreceptor inhibitor. Therefore, we compared the mechanism for engagement by vMIP-II of CCR5 to its interaction with physiologic ligands. RANTES, MIP-1 alpha, and vMIP-II bound CCR5 at high affinity, but demonstrated partial cross-competition. Characterization of 15 CCR5 alanine scanning mutants of charged extracellular amino acids revealed that alteration of acidic residues in the distal N-ter abrogated binding of RANTES, MIP-1 alpha, and vMIP-II. Whereas mutation of residues in ECL2 of CCR5 dramatically reduced the binding of RANTES and MIP-1 alpha and their ability to induce signaling, interaction with vMIP-II was not altered by any mutation in the exoloops of the receptor. Paradoxically, monoclonal antibodies to N-ter epitopes did not block chemokine binding, but those mapped to ECL2 were effective inhibitors. A CCR5 chimera with the distal N-ter residues of CXCR2 bound MIP-1 alpha and vMIP-II with an affinity similar to that of the wild-type receptor. Engagement of CCR5 by vMIP-II, but not RANTES or MIP-1 alpha blocked the binding of monoclonal antibodies to the receptor, providing additional evidence for a distinct mechanism for viral chemokine binding. Analysis of the coreceptor activity of randomly generated mouse-human CCR5 chimeras implicated residues in ECL2 between H173 and V197 in this function. RANTES, but not vMIP-II blocked CCR5 M-tropic coreceptor activity in the fusion assay. The insensitivity of vMIP-II binding to mutations in ECL2 provides a potential rationale to its inefficiency as an antagonist of CCR5 coreceptor activity. These findings suggest that the molecular anatomy of CCR5 binding plays a critical role in antagonism of coreceptor activity.

Role of disease-causing genes in sporadic pancreatic endocrine tumors: MEN1 and VHL.

Pancreatic endocrine tumors (PETs) occur in association with multiple endocrine neoplasia type 1 (MEN1) and von Hippel-Lindau (VHL) syndromes caused by germline alterations in MEN1 and VHL, respectively. It is thus expected that these genes will also be altered in a proportion of sporadic PETs. Indeed, MEN1 is altered in about 25% of nonfamilial PETs, although no mutations have been found in VHL. For all clinical subtypes, the frequency of allelic loss on chromosome arm 11q mirrors observed mutational frequencies, with the exception of nonfunctional tumors (NF-PETs), in which mutations have been reported in only 8% of cases. As allelic loss on 11q is the most frequent event found in these neoplasms, this low frequency is somewhat puzzling, particularly in light of the fact that most MEN1-associated PETs are nonfunctioning. To clarify the role of these genes in sporadic PETs, we analyzed 31 sporadic NF-PETs, nine insulinomas, and one VIPoma for alterations in MEN1 and VHL. As somatic mutations were observed in eight (26%) of the NF tumors and in one insulinoma, it would therefore appear unlikely that an additional tumor suppressor gene related to sporadic PET pathogenesis is located on 11q. One insulinoma also had a somatic mutation in VHL, and thus this gene may also be altered in these neoplasms, albeit in a small proportion of cases.

Genetic alterations in primary mediastinal B-cell lymphoma: an update.

Primary mediastinal B-cell lymphoma (PMBL) is a distinct clinical entity among non-Hodgkin's lymphoma. The malignancy has received little attention from a standpoint of basic research due in part to its rarity. However, based on recent studies consistent trends are beginning to emerge regarding the molecular and chromosomal alterations commonly observed in this disease. By both CGH and AP-PCR, genetic gains involving chromosomes 2, 5, 7, 9p, 12, and Xq are among the most frequently observed events. From a molecular standpoint, alterations in the c-myc, p16(INK4) and p53 genes have been observed in up to 30% of cases. This information along with the well-established histological, immunological, and clinical features should convince the few remaining disbelievers that PMBL is a distinct pathological entity among non-Hodgkin's lymphomas.

Molecular virology of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV), the most recently discovered human tumour virus, is the causative agent of Kaposi's sarcoma, primary effusion lymphoma and some forms of Castleman's disease. KSHV is a rhadinovirus, and like other rhadinoviruses, it has an extensive array of regulatory genes obtained from the host cell genome. These pirated KSHV proteins include homologues to cellular CD21, three different beta-chemokines, IL-6, BCL-2, several different interferon regulatory factor homologues, Fas-ligand ICE inhibitory protein (FLIP), cyclin D and a G-protein-coupled receptor, as well as DNA synthetic enzymes including thymidylate synthase, dihydrofolate reductase, DNA polymerase, thymidine kinase and ribonucleotide reductases. Despite marked differences between KSHV and Epstein-Barr virus, both viruses target many of the same cellular pathways, but use different strategies to achieve the same effects. KSHV proteins have been identified which inhibit cell-cycle regulation checkpoints, apoptosis control mechanisms and the immune response regulatory machinery. Inhibition of these cellular regulatory networks app ears to be a defensive means of allowing the virus to escape from innate antiviral immune responses. However, due to the overlapping nature of innate immune and tumour-suppressor pathways, inhibition of these regulatory networks can lead to unregulated cell proliferation and may contribute to virus-induced tumorigenesis.

Alteration of chromosome arm 6p is characteristic of primary mediastinal B-cell lymphoma, as identified by genome-wide allelotyping.

Five cases of primary mediastinal B-cell lymphoma (PMBL) each have been studied with 375 microsatellite markers from all 22 autosomes. Of the 151 genomic alterations among the 1,875 assays, only five were allelic losses. The remainder of the microsatellite alterations consisted of 114 allelic imbalances and 32 instabilities. Microsatellite alterations were found in all cases on chromosomal arms 6p and 9p. These allelic imbalances most likely are indicative of genetic amplification, a finding agreeing well with those of studies using either comparative genomic hybridization or arbitrarily primed polymerase chain reaction, in which amplification of chromosome arm 9p in PMBL has been found. The allelic imbalances on chromosome arm 6p always included marker D6S276 located at 6p21.3-p22.3, where the MHC class I genes reside. These allelic imbalances may be reflective of alterations in the expression of the MHC gene products, characteristic of PMBL. Allelic anomalies close to the MYB gene locus on 6q were detected in two cases and prompted the analysis of MYB rearrangements in a series of 30 lymphomas. One rearrangement was detected in one of 18 cases of PMBL and in none of 10 diffuse, large B-cell lymphomas and two T-cell lymphomas. Our genome-wide microsatellite analysis provides independent confirmation that PMBL is characterized by infrequent chromosomal losses and by frequent genetic alterations involving chromosomal arm 9p. For the first time, chromosomal arm 6p has been identified as a highly frequent target of genetic alterations in this tumor type. Finally, MYB may also be involved occasionally in PMBL pathogenesis.

Successful xenografting of cryopreserved primary pancreatic cancers.

In order to assess the suitability of cryopreserved neoplastic tissues for xenografting into nude (nu/nu) mice, we compared the take rate in 28 samples of pancreatic ductal carcinoma. Eleven fresh samples were implanted in nu/nu mice, and 17 were frozen in cryopreserving solution and implanted at a later time. All samples were examined for the presence of neoplastic tissue in cryostat sections. A total of 15 tumors grew in the animals; five from the freshly implanted samples and ten from those cryopreserved. Ten xenografted tumors were characterized for alterations in p53, K-ras, and p16 genes, which were found in six, eight, and nine cases, respectively. Our results demonstrate that the take rate for xenografting is comparable between cryopreserved and fresh tissue samples. The procedure allows for the exchange of tumor material between institutions and permits the establishment of centralized facilities for the storage of an array of different primary tumor samples suitable for the production of in vivo models of cancers.

Clinical significance of mutator phenotype and chromosome 17p and 18q allelic loss in gastric cancer.

BACKGROUND: Tumour stage is the only reliable prognostic factor for gastric cancer. The molecular anomalies involved in this process have the potential to serve as additional prognostic markers. METHODS: Forty-four gastric cancers, treated by surgery alone, have been analysed for chromosome 17p and 18q allelic loss and for the presence of microsatellite instability (MSI), using microsatellite markers and DNA from paraffin-embedded tumours. RESULTS: Eight cancers showed a MSI-positive (MSI+) phenotype. Among the 36 MSI-negative cancers, chromosome 17p and 18q allelic losses were found in 22 of 34 and 19 of 33 informative cases respectively. Multivariate survival analysis indicated MSI status to be an independent prognostic factor along with the tumour stage. MSI+ cancers were associated with longer patient survival, whereas MSI-negative cancers had a significantly poorer prognosis (P = 0.007), with a median actuarial survival of 24 months. CONCLUSION: MSI status is an independent prognostic factor among gastric cancers at the same stage. Chromosome 17p and 18q status added no additional prognostic information to that of tumour stage. The combined use of tumour stage and MSI status may help in deciding whether patients with advanced gastric cancer require additional therapy other than surgery alone.

Pancreatic tumours: molecular pathways implicated in ductal cancer are involved in ampullary but not in exocrine nonductal or endocrine tumorigenesis.

Alterations of K- ras, p53, p16 and DPC4/Smad4 characterize pancreatic ductal cancer (PDC). Reports of inactivation of these latter two genes in pancreatic endocrine tumours (PET) suggest that common molecular pathways are involved in the tumorigenesis of pancreatic exocrine and endocrine epithelia. We characterized 112 primary pancreatic tumours for alterations in p16 and DPC4 and immunohistochemical expression of DPC4. The cases included 34 PDC, 10 intraductal papillary-mucinous tumours (IPMT), 6 acinar carcinomas (PAC), 5 solid-pseudopapillary tumours (SPT), 16 ampulla of Vater cancers (AVC) and 41 PET. All tumours were also presently or previously analysed for K- ras and p53 mutations and allelic loss at 9p, 17p and 18q. Alterations in K- ras, p53, p16 and DPC4 were found in 82%, 53%, 38% and 9% of PDC, respectively and in 47%, 60%, 25% and 6% of AVC. Alterations in these genes were virtually absent in PET, PAC or SPT, while in IPMT only K- ras mutations were present (30%). Positive immunostaining confirmed the absence of DPC4 alterations in all IPMT, SPT, PAC and PET, while 47% of PDC and 38% of AVC were immunonegative. These data suggest that pancreatic exocrine and endocrine tumourigenesis involves different genetic targets and that among exocrine pancreatic neoplasms, only ductal and ampullary cancers share common molecular events.

High resolution allelotype of nonfunctional pancreatic endocrine tumors: identification of two molecular subgroups with clinical implications.

A high resolution allelotype for nonfunctional pancreatic endocrine tumors (NF-PETs) has been generated by microsatellite analysis of DNA from 16 frozen cases, each probed with 394 markers. Two subgroups of NF-PETs were found. Seven cases showed frequent, large allelic deletions [loss of heterozygosity (LOH)] with an average fractional allelic loss (FAL) of 0.55, whereas nine cases showed a small number of random losses with a FAL of 0.15. Designated high or low FAL, respectively, these genetic phenotypes showed correlation with the ploidy status: high-FAL tumors were aneuploid, low-FAL were diploid. Chromosomes 6q and 11q showed LOH in >60% of cases. About 50% of cases had losses on 11p, 20q, and 21. Selected LOH analysis on an additional 16 paraffin-embedded NF-PETs confirmed the high frequency of 6q and 11q LOH. The allelotype of NF-PET is markedly different from that of either ductal or acinar tumors of the pancreas as well as from that of functional-PETs. Moreover, whereas deletions involving chromosome 11 also are a feature of functional-PETs, the involvement of chromosome 6q is characteristic of NF-PETs. Survival analysis showed that none of the single chromosomal alterations was associated with outcome, whereas ploidy status is an independent factor adding prognostic information to that furnished by the proliferative index measured by Ki-67 immunohistochemistry.

Non-random chromosomal rearrangements in pancreatic cancer cell lines identified by spectral karyotyping.

The molecular events involved in pancreatic cancer are becoming increasingly well characterized, with mutations in the dominant oncogene KRAS and the tumour suppressor genes TP53, CDKN2A and MADH4 being typically observed. However, other genetic abnormalities remain to be identified and molecular cytogenetics may be useful to detect chromosomal loci involved in recurrent rearrangements. We have used spectral karyotyping to characterize cytogenetic aberrations in a panel of 20 human pancreatic carcinoma cell lines and confirmed their identities by dual and triple color fluorescence in situ hybridization. The most common partial or whole-arm gains involved 5p, 7q, 12p, 1q, 7p, 5q, 9p, 9q and 11p. The most common partial or whole-arm losses affected 9p, 11q, 18q, 3p, 2q and 1p, as well as the short arms of the acrocentric chromosomes. Spectral karyotyping allowed us to identify a number of recurrent structural aberrations, all of them unbalanced: most frequently i(5)(p10), del(11)(q23), i(12)(p10), i(1)(q10), del(7)(q22) and del(10)(p11). Spectral karyotyping mapped the complex aberrations occurring in pancreatic cancer cell lines and identified non-random patterns of chromosomal rearrangement. This comprehensive characterization should be useful to direct future investigation. The observation that loss at 11q and gains at 5p with i(5)(p10) and 12p with i(12)(p10) are more frequent changes than previously reported would justify more intensive investigation of these chromosomal regions.

Molecular characterization of pancreatic serous microcystic adenomas: evidence for a tumor suppressor gene on chromosome 10q.

Pancreatic serous microcystic adenomas (SCAs) are rare, benign tumors with a striking female preference. Virtually no information is available about chromosomal or genetic anomalies in this disease. We performed extensive molecular characterization of 21 cases of formalin-fixed, paraffin-embedded sporadic SCAs consisting in genome-wide allelic loss analysis with 79 microsatellite markers covering all 22 autosomes, assessment of microsatellite instability, and mutational analysis of the VHL, K-ras, and p53 genes in nine cases for which frozen tissue was available. Although no case showed microsatellite instability of the type seen in mismatch repair-deficient tumors, a relatively low fractional allelic loss of 0.08 was found. Losses on chromosome 10q were the most frequent event in SCAs (50% of cases), followed by allelic losses on chromosome 3p (40% of cases). Moderately frequent losses (>25% of cases) were found on chromosomes 1q, 2q, and 7q. The VHL gene, located on chromosome 3p, had somatic inactivating mutations in two of nine cases (22%), whereas no mutations were found in either K-ras or p53, in agreement with the finding that all 21 cases stained negative for p53 by immunohistochemistry. Our study indicates that the involvement of chromosomal arms 10q and 3p is characteristic of SCAs and that the VHL gene is involved in a subset of sporadic cases.

Kaposi's sarcoma-associated herpesvirus LANA2 is a B-cell-specific latent viral protein that inhibits p53.

Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, is associated with three proliferative diseases ranging from viral cytokine-induced hyperplasia to monoclonal neoplasia: multicentric Castleman's disease (CD), Kaposi's sarcoma (KS), and primary effusion lymphoma (PEL). Here we report a new latency-associated 1,704-bp KSHV spliced gene belonging to a cluster of KSHV sequences having homology to the interferon regulatory factor (IRF) family of transcription factors. ORFK10.5 encodes a protein, latency-associated nuclear antigen 2 (LANA2), which is expressed in KSHV-infected hematopoietic tissues, including PEL and CD but not KS lesions. LANA2 is abundantly expressed in the nuclei of cultured KSHV-infected B cells. Transcription of K10.5 in PEL cell cultures is not inhibited by DNA polymerase inhibitors nor significantly induced by phorbol ester treatment. Unlike LANA1, LANA2 does not elicit a serologic response from patients with KS, PEL, or CD as measured by Western blot hybridization. Both KSHV vIRF1 (ORFK9) and LANA2 (ORFK10.5) appear to have arisen through gene duplication of a captured cellular IRF gene. LANA2 is a potent inhibitor of p53-induced transcription in reporter assays. LANA2 antagonizes apoptosis due to p53 overexpression in p53-null SAOS-2 cells and apoptosis due to doxorubicin treatment of wild-type p53 U2OS cells. While LANA2 specifically interacts with amino acids 290 to 393 of p53 in glutathione S-transferase pull-down assays, we were unable to demonstrate LANA2-p53 interaction in vivo by immunoprecipitation. These findings show that KSHV has tissue-specific latent gene expression programs and identify a new latent protein which may contribute to KSHV tumorigenesis in hematopoietic tissues via p53 inhibition.

DNA array/microarrays in oncological research with focus on pancreatic cancer.

DNA array/microarray technology is becoming one of the most productive methods for characterizing physiological and pathological processes and is being used to elucidate the complex interdependence present in biological systems that cannot be carried out by classic methodologies. This technology is particularly well suited to the study of cancer and has already permitted the identification of subtypes of different tumors with clinically relevant subtypes. As for other neoplasms, an improved understanding of pancreas cancer genetics is the only means to provide new markers for earlier diagnosis and to identify potential targets for therapeutic intervention. Transcriptional profiling using DNA arrays has the potential to satisfy these requirements. To date, only a few reports have emerged regarding the analysis of common pancreatic cancer by these techniques. Initial expression profiles using macroarrays have been generated and expression profiles of the cancer cell and the desmoplastic reactions on cDNA arrays have been performed using material obtained by fine needle aspiration. More recently, microarrays have been used to identify genes abnormally expressed in chronic pancreatitis with respect to pancreatic cancer and normal pancreas. Gene expression profiles will also be invaluable for the study of other less common pancreatic tumor types such as intraductal, ampullary, serous microcystic, acinar, and endocrine tumors.

Genetic profile of 22 pancreatic carcinoma cell lines. Analysis of K-ras, p53, p16 and DPC4/Smad4.

The K-ras, p53, p16 and DPC4 genes are among those most frequently altered in pancreatic ductal carcinoma. We analyzed 22 cell lines for alterations in these genes by direct sequence analysis and methylation-specific polymerase chain reaction. These cell lines showed mutations in K-ras and p53 at frequencies of 91% and 95%, respectively. Alterations in p16INK4a were found in all cases and included nine homozygous deletions, seven mutations and promoter methylation in six cases. Eight cell lines (36%) had an alteration of DPC4, including one mutation and seven homozygous deletions. The most typical mutational profile involved K-ras, p53, and p16INK4a, concurrently aberrated in 20 cases (91%). Eight cell lines had alterations in all four genes. Inactivation of DPC4 was always accompanied by alteration of all of the other three genes. This comprehensive data regarding the cumulative genetic alterations in pancreatic carcinoma cell lines will be of great value for studies involving drug sensitivity or resistance that may be associated with inactivation of a particular gene or molecular pathway.