A functional mammalian target of rapamycin complex 1 signaling is indispensable for c-Myc-driven hepatocarcinogenesis.

Amplification and/or activation of the c-Myc proto-oncogene is one of the leading genetic events along hepatocarcinogenesis. The oncogenic potential of c-Myc has been proven experimentally by the finding that its overexpression in the mouse liver triggers tumor formation. However, the molecular mechanism whereby c-Myc exerts its oncogenic activity in the liver remains poorly understood. Here, we demonstrate that the mammalian target of rapamycin complex 1 (mTORC1) cascade is activated and necessary for c-Myc-dependent hepatocarcinogenesis. Specifically, we found that ablation of Raptor, the unique member of mTORC1, strongly inhibits c-Myc liver tumor formation. Also, the p70 ribosomal S6 kinase/ribosomal protein S6 and eukaryotic translation initiation factor 4E-binding protein 1/eukaryotic translation initiation factor 4E signaling cascades downstream of mTORC1 are required for c-Myc-driven tumorigenesis. Intriguingly, microarray expression analysis revealed up-regulation of multiple amino acid transporters, including solute carrier family 1 member A5 (SLC1A5) and SLC7A6, leading to robust uptake of amino acids, including glutamine, into c-Myc tumor cells. Subsequent functional studies showed that amino acids are critical for activation of mTORC1 as their inhibition suppressed mTORC1 in c-Myc tumor cells. In human hepatocellular carcinoma specimens, levels of c-Myc directly correlate with those of mTORC1 activation as well as of SLC1A5 and SLC7A6. CONCLUSION: Our current study indicates that an intact mTORC1 axis is required for c-Myc-driven hepatocarcinogenesis; thus, targeting the mTOR pathway or amino acid transporters may be an effective and novel therapeutic option for the treatment of hepatocellular carcinoma with activated c-Myc signaling. (Hepatology 2017;66:167-181).

Female Adnexal Tumors of Probable Wolffian Origin (FATWO): A Case Series With Next-Generation Sequencing Mutation Analysis.

Female adnexal tumors of probable Wolffian origin are rare gynecologic tumors with <90 cases reported in the current scientific literature. Their clinical features have been described extensively; less is known about the pathophysiological mechanisms and the molecular alterations underlying their development and growth. We performed a complete histopathologic examination and a systematic mutation analysis using a next-generation sequencing approach on 3 female adnexal tumors of probable Wolffian origin from the archives of our institution to detect possible genetic alterations and to explore their role in the development of these rare tumors. The 3 cases contained missense mutations in different genes belonging to distinct molecular pathways: CTNNB1 and MET mutations for the first case, PIK3CA for the second one, and BRAF and CDKN2A for the third one. Two variants with an unknown functional effect on the protein were found in KDR and TP53 genes. In conclusion, genetic heterogeneity was found in our series. No constant involvement of the most common pathways involved in tumorigenesis was found; nevertheless, further studies are necessary to confirm the results of this pilot study.

KIT and PDGFRa mutational patterns in Sardinian patients with gastrointestinal stromal tumors.

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal malignancy of the gastrointestinal tract. We provide in the present article the molecular characterization of a series of primary GISTs in a cohort of Sardinian patients (Italy), with the aim to describe the patterns of KIT and PDGFRa mutations and the corresponding clinical features. Ninety-nine Sardinian patients with histologically-proven diagnosis of GIST were included in the study. Medical records and pathology reports were used to assess the demographic and clinical features of the patients and the disease at the time of the diagnosis. Formalin-fixed, paraffin-embedded tissue samples were retrieved for each case, and mutation analysis of the KIT and PDGFRa genes was performed. KIT and PDGFRa mutations were detected in 81.8% and 5% of the cases, respectively. The most common KIT mutation was W557_K558del in exon 11, while D842V in exon 18 was the most common PDGFRa genetic alteration; V561D was the only PDGFRa mutation found in exon 12. The global "wild-type" cases, with no mutations in either the KIT or PDGFRa genes, were 13 (13.1%). The mean survival of those patients was approximately 46.9 (±43.9) months. Globally, 86.9% of Sardinian patients with GIST had a KIT or PDGFRa mutation; the former were more frequent in comparison with other Italian cohorts, while PDGFRa mutations were rare. No statistical differences in survival between mutated and wild-type cases, and between KIT and PDGFRa mutated cases were detected in our study.

Origin and distribution of the BRCA2-8765delAG mutation in breast cancer.

BACKGROUND: The BRCA2-8765delAG mutation was firstly described in breast cancer families from French-Canadian and Jewish-Yemenite populations; it was then reported as a founder mutation in Sardinian families. We evaluated both the prevalence of the BRCA2-8765delAG variant in Sardinia and the putative existence of a common ancestral origin through a haplotype analysis of breast cancer family members carrying such a mutation. METHODS: Eight polymorphic microsatellite markers (D13S1250, centromeric, to D13S267, telomeric) spanning the BRCA2 gene locus were used for the haplotype analysis. Screening for the 8765delAG mutation was performed by PCR-based amplification of BRCA2-exon 20, followed by automated sequencing. RESULTS: Among families with high recurrence of breast cancer (> or = 3 cases in first-degree relatives), those from North Sardinia shared the same haplotype whereas the families from French Canadian and Jewish-Yemenite populations presented distinct genetic assets at the BRCA2 locus. Screening for the BRCA2-8765delAG variant among unselected and consecutively-collected breast cancer patients originating from the entire Sardinia revealed that such a mutation is present in the northern part of the island only [9/648 (1.4%) among cases from North Sardinia versus 0/493 among cases from South Sardinia]. CONCLUSION: The BRCA2-8765delAG has an independent origin in geographically and ethnically distinct populations, acting as a founder mutation in North but not in South Sardinia. Since BRCA2-8765delAG occurs within a triplet repeat sequence of AGAGAG, our study further confirmed the existence of a mutational hot-spot at this genomic position (additional genetic factors within each single population might be involved in generating such a mutation).

CASC2a gene is down-regulated in endometrial cancer.

BACKGROUND: Chromosome 10q25-q26 has been strongly correlated to endometrial tumorigenesis. A novel human gene, CASC2, has previously been identified at chromosome 10q26. One out of the three alternative transcripted forms, CASC2a, has been demonstrated to be mutated at a low frequency in endometrial cancer (EC). In this study, the role of the CASC2a gene in cancer has been further defined. MATERIALS AND METHODS: Tumour and corresponding normal tissues were analysed for CASC2a mRNA expression by real-time RT-PCR and mutation status by PCR-based approaches. RESULTS: A significantly decreased level of CASC2a transcripts was observed in 13/17 (76%) EC tissues, as well as in 6/9 (67%) colorectal cancers. Exogenous expression of CASC2a in undifferentiated AN3CA endometrial cancer cells inhibited cellular growth in anchorage-independent growth assays. Finally, infrequent CASC2a mutations were able to impair the gene function. CONCLUSION: Altogether, our findings strongly suggest that CASC2a may act as a tumour suppressor gene, with both epigenetic and genetic alterations concurring to gene inactivation. Down-regulation of CASC2a may provide a growth advantage in EC cells.

Epidemiology and genetic susceptibility of malignant melanoma in North Sardinia, Italy.

The aim of this report was to study the descriptive and genetic epidemiology of malignant melanoma in North Sardinia, Italy, in the period 1992-2011. Epidemiological data were obtained from the local tumor registry, which is part of the Italian Association for Tumor Registries. Among patients included in the North Sardinia tumor registry, 316 patients first evaluated for familial recurrence of melanoma were submitted to mutation analysis in CDKN2A and CDK4 genes. The overall number of cases registered was 532. The male-to-female ratio was 1 : 1 and the mean age was 56 years for men and 55 years for women. The standardized incidence rates were 4.9/100 000 and 4.4/100 000 and the standardized mortality rates were 1.7/100 000 and 1.3/100 000 for men and women, respectively. The relative 5-year survival was 77% for men and 79% for women. In our series, 24/316 (7.6%) patients had a familial occurrence of melanoma (presence of at least one additional family member affected). Among these, one variant (Gly23Asp), reported previously as a low-frequency disease-causing mutation, was detected by mutational screening in the p16 gene only. With the exception of polymorphisms, none of either the sporadic melanoma patients or healthy controls presented a germline mutation in candidate genes. An increase in incidence and a decrease in mortality rates of malignant melanoma were registered in North Sardinia, from 1992 to 2011, whereas survival was similar to that reported in recent international reports. The high-penetrance melanoma susceptibility genes (CDKN2A and CDK4) are not involved in predisposition to melanoma in North Sardinia.

Vitamin D status and risk for malignant cutaneous melanoma: recent advances.

Cutaneous malignant melanoma, whose incidence is increasing steadily worldwide, is the result of complex interactions between individual genetic factors and environmental risk factors. Ultraviolet radiation represents the most important environmental risk factor for the development of skin cancers, including melanoma. Sun exposure and early sunburn during childhood are the principal causes of cutaneous melanoma insurgence in adults, with double the risk relative to a nonexposed population. Consequently, ultraviolet protection has long been recognized as an important measure to prevent such a malignancy. Biological and epidemiological data suggest that vitamin D status could affect the risk of cancer and play a role in cancer prevention by exerting antiproliferative effects. Solar radiations are critical for vitamin D synthesis in humans; however, uncontrolled and intensive sun exposure is dangerous to skin health and may contribute toward the development of cutaneous malignant melanoma. An optimum balance between sun protection and exposure is thus advocated. Additional research is required to confirm the preventive role of vitamin D in melanoma incidence or a positive influence on patient outcome.

Deregulated c-Myc requires a functional HSF1 for experimental and human hepatocarcinogenesis.

Deregulated activity of the c-Myc protooncogene is a frequent molecular event underlying mouse and human hepatocarcinogenesis. Nonetheless, the mechanisms sustaining c-Myc oncogenic activity in liver cancer remain scarcely delineated. Recently, we showed that the mammalian target of rapamycin complex 1 (mTORC1) cascade is induced and necessary for c-Myc dependent liver tumor development and progression. Since the heat shock factor 1 (HSF1) transcription factor is a major positive regulator of mTORC1 in the cell, we investigated the functional interaction between HSF1 and c-Myc using in vitro and in vivo approaches. We found that ablation of HSF1 restrains the growth of c-Myc-derived mouse hepatocellular carcinoma (HCC) cell lines, where it induces downregulation of c-Myc levels. Conversely, silencing of c-Myc gene in human and mouse HCC cells led to downregulation of HSF1 expression. Most importantly, overexpression of a dominant negative form of HSF1 (HSF1dn) in the mouse liver via hydrodynamic gene delivery resulted in the complete inhibition of mouse hepatocarcinogenesis driven by overexpression of c-Myc. Altogether, the present results indicate that a functional HSF1 is necessary for c-Myc-driven hepatocarcinogenesis. Consequently, targeting HSF1 might represent a novel and effective therapeutic strategy for the treatment of HCC subsets with activated c-Myc signaling.

Overexpression of h-prune in breast cancer is correlated with advanced disease status.

PURPOSE: The h-prune gene is involved in cellular motility and metastasis formation in breast cancer through interacting with the nm23-H1 protein. The aim of this study was to better define the clinical and pathologic role of h-prune in breast cancer patients. EXPERIMENTAL DESIGN: Using immunohistochemistry, we assessed h-prune and nm23-H1 protein expression in two series of breast cancer patients: (i) in 2,109 cases with pathologic reports on primary tumors and (ii) in 412 cases with detailed clinical information. To assess the role of DNA amplification in gene activation, the h-prune copy number was evaluated by fluorescence in situ hybridization analysis in 1,016 breast cancer cases. RESULTS: In the patients tested (n = 2,463), 1,340 (54%) had an increased level of h-prune expression; a positive immunostaining for nm23-H1 was observed in 615 of 2,061 (30%) cases. Overexpression of h-prune was associated with multiple gene copy number at chromosome 1q21.3 in a very limited fraction of cases (68 of 1,016; 6.7%), strongly indicating that alternative pathways induce h-prune activation in breast cancer. Multivariate Cox regression analysis showed that neither h-prune overexpression nor decreased nm23-H1 immunostaining is independent prognostic factors. However, a significant association of h-prune overexpression with either advanced lymph node status (P = 0.017) or presence of distant metastases (P = 0.029) was observed. CONCLUSIONS: Although not significantly correlated with overall survival, positive h-prune immunostaining identifies subsets of breast cancer patients with higher tumor aggressiveness. Further investigations using larger collections of advanced breast cancer patients are required for assessing the predictive role of h-prune in breast cancer.

Identification of predictive factors for the occurrence of predisposing MLH1 and MSH2 germline mutations among Sardinian patients with colorectal carcinoma.

Factors predictive of carrying MLH1 and MSH2 germline mutations in patients with colorectal cancer (CRC) are as yet unknown. The aim of this population-based study, was to further define the role of MLH1/MSH2 mutations through an evaluation clinic program with 362 consecutive Sardinian CRC patients. Eight MLH1/MSH2 germline mutations were detected in 21 (6%) patients. Examining family cancer history, MLH1/MSH2 mutations were found in 14/48 (29.2%) probands from CRC families and, among them, in 10/13 (76.9%) families fulfilling the Amsterdam criteria. The patients with low familial recurrence (two CRCs in the family) presented a much lower frequency of MLH1/MSH2 mutations (2/55; 3.6%). Significantly higher rates of MLH1/MSH2 mutations were found in patients with age of onset 45 years (P=0.012) or with 3 affected family members (P=0.009). While no significant predictive value was found for the presence of endometrial cancer within the family, earlier age of diagnosis and/or familial CRC recurrence should be considered as strong predictors for the occurrence of MLH1/MSH2 mutations, and therefore useful in recommending CRC patients for genetic testing.

Analysis of candidate genes through a proteomics-based approach in primary cell lines from malignant melanomas and their metastases.

Proteomics provides a powerful approach for screening alterations in protein expression and post-translational modification associated with particular human diseases. In this study, the analysis of protein expression was focused on malignant melanoma in order to determine the candidate genes involved in tumour progression. The proteomes of cultured melanocytes and of cell lines from primary and metastatic lesions of one malignant melanoma patient were profiled using two-dimensional electrophoresis (2-DE) and mass spectrometry. Differentially expressed proteins were confirmed by 2-DE and mass spectrometry on an additional four malignant melanoma cell lines. Total RNA from the first subset of cell lines was used for quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) of the candidate genes identified after proteomics analysis. A very high similarity was observed in the 2-DE maps of two malignant melanoma cell lines derived from primary and secondary lesions of the same patient. Mass spectrometry identified 37 proteins which were found to be more abundant in tumour cells in comparison with control melanocytes (as confirmed on additional cell lines), with a relatively high prevalence of stress proteins. Eight candidate genes (PRDX2, HSP27, HSP60, HSPA8, HSP9B, STIP1, PDI and P4HB) were further characterized by evaluating their messenger RNA expression levels through real-time RT-PCR analysis. Overexpression of HSP27, HSP60 and HSPA8 and downregulation of PRDX2 were observed in cells from metastatic malignant melanoma in comparison with those from primary melanoma. Although further investigations with larger numbers of paired normal and tumour samples are needed, our findings strongly suggest that the dysregulation of stress pathways may be involved in melanoma progression.

Identification of a novel candidate gene, CASC2, in a region of common allelic loss at chromosome 10q26 in human endometrial cancer.

Allelic deletions, which are suggestive for the presence of tumor suppressor genes, represent a common event in endometrial cancer (EC). Previous loss-of-heterozygosity studies for human chromosome 10q identified a candidate deletion interval at 10q25-q26, which we further narrowed to a 160-kb region at 10q26, bounded by markers D10S1236 and WIAF3299. Using a positional candidate approach, we identified three alternative transcripts of a novel human gene, CASC2 (cancer susceptibility candidate 2; formely C10orf5). One of such transcripts, CASC2a, encodes a short protein of 102 amino acids with no similarity to any other known gene product. Three (7%) CASC2a mutations were identified in tumor DNA from 44 EC patients. While c.-156G>T and c.22C>T (p.Pro8Ser) are sequence variants with unknown functional significance, c.84delA is a mutation with a truncation effect on the predicted protein (p. Asn28fsX50). Expression studies by real-time RT-PCR on several normal and tumor cells revealed that CASC2a mRNA is downregulated in cancer, suggesting that it may act as a potential tumor suppressor gene. The very low mutation rate seems to also indicate that inactivation of CASC2a might probably be due to mechanisms different from genetic alterations.

Mutation analysis of candidate genes in melanoma-prone families: evidence of different pathogenetic mechanisms at chromosome 9P21.

Putative tumour suppressor genes CDKN2A and CDKN2B (on chromosome 9p21) and CDKN2A-interacting cell growth regulatory genes CDK4 and Id-1 have been demonstrated to be involved in the pathogenesis of malignant melanoma (MM). Mutation analysis of these candidate genes was performed in MM families from southern Italy with three or more affected members or two affected members and one or more relative with histologically diagnosed atypical naevus. Two CDKN2A mutations, Arg24Pro and 1-292 G>A, were observed in two (15%) families; except for CDKN2A and Id-1 polymorphisms, no sequence variations were detected in the remaining genes. Screening among 119 sporadic MM cases revealed two additional CDKN2A mutations at very low prevalences. Identification of a large shared haplotype at 9p21 in some MM families negative for CDKN germline mutations suggests that other CDKN-inactivating mechanisms may be responsible for MM predisposition or, alternatively, additional susceptibility gene(s) may be present on chromosome 9p21. Fluorescence in situ hybridization analysis of a subset of MM tissue sections seemed to indicate that the D9S171 locus may be involved in MM pathogenesis.

CDKN2A and MC1R analysis in amelanotic and pigmented melanoma.

Amelanotic melanoma (AM) is a rare subtype of melanoma with little or no clinically visible pigment; it is more difficult to diagnose than pigmented melanoma (PM), and has a worse prognosis. In the attempt to find a genetic explanation for the distinction between AM and PM, we conducted a case-case study, matching AM and PM patients, and testing them for germline mutations in high- (p16INK4A, p14ARF, CDK4) and low-penetrance (MC1R) melanoma susceptibility genes. Similar CDKN2A mutations were found in both sets of melanomas. A p14ARF splice germline mutation was detected for the first time in an Italian family with AM. This rare mutation, which has been described only once previously, may be involved in predisposition to the amelanotic phenotype in combination with germline MC1R variants and coordinate somatic expression of pigmentation genes and their regulators.