A 200-300 kb region of chromosome 3p14.2, including the fragile site locus FRA3B, is homozygously deleted in multiple tumor-derived cell lines. Exon amplification from cosmids covering this deleted region allowed identification of the human FHIT gene, a member of ther histidine triad gene family, which encodes a protein with 69% similarity to an S. pombe enzyme, diadenosine 5', 5''' P1, P4-tetraphosphate asymmetrical hydrolase. The FHIT locus is composed of ten exons distributed over at least 500 kb, with three 5' untranslated exons centromeric to the renal carcinoma-associated 3p14.2 breakpoint, the remaining exons telomeric to this translocation breakpoint, and exon 5 within the homozygously deleted fragile region. Aberrant transcripts of the FHIT locus were found in approximately 50% of esophageal, stomach, and colon carcinomas.

Pancreatic ductal adenocarcinoma (PDA) has a dismal prognosis and insights into both disease etiology and targeted intervention are needed. A total of 109 micro-dissected PDA cases were subjected to whole-exome sequencing. Microdissection enriches tumour cellularity and enhances mutation calling. Here we show that environmental stress and alterations in DNA repair genes associate with distinct mutation spectra. Copy number alterations target multiple tumour suppressive/oncogenic loci; however, amplification of MYC is uniquely associated with poor outcome and adenosquamous subtype. We identify multiple novel mutated genes in PDA, with select genes harbouring prognostic significance. RBM10 mutations associate with longer survival in spite of histological features of aggressive disease. KRAS mutations are observed in >90% of cases, but codon Q61 alleles are selectively associated with improved survival. Oncogenic BRAF mutations are mutually exclusive with KRAS and define sensitivity to vemurafenib in PDA models. High-frequency alterations in Wnt signalling, chromatin remodelling, Hedgehog signalling, DNA repair and cell cycle processes are observed. Together, these data delineate new genetic diversity of PDA and provide insights into prognostic determinants and therapeutic targets.

Increased albuminuria is associated with obesity and diabetes and is a risk factor for cardiovascular and renal disease. However, the link between early albuminuria and adiposity remains unclear. To determine whether adiponectin, an adipocyte-derived hormone, is a communication signal between adipocytes and the kidney, we performed studies in a cohort of patients at high risk for diabetes and kidney disease as well as in adiponectin-knockout (Ad -/-) mice. Albuminuria had a negative correlation with plasma adiponectin in obese patients, and Ad -/-mice exhibited increased albuminuria and fusion of podocyte foot processes. In cultured podocytes, adiponectin administration was associated with increased activity of AMPK, and both adiponectin and AMPK activation reduced podocyte permeability to albumin and podocyte dysfunction, as evidenced by zona occludens-1 translocation to the membrane. These effects seemed to be caused by reduction of oxidative stress, as adiponectin and AMPK activation both reduced protein levels of the NADPH oxidase Nox4 in podocytes. Ad -/-mice treated with adiponectin exhibited normalization of albuminuria, improvement of podocyte foot process effacement, increased glomerular AMPK activation, and reduced urinary and glomerular markers of oxidant stress. These results suggest that adiponectin is a key regulator of albuminuria, likely acting through the AMPK pathway to modulate oxidant stress in podocytes.

Diabetic nephropathy is increasing in incidence and is now the number one cause of end-stage renal disease in the industrialized world. To gain insight into the genetic susceptibility and pathophysiology of diabetic nephropathy, an appropriate mouse model of diabetic nephropathy would be critical. A large number of mouse models of diabetes have been identified and their kidney disease characterized to various degrees. Perhaps the best characterized and most intensively investigated model is the db/db mouse. Because this model appears to exhibit the most consistent and robust increase in albuminuria and mesangial matrix expansion, it has been used as a model of progressive diabetic renal disease. In this review, we present the findings from various studies on the renal pathology of the db/db mouse model of diabetes in the context of human diabetic nephropathy. Furthermore, we discuss shortfalls of assessing functional renal disease in mouse models of diabetic kidney disease.

Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear enzyme that modifies substrates by poly(ADP-ribose)-ylation. PARP-1 has well-described functions in DNA damage repair, and also functions as a context-specific regulator of transcription factors. Using multiple models, data demonstrate that PARP-1 elicits pro-tumorigenic effects in androgen receptor (AR)-positive prostate cancer (PCa) cells, both in the presence and absence of genotoxic insult. Mechanistically, PARP-1 is recruited to sites of AR function, therein promoting AR occupancy and AR function. It was further confirmed in genetically-defined systems that PARP-1 supports AR transcriptional function, and that in models of advanced PCa, PARP-1 enzymatic activity is enhanced, further linking PARP-1 to AR activity and disease progression. In vivo analyses demonstrate that PARP-1 activity is required for AR function in xenograft tumors, as well as tumor cell growth in vivo and generation and maintenance of castration-resistance. Finally, in a novel explant system of primary human tumors, targeting PARP-1 potently suppresses tumor cell proliferation. Collectively, these studies identify novel functions of PARP-1 in promoting disease progression, and ultimately suggest that the dual functions of PARP-1 can be targeted in human PCa to suppress tumor growth and progression to castration-resistance.

The candidate tumor suppressor gene, FHIT, encompasses the common human chromosomal fragile site at 3p14.2, the hereditary renal cancer translocation breakpoint, and cancer cell homozygous deletions. Fhit hydrolyzes dinucleotide 5,5ٟ-P 1 ,P 3 -triphosphate in vitro and mutation of a central histidine abolishes hydrolase activity. To study Fhit function, wild-type and mutant FHIT genes were transfected into cancer cell lines that lacked endogenous Fhit. No consistent effect of exogenous Fhit on growth in culture was observed, but Fhit and hydrolase ''dead'' Fhit mutant proteins suppressed tumorigenicity in nude mice, indicating that 5,5ٟ-P 1 ,P 3 -triphosphate hydrolysis is not required for tumor suppression.The structure and expression of the FHIT gene encompassing the FRA3B common fragile site frequently are altered in primary or cultured esophageal, head and neck, lung, gastric, breast, and cervical carcinomas (1-8). Structural alterations tend to be because of deletion within both FHIT alleles, resulting in loss of exons and concomitant absence of full-length FHIT transcript and protein (ref. 6; for review, ref. 9). It has been argued that the FHIT gene may be altered in cancer cells simply because it encompasses the fragile region and is likely to be very susceptible to breakage (7). We agree that the locus is highly susceptible to carcinogen damage, explaining why deletion is much more frequent than point mutation in the gene, but we argue that loss of Fhit function provides a selective advantage for the tumor cell; otherwise, frequent expansion of the deleted FHIT clones in tumors and tumor-derived cell lines would be difficult to explain.Fhit-related proteins have been found in mammals and yeasts (1, 10, 11) and constitute a branch of the histidine triad (HIT) superfamily (12). The Fhit branch includes the Schizosaccharomyces pombe diadenosine tetraphosphate hydrolase [dinucleoside 5Ј,5ٞ-P 1 ,P 4 -tetraphosphate (Ap 4 A) hydrolase] (10) to which Fhit is similar. Barnes et al. (13) have shown that Fhit behaves in vitro as a typical dinucleoside 5Ј,5ٞ-P 1 ,P 3 -triphosphate (Ap 3 A) hydrolase (EC 3.6.1.29); site-directed mutagenesis of FHIT demonstrated that the conserved histidines are required for full activity, and the central histidine of the triad is essential for Ap 3 A hydrolase activity.To investigate mechanisms for a selective growth advantage of Fhit negative tumors, we have prepared vectors for expression of Fhit in cancer-derived cells and have examined the phenotypes of the Fhit-expressing clones relative to the Fhit negative parental cells. To determine if the in vitro enzymatic activity was associated with a role in tumor suppression, the hydrolase ''dead'' mutant gene, FHITH96N, with the central histidine codon of the HIT changed to an asparagine codon, also was expressed in Fhit negative cancer cells. MATERIALS AND METHODSCells. The MKN74 cell line (kindly provided by Eiichi Tahara, University of Hiroshima, Japan), was derived from a gastric carcinoma (14) and forms tumors rapidly ...

Decreased expression of specific microRNAs (miRNAs) occurs in human tumors, which suggests a function for miRNAs in tumor suppression. Herein, levels of the miR-17-5p/miR-20a miRNA cluster were inversely correlated to cyclin D1 abundance in human breast tumors and cell lines. MiR-17/20 suppressed breast cancer cell proliferation and tumor colony formation by negatively regulating cyclin D1 translation via a conserved 3′ untranslated region miRNA-binding site, thereby inhibiting serum-induced S phase entry. The cell cycle effect of miR-17/20 was abrogated by cyclin D1 siRNA and in cyclin D1–deficient breast cancer cells. Mammary epithelial cell–targeted cyclin D1 expression induced miR-17-5p and miR-20a expression in vivo, and cyclin D1 bound the miR-17/20 cluster promoter regulatory region. In summary, these studies identify a novel cyclin D1/miR-17/20 regulatory feedback loop through which cyclin D1 induces miR-17-5p/miR-20a. In turn, miR-17/20 limits the proliferative function of cyclin D1, thus linking expression of a specific miRNA cluster to the regulation of oncogenesis.

Seven immunocompetent, revaccinated patients with surgically incurable cutaneous melanoma underwent treatment of dermal and/or subcutaneous metastases with twice-weekly intratumoral injections of escalating doses (10 4 Ϫ2 ϫ 10 7 plaque-forming units (PFU)/lesion; 10 4 Ϫ8 ϫ 10 7 PFU/session) of a vaccinia/GM-CSF recombinant virus for 6 weeks. Patients with stable or responding disease were maintained on treatment until tumor resolution or progression. Systemic toxicity was infrequent, dose-related, and limited to mild flu-like symptoms that resolved within 24 hours. Local inflammation, at times with pustule formation, was consistently seen with doses of Ն10 7 PFU/lesion. Chronically treated lesions showed a dense infiltration, with CD4 ϩ and CD8 ϩ lymphocytes, histiocytes, and eosinophils. All seven patients developed an antivaccinia humoral immune response 14 -21 days following revaccination. Despite the presence of these antivaccinia antibodies, the reporter gene was expressed, as judged by the development of anti-␤-galactosidase antibodies in all patients. Passenger cytokine gene function was evidenced by the presence of virally encoded GM-CSF mRNA at injection sites both early (weeks 1 and 5) and late (week 31) in the course of treatment. Eosinophilia at treatment sites indicated that physiologically significant levels of functional cytokine were generated. However, there were no changes in the total number of peripheral white blood cells or in the numbers or percentages of polymorphonuclear leukocytes, monocytes, or eosinophils. GM-CSF was not detected in the sera. The two patients with the largest tumor burdens failed to respond even at treatment sites. Three patients had mixed responses, with regression of treated and untreated dermal metastases and progression of disease elsewhere. One patient had a partial response, with regression of injected and uninjected regional dermal metastases. Residual melanoma was excised, rendering the patient disease free. One patient with only dermal metastases confined to the scalp achieved a complete remission. Sequential administration of escalating doses of a GM-CSF recombinant vaccinia virus is safe, effective at maintaining passenger gene function, and can induce tumor regression.