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Breast cancer is the cancer most often diagnosed in women. MicroRNAs (MIRs) are short RNA molecules that bind mRNA resulting in their downregulation. MIR21 has been shown to be an oncomiR in most cancer types, including breast cancer. Most of the effects of miR-21 have been attributed to hsa-miR-21-5p that is transcribed from the leading strand of MIR21, but hsa-miR-21-3p (miR-21-3p), transcribed from the lagging strand, is much less studied. The aim of the study is to analyze whether expression of miR-21-3p is prognostic for breast cancer. MiR-21-3p association with survival, clinical and pathological characteristics was analyzed in a large breast cancer cohort and validated in three separate cohorts, including TCGA and METABRIC. Analytical tools were also used to infer miR-21-3p function and to identify potential target genes and functional pathways. The results showed that in the exploration cohort, high miR-21-3p levels associated with shorter survival and lymph node positivity. In the three validation cohorts, high miR-21-3p levels associated with pathological characteristics that predict worse prognosis. Specifically, in the largest validation cohort, METABRIC (n = 1174), high miR-21-3p levels associated with large tumors, a high grade, lymph node and HER2 positivity, and shorter breast-cancer-specific survival (HR = 1.38, CI 1.13–1.68). This association remained significant after adjusting for confounding factors. The genes with expression levels that correlated with miR-21-3p were enriched in particular pathways, including the epithelial-to-mesenchymal transition and proliferation. Among the most significantly downregulated targets were MAT2A and the tumor suppressive genes STARD13 and ZNF132. The results from this study emphasize that both 3p- and 5p-arms from a MIR warrant independent study. The data show that miR-21-3p overexpression in breast tumors is a marker of worse breast cancer progression and it affects genes in pathways that drive breast cancer by down-regulating tumor suppressor genes. The results suggest miR-21-3p as a potential biomarker.

Mutations in BRCA1 result in predisposal to breast and ovarian cancers, but many variants exist with unknown clinical significance (VUS). One is BRCA1 c.4096+3A>G, which affects production of the full-length BRCA1 transcript, while augmenting transcripts lacking most or all of exon 11. Nonetheless, homozygosity of this variant has been reported in a healthy woman. We saw this variant cosegregate with breast and ovarian cancer in several family branches of four Icelandic pedigrees, with instances of phenocopies and a homozygous woman with lung cancer. We found eight heterozygous carriers (0.44%) in 1820 unselected breast cancer cases, and three (0.15%) in 1968 controls (p = 0.13). Seeking conclusive evidence, we studied tumors from carriers in the pedigrees for wild-type-loss of heterozygosity (wtLOH) and BRCA1-characteristic prevalence of estrogen receptor (ER) negativity. Of 15 breast and six ovarian tumors, wtLOH occurred in nine breast and all six ovarian tumours, and six of the nine breast tumors with wtLOH were ER-negative. These data accord with a pathogenic BRCA1-mutation. Our findings add to the current knowledge of BRCA1, and the role of its exon 11 in cancer pathogenicity, and will be of use in clinical genetic counselling.

BackgroundFusion genes result from genomic structural changes, which can lead to alterations in gene expression that supports tumor development. The aim of the study was to use fusion genes as a tool to identify new breast cancer (BC) genes with a role in BC progression.MethodsFusion genes from breast tumors and BC cell lines were collected from publications. RNA-Seq data from tumors and cell lines were retrieved from databanks and analyzed for fusions with SOAPfuse or the analysis was purchased. Fusion genes identified in both tumors (n = 1724) and cell lines (n = 45) were confirmed by qRT-PCR and sequencing. Their individual genes were ranked by selection criteria that included correlation of their mRNA level with copy number. The expression of the top ranked gene was measured by qRT-PCR in normal tissue and in breast tumors from an exploratory cohort (n = 141) and a validation cohort (n = 277). Expression levels were correlated with clinical and pathological factors as well as the patients' survival. The results were followed up in BC cohorts from TCGA (n = 818) and METABRIC (n = 2509).ResultsVacuole membrane protein 1 (VMP1) was the most promising candidate based on specific selection criteria. Its expression was higher in breast tumor tissue than normal tissue (p = 1x10-4), and its expression was significantly higher in HER2 positive than HER2 negative breast tumors in all four cohorts analyzed. High expression of VMP1 associated with breast cancer specific survival (BCSS) in cohort 1 (hazard ratio (HR) = 2.31, CI 1.27-4.18) and METABRIC (HR = 1.26, CI 1.02-1.57), and also after adjusting for HER2 expression in cohort 1 (HR = 2.03, CI 1.10-3.72). BCSS was not significant in cohort 2 or TCGA cohort, which may be due to differences in treatment regimens.ConclusionsThe results suggest that high VMP1 expression is a potential marker of poor prognosis in HER2 positive BC. Further studies are needed to elucidate how VMP1 could affect pathways supportive of tumorigenesis.

Structural similarity search of commercially available analogues of thieno[2,3- b ]pyridine and 1 H -pyrazole derivatives, known anticancer agents, resulted in 717 hits. These were docked into the phosphoinositide specific-phospholipase C (PLC) binding pocket, the putative target of the compounds, to further focus the selection. Thirteen derivatives of the thieno[2,3- b ]pyridines were identified and tested against the NCI60 panel of human tumour cell lines. The most active derivative 1 was most potent against the MDA-MB-435 melanoma cell line with GI 50 at 30 nM. Also, it was found that a piperidine moiety is tolerated on the thieno[2,3- b ]pyridine scaffold with GI 50 = 296 nM (MDA-MB-435) for derivative 10 considerably expanding the structure activity relationship for the series. For the 1 H -pyrazoles four derivatives were identified using the in silico approach and additionally ten were synthesised with various substituents on the phenyl moiety to extend the structural activity relationship but only modest anticancer activity was found.

MIR21 is a well known oncomir in many cancer types including breast cancer. Most studies that have described the oncogenic properties of MIR21 have focused on the leading strand miR-21-5p. Fewer studies have focused on the passenger strand miR-21-3p, but in a few types of cancer it has been shown to have oncogenic properties. A handful of studies suggest that miR-21-3p could be a biomarker in breast cancer. MIR21 is located at 17q23.1, a region which is often amplified in breast tumors. It overlaps the 3´ end of VMP1 but it has its own distinct promoter. The aim of this study was to analyze whether miR-21-3p associated with prognostic factors in breast cancer. The levels of miR-21-5p and miR-21-3p were measured by qPCR in 144 breast tumors from a cohort of breast cancer patients (cohort 1). The association of expression levels with clinical and pathological factors was analyzed by Student´s t-test and Anova, and with survival by Kaplan-Meier, the log-rank test, and the Cox proportional hazards test. To confirm the results, available data were used from breast cancer cohorts from The Cancer Genome Atlas (TCGA) (n=256) and METABRIC (n=1286). The mean expression levels of miR-21-5p were significantly higher than those of miR-21-3p in breast tumors from cohort 1 (p=2.2e−10). However, only miR-21-3p was more highly expressed in tumors than normal breast tissue (p=0.003), and in all three cohorts there was a strong positive correlation between MIR21 copy number and miR-21-3p expression levels. There was a significant higher expression of miR-21-3p in HER2 positive tumors compared to HER2 negative tumors in the two larger cohorts (p < 0.05), and high miR-21-3p levels associated with higher histograde in the largest cohort (9.2e−14). A shorter breast cancer specific survival was associated with higher miR-21-3p expression in the METABRIC cohort (HR 1.338, CI 1.104-1.622, p=0.003). HER2 was the largest confounding factor but even after adjustment for HER2 positivity the effect remained significant (HR 1.260, CI 1.036-1.532, p = 0.021). Our data show that miR-21-3p is expressed in breast tissue and tumors. Furthermore, our preliminary data suggest that miR-21-3p may be an indicator of worse outcome for breast cancer patients. However, additional work is needed to elucidate the association of miR-21-3p expression with distinct prognostic factors in breast cancer. Citation Format: Arsalan Amirfallah, Adalgeir Arason, Bjarni A. Agnarsson, Oskar Th Johannsson, Bylgja Hilmarsdottir, Rosa Bjork Barkardottir, Inga Reynisdottir. miR-21-3p: A potential oncomir in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3709.

Publisher's version (útgefin grein)., Mutations in BRCA1 result in predisposal to breast and ovarian cancers, but many variants exist with unknown clinical significance (VUS). One is BRCA1 c.4096+3A>G, which affects production of the full-length BRCA1 transcript, while augmenting transcripts lacking most or all of exon 11. Nonetheless, homozygosity of this variant has been reported in a healthy woman. We saw this variant cosegregate with breast and ovarian cancer in several family branches of four Icelandic pedigrees, with instances of phenocopies and a homozygous woman with lung cancer. We found eight heterozygous carriers (0.44%) in 1820 unselected breast cancer cases, and three (0.15%) in 1968 controls (p = 0.13). Seeking conclusive evidence, we studied tumors from carriers in the pedigrees for wild-type-loss of heterozygosity (wtLOH) and BRCA1-characteristic prevalence of estrogen receptor (ER) negativity. Of 15 breast and six ovarian tumors, wtLOH occurred in nine breast and all six ovarian tumours, and six of the nine breast tumors with wtLOH were ER-negative. These data accord with a pathogenic BRCA1-mutation. Our findings add to the current knowledge of BRCA1, and the role of its exon 11 in cancer pathogenicity, and will be of use in clinical genetic counselling., This research was funded by the Landspitali University Hospital Research Fund, grant A-2019-001, and by the Icelandic association "Walking for Breast Cancer Research" (Göngum saman).

Amplification of 8p12-p11 is relatively common in breast cancer and several genes within the region have been suggested to affect breast tumor progression. The aim of the study was to map the amplified 8p12-p11 region in a large set of breast tumors in an effort to identify the genetic driver and to explore its impact on tumor progression and prognosis. Copy number alterations (CNAs) were mapped in 359 tumors, and gene expression data from 577 tumors (359 tumors included) were correlated with CNA, clinical–pathological factors, and protein expression (39 tumors). 8p12-p11 was amplified in 11.4% of tumors. The smallest region of amplification harbored one full-length gene, ZNF703. ZNF703 mRNA expression was significantly higher in estrogen receptor (ER)-positive than ER-negative tumors (P = 2 × 10−16), a reflection of high expression in luminal tumors. Forty-eight percent of tumors with ZNF703 amplification were luminal B tumors in which the best correlation between DNA copy number and mRNA was seen (P = 1.2 × 10−7) as well as correlation between mRNA and protein expression (P = 0.02). High ZNF703 mRNA correlated with poor survival in patients with ER-positive luminal B tumors (log rank P = 0.04). Furthermore, high ZNF703 mRNA expression correlated with poor outcome in patients with ZNF703 copy number neutral, ER-positive, luminal B tumors (log rank P = 0.004). The results support ZNF703 as the driver gene of the 8p12 amplification and suggest that independent of amplification, high expression of the gene affects prognosis in luminal B tumors. Our mapping of 8p12-p11 and analyses of ZNF703 mRNA and protein expression in breast tumors support ZNF703 as an oncogene in luminal B tumors. High ZNF703 expression, independent of the amplification, correlated with worse prognosis for the breast cancer patients with ER-positive luminal tumors, particularly of the luminal B subtype.

Introduction miR-21 is an oncomir that is overexpressed in many types of solid tumours. According to some studies miR-21 expression is higher in breast tumours than normal breast tissue and it is significantly correlated with shorter survival of patients. Many studies have focused on miR-21–5 p and its correlation with clinic pathological features and survival of breast tumours. In some of the studies miR-21–5 p expression has been shown to be associated with ER status, PR status, HER2 status, clinical stage, lymph node, tumour grade and size. The purpose of this study was to compare the expression of miR-21–3 p and miR-21–5 p in progression of breast tumours and their association with corresponding clinic pathological data from the breast cancer patients. Material and methods Six non-neoplastic breast tissues and 144 tumours, from breast cancer patients diagnosed 1987–2003, were obtained from the Department of Pathology at Landspitali University Hospital. The expression levels of miR-21–3 p and miR-21–5 p were measured with StepOnePlus Real-Time PCR System by applying EXIQON primer sets and SYBR Green master mix using miR-16–5 p as endogenous control. Results and discussions miR-21–5 p was expressed at significantly higher levels than miR-21–3 p in both normal (p=0.0003) and tumour tissues (p=6.378e-12). A significant difference was not observed between miR-21–5 p expression in normal breast tissue and tumours. High miR-21–5 p expression correlated significantly with HER2 expression (p=0.001) but it did not correlate with other clinical and pathological factors or survival. miR-21–3 p was expressed at significantly higher levels in tumour tissues than normal breast tissue (p=0.003). Its expression was significantly higher in tumours from patients with metastasis than without metastasis (p=0.02). Furthermore, higher levels of miR-21–3 p correlated with shorter disease free survival (p=0.03, HR=1.72, 95%CI=1.03–2.9). Conclusion Although miR-21 is one of the most studied and upregulated miRNAs in human solid tumours the comparison of miR-21–3 p expression with miR-21–5 p expression in breast tumours has not been well studied. Our results suggest that miR-21–3 p could be a better indicator of breast cancer progression than miR-21–5 p. More studies in larger breast cohorts and functional cell based assays will be needed to shed light on the potential role of miR-21–3 p in breast tumours progression.

Common human diseases result from the interplay of many genes and environmental factors. Therefore, a more integrative biology approach is needed to unravel the complexity and causes of such diseases. To elucidate the complexity of common human diseases such as obesity, we have analysed the expression of 23,720 transcripts in large population-based blood and adipose tissue cohorts comprehensively assessed for various phenotypes, including traits related to clinical obesity. In contrast to the blood expression profiles, we observed a marked correlation between gene expression in adipose tissue and obesity-related traits. Genome-wide linkage and association mapping revealed a highly significant genetic component to gene expression traits, including a strong genetic effect of proximal (cis) signals, with 50% of the cis signals overlapping between the two tissues profiled. Here we demonstrate an extensive transcriptional network constructed from the human adipose data that exhibits significant overlap with similar network modules constructed from mouse adipose data. A core network module in humans and mice was identified that is enriched for genes involved in the inflammatory and immune response and has been found to be causally associated to obesity-related traits.

We recently described an association between risk of type 2diabetes and variants in the transcription factor 7-like 2 gene (TCF7L2; formerly TCF4), with a population attributable risk (PAR) of 17%-28% in three populations of European ancestry. Here, we refine the definition of the TCF7L2 type 2diabetes risk variant, HapB(T2D), to the ancestral T allele of a SNP, rs7903146, through replication in West African and Danish type 2 diabetes case-control studies and an expanded Icelandic study. We also identify another variant of the same gene, HapA, that shows evidence of positive selection in East Asian, European and West African populations. Notably, HapA shows a suggestive association with body mass index and altered concentrations of the hunger-satiety hormones ghrelin and leptin in males, indicating that the selective advantage of HapA may have been mediated through effects on energy metabolism.

We conducted a genome-wide association study for type 2 diabetes (T2D) in Icelandic cases and controls, and we found that a previously described variant in the transcription factor 7-like 2 gene (TCF7L2) gene conferred the most significant risk. In addition to confirming two recently identified risk variants(1), we identified a variant in the CDKAL1 gene that was associated with T2D in individuals of European ancestry (allele-specific odds ratio (OR) = 1.20 (95% confidence interval, 1.13 - 1.27), P = 7.7 x 10(-9)) and individuals from Hong Kong of Han Chinese ancestry (OR = 1.25 (1.11 - 1.40), P = 0.00018). The genotype OR of this variant suggested that the effect was substantially stronger in homozygous carriers than in heterozygous carriers. The ORs for homozygotes were 1.50 (1.31 - 1.72) and 1.55 (1.23 - 1.95) in the European and Hong Kong groups, respectively. The insulin response for homozygotes was approximately 20% lower than for heterozygotes or noncarriers, suggesting that this variant confers risk of T2D through reduced insulin secretion.

Background & Aims: Both genetic and environmental factors play a role in the development of Crohn's disease (CD) and ulcerative colitis (UC), collectively known as inflammatory bowel disease (IBD). The aim of this study was to estimate the genetic component in IBD in Iceland. Methods: A population-based sample, representing everyone diagnosed with IBD in Iceland from 1950 to 1996, was studied using a computerized population-wide genealogic database. The relationships among the patients were analyzed by calculating the kinship coefficient and the relative risk. Results: The kinship coefficients for the patients were significantly greater than the mean kinship coefficient for the controls ( P −6 ). The risk ratio for siblings of IBD, UC, and CD patients was 5.0 ( P P P = 0.033), respectively. The cross-risk ratio for siblings of UC patients developing CD (or vice versa) was 2.6 ( P = 0.015). Conclusions: The results indicate that the IBD patients are more closely related than the controls, which strongly supports the involvement of a genetic component in the development of IBD in Icelandic patients. We find that the increase in risk for relatives of UC probands to develop UC, or relatives of CD probands to develop CD, is greater than the increase in risk for relatives of UC probands to develop CD, or relatives of CD probands to develop UC.

We report a genomewide linkage study of type 2 diabetes (T2D [MIM 125853]) in the Icelandic population. A list of type 2 diabetics was cross-matched with a computerized genealogical database clustering 763 type 2 diabetics into 227 families. The diabetic patients and their relatives were genotyped with 906 microsatellite markers. A nonparametric multipoint linkage analysis yielded linkage to 5q34-q35.2 (LOD = 2.90, P=1.29 x 10(-4)) in all diabetics. Since obesity, here defined as body mass index (BMI)or =30 kg/m(2), is a key risk factor for the development of T2D, we studied the data either independently of BMI or by stratifying the patient group as obese (BMIor =30) or nonobese (BMI30). A nonparametric multipoint linkage analysis yielded linkage to 5q34-q35.2 (LOD = 3.64, P=2.12 x (10)-5) in the nonobese diabetics. No linkage was observed in this region for the obese diabetics. Linkage analysis conditioning on maternal transmission to the nonobese diabetics resulted in a LOD score of 3.48 (P=3.12 x 10(-5)) in the same region, whereas conditioning on paternal transmission led to a substantial drop in the LOD score. Finally, we observed potential interactions between the 5q locus and two T2D susceptibility loci, previously mapped in other populations.

One approach to unraveling the genetics of complex inherited disease, such as Crohn's disease, is to search for subclinical disease markers among unaffected family members. We assessed the possible presence, prevalence, and inheritance pattern of subclinical intestinal inflammation in apparently healthy relatives of patients with Crohn's disease.A total of 49 patients with Crohn's disease, 16 spouses, and 151 (58%) of 260 available first-degree relatives underwent a test for intestinal inflammation (fecal calprotectin concentration). The mode of inheritance was assessed from 36 index patients (by variance component analysis) when more than 50% of relatives were studied.Fecal calprotectin concentrations in patients with Crohn's disease (47 mg/L; confidence interval [CI], 27-95 mg/L) and relatives (11 mg/L; CI, 9-14 mg/L) differed significantly (P0.0001) from controls (4 mg/L; CI, 3-5 mg/L), whereas that of the spouses did not (4 mg/L; CI, 3-6 mg/L; P0.5). Fecal calprotectin concentration was increased in 49% of all relatives studied. The increased fecal calprotectin concentration among the relatives of the 36 index patients had an inheritance pattern that was most consistent with an additive inheritance pattern.There is a high prevalence of subclinical intestinal inflammation in first-degree relatives of patients with Crohn's disease that conforms best to an additive inheritance pattern. The genetic basis for this abnormality may represent a risk factor for Crohn's disease.

The phosphoinositide specific-phospholipase C–γ (PLC-γ1 and 2) enzymes are plausible anticancer targets implicated in cell motility important to invasion and dissemination of tumour cells. A host of known PLC-γ2 inhibitors were tested against the NCI60 panel of human tumour cell lines as well as their commercially available structural derivatives. A class of thieno[2,3- b ]pyridines showed excellent growth arrest with derivative 3 giving GI 50 = 58 nM for the melanoma MDA-MB-435 cell line. The PLC-γ2 is uniquely expressed in haematopoietic cells and the leukaemia tumour cell lines were growth restricted on average GI 50 = 275 nM by derivative 3 indicating a specific interaction with this isoform. Furthermore, a moderate growth inhibition was found for compound classes of indoles and 1 H -pyrazoles. It is likely that the active compounds do not only inhibit the PLC-γ2 isoform but other PLCs as well due to their conserved binding site. The compounds tested were identified by applying the tools of chemoinformatics, which supports the use of in silico methods in drug design.

ATP induces structural alterations in SV40 large T antigen and promotes changes in its interaction with the viral replication origin. We have analyzed nucleotide-induced changes in T antigen structure in the absence of origin DNA. Most preparations of immunopurified T antigen contain several discrete species ranging in size from monomers through oligomers larger than hexamers. The predominant species consist of monomers and dimers. Incubation of T antigen with ATP or dATP leads to a dramatic and rapid increase in the appearance of T antigen hexamers. Weakly and nonhydrolyzable analogs of ATP are effective as well, indicating that hexamer formation does not require active ATP hydrolysis. After incubation of T antigen with [gamma-35S]ATP, stable association of the labeled nucleotide with all detectable forms occurs. Removal of greater than 80% of the T antigen phosphate residues does not significantly affect the formation of T antigen hexamers, although changes in the distribution and mobility of the other species of T antigen are apparent. Furthermore, T antigen synthesized in and purified from Escherichia coli and, therefore, presumably un- or underphosphorylated, is capable of forming hexamers. Nucleotide-induced T antigen hexamer formation thus appears to require neither protein phosphorylation nor active ATP hydrolysis.

The mutation in the temperature-sensitive tsA58 mutant T antigen (Ala-438----Val) lies within the presumptive ATP-binding fold. We have constructed a recombinant baculovirus that expresses large quantities of the tsA58 T antigen in infected insect cells. The mutant T antigen mediated simian virus 40 origin-containing DNA (ori-DNA) synthesis in vitro to nearly the same extent as similar quantities of wild-type T antigen at 33 degrees C. However, if wild-type and tsA58 T antigens were heated at 41 degrees C in replication extracts prior to addition of template DNA, the tsA58 T antigen but not the wild type was completely inactivated. The mutant protein displayed greater thermosensitivity for many of the DNA replication activities of T antigen than did the wild-type protein. Some of the replication functions of tsA58 T antigen were differentially affected depending on the presence or absence of ATP during the preheating period. When tsA58 T antigen was preheated in the presence of ATP at 41 degrees C for a time sufficient to completely inactivate its ability to replicate ori-DNA in vitro, it displayed substantial ATPase and normal DNA helicase activities. Conversely, when preheated in the absence of nucleotide, it completely lost both ATPase and helicase activities. Preheating tsA58 T antigen, even in the presence of ATP, led to drastic reductions in its ability to bind to and unwind DNA containing the replication origin. The mutant T antigen also displayed thermosensitivity for binding to and unwinding nonspecific double-stranded DNA in the presence of ATP. Our results suggest that the interactions of T antigen with ATP that are involved in T-antigen DNA binding and DNA helicase activities are different. Moreover, we conclude, consistent with its phenotype in vivo, that the tsA58 T antigen is defective in the initiation but not in the putative elongation functions of T antigen in vitro.

In our genome wide association study on type 2 diabetes (T2D), a variant we had previously identified in TCF7L2 stood out as the most significant finding. In addition, a variant of the CDKAL1 gene was found to be associated with increased risk of T2D in a nearly recessive manner, with genotype odds ratio for the homozygous carrier 1.45 and 1.55 for individuals of European and Asian ancestry respectively. The function of the CDKAL1 gene product is unknown but it is similar to another protein, CDK5RAP1, an inhibitor of the CDK5/p35 complex in neuronal tissue. This complex is also expressed in pancreatic beta cells and, in the presence of its active form, insulin expression is decreased under glucotoxic conditions. This led to the suggestion that CDKAL1 may be an inhibitor of the CDK5/p35 complex in beta cells, similar to the role of CDK5RAP1 in neuronal tissue. Furthermore, we have shown that the risk variant of CDKAL1 is associated with reduced insulin secretion and this effect is mostly seen for the homozygote where a 24% reduction in insulin response is observed compared to the heterozygous carriers or non-carriers. This is in line with the nearly recessive mode of inheritance observed for this variant with respect to disease risk. The aim of this study was to gain further insight into the functional role of CDKAL1 using the rat pancreatic beta cell line INS-1. The rat pancreatic beta cell line INS-1 was cultured in the presence of variable glucose concentration, ranging from 2.5–30 mM, to evaluate whether CDKAL1 expression is regulated by glucose concentration. We demonstrated that the expression of CDKAL1 in rat INS-1 cells varied according to glucose concentration in the culture medium with reduced expression detected under glucotoxic conditions compared to normal glucose concentration. This indicates that CDKAL1 expression in pancreatic beta cells is sensitive to glucose concentration. It is possible that this response to glucose may be affected in individuals carrying the variant of CDKAL1 that is associated to T2D. This could explain the reduced insulin secretion observed for these individuals in response to an oral glucose tolerance test.

We have previously reported suggestive linkage of type 2 diabetes mellitus to chromosome 10q. We genotyped 228 microsatellite markers in Icelandic individuals with type 2 diabetes and controls throughout a 10.5-Mb interval on 10q. A microsatellite, DG10S478, within intron 3 of the transcription factor 7-like 2 gene (TCF7L2; formerly TCF4) was associated with type 2 diabetes (P = 2.1 x 10(-9)). This was replicated in a Danish cohort (P = 4.8 x 10(-3)) and in a US cohort (P = 3.3 x 10(-9)). Compared with non-carriers, heterozygous and homozygous carriers of the at-risk alleles (38% and 7% of the population, respectively) have relative risks of 1.45 and 2.41. This corresponds to a population attributable risk of 21%. The TCF7L2 gene product is a high mobility group box-containing transcription factor previously implicated in blood glucose homeostasis. It is thought to act through regulation of proglucagon gene expression in enteroendocrine cells via the Wnt signaling pathway.

Background & Aims: It has been suggested that subclinical intestinal inflammation plays a pathogenic role in the spondylarthropathy of ankylosing spondylitis (AS). We assessed the possible presence and inheritance pattern of subclinical intestinal inflammation in first-degree relatives of patients with AS. The relationship between this inflammation and the subjects' HLA-B27 genotype as well as computerized tomographic sacroiliac abnormalities was also assessed. Methods: A total of 124 of 213 (58%) available first-degree relatives of 47 patients with AS in Iceland underwent investigation for intestinal inflammation (fecal calprotectin concentration), HLA-B27 genotyping, and computerized tomography of the sacroiliac joints. Results: A total of 41% of the first-degree relatives had subclinical intestinal inflammation, whereas 15 of 17 spouses were normal. Variance components analyses suggest that the inheritance pattern of this inflammation is affected by a major additive gene. Some sacroiliac changes, suggestive of early AS, differed significantly between subjects with and without subclinical intestinal inflammation (mean diameter of subchondral cysts [2.9 vs. 1.2 mm; P = 0.026] and blurring of joint margins [9 of 44 (20%) vs. 1 of 41 (2%); P = 0.02]). Intestinal inflammation and sacroiliac changes did not relate to the subjects' HLA-B27 status. Conclusions: Many first-degree relatives of patients with AS appear to have an inherited abnormality that leads to subclinical intestinal inflammation. The association between the presence of this inflammation and the sacroiliac changes suggests that it may play a pathogenic role in the spondylarthropathy of AS.

The retinoblastoma tumor suppressor protein (pRb) can associate with the transforming proteins of several DNA tumor viruses, including the large T antigen encoded by polyomavirus (Py T Ag). Although pRb function is critical for regulating progression from G 1 to S phase, a role for pRb in S phase has not been demonstrated or excluded. To identify a potential effect of pRb on DNA replication, pRb protein was added to reaction mixtures containing Py T Ag, Py origin-containing DNA (Py ori-DNA), and murine FM3A cell extracts. We found that pRb strongly represses Py ori-DNA replication in vitro. Unexpectedly, however, this inhibition only partially depends on the interaction of pRb with Py T Ag, since a mutant Py T Ag ( dl 141) lacking the pRb interaction region was also significantly inhibited by pRb. This result suggests that pRb interferes with or alters one or more components of the murine cell replication extract. Furthermore, the ability of Py T Ag to be phosphorylated in such extracts is markedly reduced in the presence of pRb. Since cyclin-dependent kinase (CDK) phosphorylation of Py T Ag is required for its replication function, we hypothesize that pRb interferes with this phosphorylation event. Indeed, the S-phase CDK complex (cyclin A-CDK2), which phosphorylates both pRb and Py T Ag, alleviates inhibition caused by pRb. Moreover, hyperphosphorylated pRb is incapable of inhibiting replication of Py ori-DNA in vitro. We propose a new requirement for maintaining pRb phosphorylation in S phase, namely, to prevent deleterious effects on the cellular replication machinery.

In dividing cells, p27(Kip1) is predominantly bound to cyclin D-cdk4 without inhibiting this kinase. Upon being induced by TGF-beta or with a conditional expression system in lung epithelial cells, p15(Ink4b) binds to and inhibits the cyclin D-dependent kinases, prevents p27 binding to these cdk complexes, and promotes p27 binding and inhibition of cyclin-cdk2. In vitro, however, p15 prevents p27 binding only if it has access to cyclin D-cdk4 first. We present evidence that the different subcellular location of p15 and p27 ensures the prior access of p15 to cdk4. In the cell, p15 is localized mostly in the cytoplasm, whereas p27 is nuclear. p15 prevails over p27 or a p27 construct consisting of the cdk inhibitory domain tagged with a nuclear localization signal. However, when p15 and p27 are forced to reside in the same subcellular location, either the cytoplasm or the nucleus, p15 no longer prevents p27 from binding to cdk4. These properties allow p15 and p27 to coordinately inhibit cdk4 and cdk2.

Breast cancer is the most common cancer in women. Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) that increase the risk of breast cancer. The minor allele of SNP rs3803662 has been shown to correlate with increased breast cancer risk in women of European descent as well as with lower expression of TOX3. The SNP is located on chromosome 16q12.1 approximately 4 kb upstream of the gene. The aim of the study was to examine the association of the risk allele with the expression of TOX3 as well as the correlation of the mRNA levels and genotype with clinical and pathological characteristics. The SNP was genotyped in normal DNA from 160 breast cancer patients. The mRNA levels were measured in breast tumors by gene expression microarrays and confirmed by quantitative real-time (qRT)-PCR. The clinical and tumor characteristics were retrieved from hospital records and their association with the level of mRNA and the SNP was analyzed. Statistical and survival analyses were performed in R. The association of the risk allele of rs3803662 with lower TOX3 expression was confirmed in estrogen receptor (ER) positive tumors. It was more often observed in lobular than ductal tumors (p = 0.04), and carriers of the risk allele who had been diagnosed with luminal A tumors had shorter overall survival than carriers of the non-risk allele (p = 0.009). Correlation analysis with tumor histopathology showed that low TOX3 mRNA levels correlated with high Ki67 levels (p = 0.026) and tumors of the basal subtype (p < 0.001), whereas high expression correlated with ER (p = 0.004) and progesterone receptor (p = 0.005) positivity. In addition, high TOX3 mRNA levels correlated with positive lymph nodes at time of diagnosis (p < 0.001). Patients with ER positive tumors and high levels of TOX3 mRNA had shorter overall- and distant metastasis free-survival (p = 0.017 and p = 0.021, respectively), an effect that was mostly attributable to patients with tumors of the luminal B subtype. In conclusion, the results suggest that the risk allele of rs3803662 has the strongest effect in patients with tumors of the luminal A subtype. They also imply that the expression levels of TOX3 affect the progression of breast cancer but the effect may depend on the subtype. Citation Format: Eydis Th. Gudmundsdottir, Rosa B. Barkardottir, Adalgeir Arason, Haukur Gunnarsson, Laufey Th. Amundadottir, Bjarni A. Agnarsson, Oskar Th. Johannsson, Inga Reynisdottir. Expression levels of TOX3 and the risk allele of the associated SNP rs3803662 predict adverse outcome for breast cancer patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3461. doi:10.1158/1538-7445.AM2013-3461

G1 progression in mammalian cells requires the activity of the cyclin D-dependent kinases Cdk4 and/or Cdk6 and the cyclin E-dependent kinase Cdk2. Proliferating Mv1Lu mink lung epithelial cells and human keratinocytes contain high levels of the universal Cdk inhibitor p27Kip1 distributed in complexes with Cdk2, Cdk4, and Cdk6. Addition of the antimitogenic cytokine transforming growth factor-beta (TGF-beta) elevates expression of the Cdk4/6-specific inhibitor p15Ink4B and induces the release of p27 from Cdk4 and Cdk6. In Mv1Lu cells, this release of p27 coincides with increased binding of p27 to Cdk2. Recombinant p15 inhibits p27 binding to Cdk4 in vitro, and p15 overexpression induces the transfer of p27 from Cdk4 to Cdk2 in vivo, suggesting that the release of Cdk4-bound p27 in TGF-beta-treated cells is caused by the surge in p15 levels. In keratinocytes, TGF-beta increases not only p15 but also p21Cip1, which binds to Cdk2. These events correlate with Cdk2 inhibition and cell cycle arrest and occur without a loss of G1 Cdk components. The results suggest that TGF-beta induces G1 arrest in these two epithelial cell types by inhibiting various cyclin-Cdk kinases through the cooperative action of an Ink4 Cdk inhibitor and a Cip/Kip Cdk inhibitor. Subsequent to cell cycle arrest, Cdk2 and Cdk4 levels decline as part of a second set of events that may represent a program of cell adaptation to the quiescent state.

Progression through the cell cycle is catalyzed by cyclin-dependent kinases (CDKs) and is negatively controlled by CDK inhibitors (CDIs). We have isolated a new member of the p21CIP1/p27KIP1 CDI family and named it p57KIP2 to denote its apparent molecular mass and higher similarity to p27KIP1. Three distinct p57 cDNAs were cloned that differ at the start of their open reading frames and correspond to messages generated by the use of distinct splice acceptor sites. p57 is distinguished from p21 and p27 by its unique domain structure. Four distinct domains follow the heterogeneous amino-terminal region and include, in order, a p21/p27-related CDK inhibitory domain, a proline-rich (28% proline) domain, an acidic (36% glutamic or aspartic acid) domain, and a carboxy-terminal nuclear targeting domain that contains a putative CDK phosphorylation site and has sequence similarity to p27 but not to p21. Most of the acidic domain consists of a novel, tandemly repeated 4-amino acid motif. p57 is a potent inhibitor of G1- and S-phase CDKs (cyclin E-cdk2, cyclin D2-cdk4, and cyclin A-cdk2) and, to lesser extent, of the mitotic cyclin B-Cdc2. In mammalian cells, p57 localizes to the nucleus, associates with G1 CDK components, and its overexpression causes a complete cell cycle arrest in G1 phase. In contrast to the widespread expression of p21 and p27 in human tissues, p57 is expressed in a tissue-specific manner, as a 1.5-kb species in placenta and at lower levels in various other tissues and a 7-kb mRNA species observed in skeletal muscle and heart. The expression pattern and unique domain structure of p57 suggest that this CDI may play a specialized role in cell cycle control.

The C11A mutant of SV40 large T antigen is unable to support the replication of viral origin containing DNA (ori-DNA) in vivo or in vitro . The mutation within C11A at residue 522 (pro → ser) is located within the presumptive ATPase region of T antigen. While C11A T antigen was previously reported to be defective in ATPase and DNA helicase activities, it was shown to be capable of binding specifically to DNA containing the viral replication origin. As the positions of many conditional mutations of SV40 T antigen are located within the ATPase domain we asked whether C11A might also exhibit temperature-sensitive defects. We found that several activities of C11A T antigen are conditionally defective. C11A T antigen was able to hydrolyze ATP, assemble into hexamers, and display ATP-dependent alterations in DNA binding and ori-DNA structure at 33° but not 41°. Wild-type T antigen did not exhibit temperature-sensitive defects in these activities. C11A T antigen was completely unable to unwind ori-DNA at either temperature. This defect in unwinding was trans-dominant; C11A T antigen inhibited ori-DNA unwinding by wild-type T antigen. These data show that a mutant displaying a nonconditional defective phenotype may contain a subset of relevant properties that are temperature sensitive.

We have characterized the simian virus 40 (SV40) origin-containing DNA (ori-DNA) replication functions of two SV40 conditional mutant T antigens: tsA438 A-V (tsA58) and tsA357 R-K (tsA30). Both tsA mutant T antigens, immunopurified from recombinant baculovirus-infected insect cells, mediated replication of SV40 ori-DNA in vitro to similar extents as did wild-type T antigen in reactions at 33 degrees C. However, at 41 degrees C, the restrictive temperature, while tsA438 T antigen still generated substantial levels of replication products, tsA357 T antigen did not support any detectable DNA synthesis. Furthermore, preincubation for approximately fourfold-longer time periods at 41 degrees C was required to heat inactivate tsA438 T antigen than to heat inactivate tsA357 T antigen. Unexpectedly, results of analyses of the various DNA replication activities of the two mutant T antigens did not correlate with results from ori-DNA replication reactions. In particular, although tsA357 T antigen was incapable of mediating replication at 41 degrees C at all protein concentrations examined, it displayed either wild-type levels or only partial reductions of the several T-antigen replication-associated activities. These data suggest either that tsA357 T antigen is defective in an as yet unidentified replication function of T antigen or that the combination of its partial defects result in a protein that is unable to support replication. The data also show that two conditional mutant T antigens can be markedly different with respect to thermal sensitivity.

Wild-type p53 protein was shown to bind specifically to DNA sequences within SV40 (Bargonetti et al. 1991), the human ribosomal gene cluster (RGC) (Kern et al. 1991a), and the murine muscle creatine kinase gene (MCK) (Zambetti et al. 1992). However, a direct comparison of these three sites was not performed. Here we demonstrate, by filter binding and gel mobility-shift assays, that wild-type p53 binds with similar affinities to MCK and RGC sites but less tightly to the SV40 site. We examined the effects of two candidate regulators of p53 function, SV40 large T antigen and oncogenic mutant p53, on the binding of wild-type p53 to RGC DNA. We show that wild-type T antigen prevents p53 from binding to the RGC site under all conditions tested. Moreover, two temperature-sensitive mutant SV40 T antigens, which fail to transform cells at the nonpermissive temperature, prevent p53 from binding to the RGC site at the permissive, but not at the restrictive, temperature. The ability of complexes containing wild-type p53 and tumor-derived mutant p53 proteins to bind to RGC DNA varies according to the position of the mutation. Complexes containing wild-type and either his175 or his273 mutant p53 proteins are completely unable to bind to the RGC DNA sequence. Interestingly, a complex containing wild-type p53 and the trp248 mutant p53 characteristic of Li-Fraumeni syndrome patients displays nearly wild-type levels of binding. Perhaps this mutant allele can be tolerated in these individuals because the wild-type mutant p53 complex maintains the ability to bind to DNA. Our data indicate that the oncogenic potential of both T antigen and some mutant p53 proteins is the result of their ability to block binding of wild-type p53 to DNA.

Background The minor allele of SNP rs3803662 has been shown to correlate with increased breast cancer risk and with lower expression of TOX3. The SNP is closely located to TOX3 residing within an uncharacterised gene LOC643714. The aim of the study was to examine the association of the risk allele with expression of TOX3 and LOC643714, and of mRNA levels and genotype with clinical and pathological characteristics. Methods The SNP was genotyped in DNA isolated from blood and normal tissue from 160 breast cancer patients and mRNA levels were measured by microarrays and quantitative real-time (qRT)-PCR in breast tumours. Association with clinical and pathological characteristics was analysed by parametric tests. Results An association of the risk allele of rs3803662 with lower TOX3 expression was confirmed in oestrogen receptor (ER) positive tumours. It was more often observed in lobular tumours (p = 0.04), and carriers of the risk allele who had been diagnosed with luminal A tumours had shorter overall survival (OS) than carriers of the non-risk allele (p = 0.01). Positive correlation between the mRNA levels of TOX3 and LOC643714 was observed (r = 0.44 and p < 0.001). Association analysis with tumour pathology showed that low TOX3 and LOC643714 expression correlated with high Ki67 levels (p = 0.026 and p = 0.002) and the basal subtype (p < 0.001 and p < 0.001), whereas high expression correlated with ER (p = 0.004 and p < 0.001) and progesterone receptor (PgR) (p = 0.005 and p < 0.001) expression. Furthermore, high TOX3 and LOC643714 correlated with positive lymph nodes (p < 0.001 and p = 0.01). Patients with ER positive tumours and high levels of TOX3 mRNA had shorter overall- and distant metastasis free-survival (p = 0.017 and p = 0.021), an effect mostly attributable to patients with luminal B tumours. Conclusions The results suggest that the effect of the risk allele of rs3803662 is strongest in luminal A tumours and that the expression levels of TOX3 and/or LOC643714 affect the progression of breast cancer. The effect may vary depending on the subtype and developmental stage of the tumour.