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PDF file - 3639K, Representative images of pancreatic lesions in wild type mice implanted with DMBA, or in Pdx1-Cre; LSL-Kras G12D mice.

PDF file - 56K, Proliferation rate analysis.

PDF file - 39K, Pathologic score of PanIN lesions in Pdx1-Cre; LSL-KRAS G12D mice, wild type (n=4 mice) or heterozygous (n=7) for the phospho-rpS6 mutant.

PDF file - 66K

PDF file - 44K, Staining for γH2AX (brown) in sections of the intestine from a CK1α-deficient mouse

PDF file - 47K, Mosaic phosphorylation of rpS6 in wild type islets.

SUMMARYRecent studies demonstrated that human trophoblast stem-like cells (hTS-like cells) can be derived from naïve embryonic stem cells or be induced from somatic cells by the pluripotency factors, OSKM. This raises two main questions; (i) whether human induced TSCs (hiTSCs) can be generated independently to pluripotent state or factors and (ii) what are the mechanisms by which hTSC state is established during reprogramming. Here, we identify GATA3, OCT4, KLF4 and MYC (GOKM) as a pluripotency-independent combination of factors that can generate stable and functional hiTSCs, from both male and female fibroblasts. By using single and double knockout (KO) fibroblasts for major pluripotency genes (i.e. SOX2 or NANOG/PRDM14) we show that GOKM not only is capable of generating hiTSCs from the KO cells, but rather that the efficiency of the process is increased. Through H3K4me2 and chromatin accessibility profiling we demonstrate that GOKM target different loci and genes than OSKM, and that a significant fraction of them is related to placenta and trophoblast function. Moreover, we show that GOKM exert a greater pioneer activity compared to OSKM. While GOKM target many specific hTSC loci, OSKM mainly target hTSC loci that are shared with hESCs. Finally, we reveal a gene signature of trophoblast-related genes, consisting of 172 genes which are highly expressed in blastocyst-derived TSCs and GOKM-hiTSCs but absent or mildly expressed in OSKM-hiTSCs.Taken together, these results imply that not only is the pluripotent state, and SOX2 specifically, not required to produce functional hiTSCs, but that pluripotency-specific factors actually interfere with the acquisition of the hTSC state during reprogramming.

Following fertilization, it is only at the 32-64-cell stage when a clear segregation between cells of the inner cell mass and trophectoderm is observed, suggesting a ‘T’-shaped model of specification. Here, we examine whether the acquisition of these two states in vitro, by nuclear reprogramming, share similar dynamics/trajectories. Using a comparative parallel multi-omics analysis (i.e., bulk RNA-seq, scRNA-seq, ATAC-seq, ChIP-seq, RRBS and CNVs) on cells undergoing reprogramming to pluripotency and TSC state we show that each reprogramming system exhibits specific trajectories from the onset of the process, suggesting ‘V’-shaped model. We describe in detail the various trajectories toward the two states and illuminate reprogramming stage-specific markers, blockers, facilitators and TSC subpopulations. Finally, we show that while the acquisition of the TSC state involves the silencing of embryonic programs by DNA methylation, during the acquisition of pluripotency these regions are initially defined but retain inactive by the elimination of H3K27ac.

ObjectiveCellular senescence limits tumourigenesis by blocking the proliferation of premalignant cells. Additionally, however, senescent cells can exert paracrine effects influencing tumour growth. Senescent cells are present in premalignant pancreatic intraepithelial neoplasia (PanIN) lesions, yet their effects on the disease are poorly characterised. It is currently unknown whether senolytic drugs, aimed at eliminating senescent cells from lesions, could be beneficial in blocking tumour development.DesignTo uncover the functions of senescent cells and their potential contribution to early pancreatic tumourigenesis, we isolated and characterised senescent cells from PanINs formed in a Kras-driven mouse model, and tested the consequences of their targeted elimination through senolytic treatment.ResultsWe found that senescent PanIN cells exert a tumour-promoting effect through expression of a proinflammatory signature that includes high Cox2 levels. Senolytic treatment with the Bcl2-family inhibitor ABT-737 eliminated Cox2-expressing senescent cells, and an intermittent short-duration treatment course dramatically reduced PanIN development and progression to pancreatic ductal adenocarcinoma.ConclusionsThese findings reveal that senescent PanIN cells support tumour growth and progression, and provide a first indication that elimination of senescent cells may be effective as preventive therapy for the progression of precancerous lesions.

Telomeric repeats in fungi of the subphylum Saccharomycotina exhibit great inter- and intra-species variability in length and sequence. Such variations challenged telomeric DNA-binding proteins that co-evolved to maintain their functions at telomeres. Here, we compare the extent of co-variations in telomeric repeats, encoded in the telomerase RNAs (TERs), and the repeat-binding proteins from 13 species belonging to the Yarrowia clade. We identified putative TER loci, analyzed their sequence and secondary structure conservation, and predicted functional elements. Moreover, in vivo complementation assays with mutant TERs showed the functional importance of four novel TER substructures. The TER-derived telomeric repeat unit of all species, except for one, is 10 bp long and can be represented as 5′-TTNNNNAGGG-3′, with repeat sequence variations occuring primarily outside the vertebrate telomeric motif 5′-TTAGGG-3′. All species possess a homologue of the Yarrowia lipolytica Tay1 protein, YlTay1p. In vitro, YlTay1p displays comparable DNA-binding affinity to all repeat variants, suggesting a conserved role among these species. Taken together, these results add significant insights into the co-evolution of TERs, telomeric repeats and telomere-binding proteins in yeasts.

The signaling pathways that mediate the development of pancreatic ductal adenocarcinoma (PDAC) downstream of mutant Kras remain incompletely understood. Here, we focus on ribosomal protein S6 (rpS6), an mTOR effector not implicated previously in cancer. Phosphorylation of rpS6 was increased in pancreatic acinar cells upon implantation of the chemical carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) or transgenic expression of mutant Kras. To examine the functional significance of rpS6 phosphorylation, we used knockin mice lacking all five phosphorylatable sites in rpS6 (termed rpS6P−/− mice). Strikingly, the development of pancreatic cancer precursor lesions induced by either DMBA or mutant Kras was greatly reduced in rpS6P−/− mice. The rpS6 mutants expressing oncogenic Kras showed increased p53 along with increased staining of γ-H2AX and 53bp1 (Trp53bp1) in areas of acinar ductal metaplasia, suggesting that rpS6 phosphorylation attenuates Kras-induced DNA damage and p53-mediated tumor suppression. These results reveal that rpS6 phosphorylation is important for the initiation of pancreatic cancer. Cancer Res; 73(6); 1811–20. ©2012 AACR.

Although 90–95% of adults recover completely from Hepatitis B (HBV) infection, a minority are unable to clear the virus.[1][1] Epidemiological studies have demonstrated an increased risk of B-NHL among those with persistent HBV and B-NHL.[2][2]–[5][3] However, the roles of exposure per se

Cannabinoid 2 (CB2) receptors expressed on immune cells are considered to be antifibrogenic. Hepatic stellate cells (HSCs) directly interact with phagocytosis lymphocytes, but the nature of this interaction is obscure. We aimed to study the effects of CB2 receptors on hepatic fibrosis via their role in mediating immunity. Hepatic fibrosis was induced by carbon-tetrachloride (CCl4) administration in C57BL/6 wild-type (WT) and CB2 knockout (CB2−/−) mice. Irradiated animals were reconstituted with WT or CB2−/−lymphocytes. Lymphocytes from naïve/fibrotic WT animals and healthy/cirrhotic hepatitis C virus were preincubated in vitro with or without CB2 antagonist, evaluated for proliferation and apoptosis, and then cocultured with primary mouse HSCs or a human HSC line (LX2), respectively. Lymphocyte phagocytosis was then evaluated. Following CCl4-administration, CB2−/−mice developed significant hepatic fibrosis but less necroinflammation. WT mice harbored decreased liver CD4+and NK+cells but increased CD8+subsets. Naïve CB2−/−mice had significantly decreased T cell subsets. Adoptive transfer of CB2−/−lymphocytes led to decreased fibrosis in the irradiated WT recipient compared with animals receiving WT lymphocytes. Moreover, necroinflammation also tended to decrease. In vitro, a CB2-antagonist directly increased human HSC activation and increased apoptosis and decreased proliferation of mice/human T cells (healthy/fibrotic) and their phagocytosis. We concluded that CB2−/−lymphocytes exert an antifibrotic activity, whereas lack of CB2 receptor in HSCs promotes fibrosis. These findings broaden our understanding of cannabinoid signaling in hepatic fibrosis beyond their activity solely in HSCs.

Gastro-intestinal stromal tumors (GISTs) of the appendix are a rare entity. To date, only a handful has been described in the literature, all of which have been of the benign type. We present the first reported case of a malignant appendiceal GIST. The tumor was discovered when the patient presented with a peri-appendiceal abscess which appeared suspicious on CT. The abscess was drained and managed medically. The patient responded to antibiotic treatment but subsequent CT and biopsy confirmed the diagnosis of appendiceal GIST, and the patient was started on treatment with imatinab mesylate. One week after initiation of therapy, the patient returned with frank peritonitis necessitating surgery. Abdominal exploration revealed an appendiceal GIST locally invading and perforating adjacent bowel. We describe the complex presentation and course of the case as well as a literature review of the appendiceal GISTs and the current approach to treatment.

Background: Risk factors for B-cell non-Hodgkin lymphoma (B-NHL) have not been assessed among Palestinian Arabs (PA) and Israeli Jews (IJ). Methods: In a case-control study we investigated self-reported medical and lifestyle exposures, reporting odds ratios (ORs) and 95% confidence intervals [CIs], by ethnicity, for overall B-NHL and subtypes. Results: We recruited 823 cases and 808 healthy controls. Among 307 PA/516 IJ B-NHL cases (mean age at diagnosis = 51 [±17] versus 60 [±15] years, respectively) subtype distributions differed, with diffuse large B-cell lymphoma (DLBCL) being prominent among PA (71%) compared to IJ (41%); follicular lymphoma (FL), was observed in 14% versus 28%, and marginal zone lymphoma, in 2% versus 14%, respectively. Overall B-NHL in both populations was associated with recreational sun exposure OR = 1.43 [CI:1.07-1.91], black hair-dye use OR = 1.70 [CI:1.00-2.87], hospitalization for infection OR = 1.68 [CI:1.34-2.11], and first-degree relative with hematopoietic cancer, OR = 1.69 [CI:1.16-2.48]. An inverse association was noted with alcohol use, OR = 0.46 [CI:0.34-0.62]. Subtype-specific exposures included smoking (FL, OR = 1.46 [CI:1.01-2.11]) and >monthly indoor pesticide use (DLBCL, OR = 2.01 [CI:1.35-3.00]). Associations observed for overall B-NHL in PA only included: gardening OR = 1.93 [CI:1.39-2.70]; history of herpes, mononucleosis, rubella, blood transfusion (OR>2.5, P

27 Background: According to data from the Palestinian Ministry of Health, breast cancer is the most common malignancy affecting Palestinian women, being 30% of all cancers affecting female patients, 60% of them present with metastasis, very few studies were conducted to explore characteristics of this cancer in this population. Triple-negative breast cancer (TNBC) has distinct clinical and pathologic features. The fact that this type is more aggressive despite lacking the expression of growth promotion receptors, suggest other players, as defect in tumor suppressors such as PTEN gene. To our knowledge this is the first study in English literature correlating TNBC to loss of PTEN protein expression. The aim of this study is to establish data about the clinicopathological characteristics of breast cancer in Palestinian women, and to study the status of PTEN protein expression by immunohistochemistry in breast cancer and specifically in TNBC. Methods: 100 confirmed cases of breast cancer, were collected from different pathology centers, clinicopathological data; age, grade, stage for each case were specified. Each case was evaluated for the expression of hormone receptors, Her-2 status, and for PTEN protein expression by immunohistochemistry (IHC) to see if a certain pattern is noted. Results: Out of the 100 cases, the mean age at presentation was 53.3 years, 69.84% of the cases presented with advanced stage and 53% were high grade, 58% were ER positive and 46% were PR positive. HER-2 positive breast cancer were 26%. 30% of all breast cancer cases were TNBC subtype. Loss of PTEN expression was seen in 44% of cases. 60% of TNBC cases lost PTEN expression, which was statistically significant (p + 0.035), no significant correlation between PTEN loss and ER, PR, HER-2, grade or stage. Conclusions: Palestinian women have high incidence of TNBC in comparison to White population, and presented with high grade, and advanced stage tumors. There is a significant correlation between PTEN loss and TNBC that needs more research and study.

Background: Carcinoma of the pancreas kills nearly all of the 230,000 people diagnosed with the disease annually worldwide. This high mortality results from poor detection, metastatic disease at the time of detection, and lack of specific or effective therapy. Early precursor lesions termed pancreatic intraepithelial neoplasia (PanIN) are thought to be the initial step in pancreatic ductal carcinoma. The PI3K signaling pathway is a central transducer of information from extracellular signals to the cell, impacting diverse cellular function such as replication, survival, migration, protein synthesis, and more. Ribosomal protein s6 (rps6) is a component of the protein translation machinery, identified originally in the regenerating liver. It is phosphorylated at specific residues by an upstream kinase, s6K, which itself is activated by mTOR and the PI3K-AKT pathway. No functional link is currently known between rps6 phosphorylation and pancreatic cancer. Methods: Pellets soaked with 7,12-dimethylbenz(a)anthracene(DMBA) were implanted in the body of wild-type mouse pancreas (n-12) and in the pancreas of mutant mice lacking rps6 phosphorylation sites (rps6ki/ki) (n=11). Three to five months later, the mice were scarified and pathological analysis and scoring for PanIN lesions were performed. To examine in a crude way if mTOR signaling is important for the development of DMBA-induced PanIN lesions, we administered rapamycin to DMBA-treated mice, and have determined the effect on PanIN score. We then examined the role of rps6 activation in a genetic model of PanIN lesions. Using extensive breeding, we generated cohorts of pdx1-Cre; LSL-KRAS G12D; rps6 ki/ki mice, in order to examine the effect of rps6 phosphorylation on the development of PanIN lesions, in comparison to LSL-KRAS wild rps6, after 3–5 months the mice were sacrificed and the same analysis and scoring were performed. In both models, rps6 pattern of staining was evaluated in all groups. Results: In the DMBA model, we observed the development of mouse PanIN lesions at all levels (PanIN 1–3) in all wild-type mice examined. Rapamycin-treated mice had a significant reduction in the score of PanIN lesions, supporting the hypothesis that mTOR signaling downstream of PI3K, participated in the formation of PanIN lesions. In mutant mice, strikingly, PanIN lesions observed were greatly reduced. These results suggest that rps6 phosphorylation is an essential event in the development of pancreatic cancer, in the DMBA carcinogenesis model. In the KRAS model, mice developed PanIN lesions, as previously described, KRAS mice lacking rps6 phosphorylation developed fewer PanIN lesions that had lower score, similar to DMBA carcinogenesis model. This shows that development of PanIN lesions driven by mutant KRAS requires at least partially phosphorylation of rps6. The expressive pattern of rps6 in normal mice revealed that rps6 phosphorylation is limited to a subset of islet cells and to a few acinar cells surrounding the islets, with minimal or no activity in ducts. In PanIN lesions generated by implantation of DMBA in the pancreas, as well in Pdx1-Cre; LSL-KRAS G12D mice, strong phosphorylation of rps6 is observed in malignant epithelial cells. This suggests that oncogenic signaling in the pancreas, and specifically KRAS signaling, activates PI3K signaling, including S6 kinase which phosphorylates rps6. Conclusion: rps6 is activated (phosphorylated) in mouse models of PanIN lesions and pancreatic cancer, and development of PanIN lesions requires at least partial phosphorylation of rps6. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A51.