These findings offer a potential for future pre-clinical or clinical investigation for the control of PIN lesions. It is projected from the findings of this study that inhibition of survivin activity may retard or block progression of the PIN lesions

A similar pattern of XIAP expression was also noticed when samples had been obtained from in ten and 21 week- previous teams. (B) Detection of better stages of cleaved-caspase 3 expression, indicated by arrows, in the PIN lesions from the conditional Pten deleted prostate tumors missing solitary or equally alleles of Survivin as opposed to tumors with intact Survivin. P < 0.05 P <0.01. (C) Illustration of senescence-activated -galactosidase staining results. An increase in senescence was indicated in the conditional Pten deleted prostate samples with complete inactivation of Survivin especially at the high-grade PIN stage compared to samples with intact and monoallelic deletion of Survivin. Note only low level of senescence in prostate adenocarcinoma tissues of conditional Pten knockout mice with either with intact and single deletion of Survivin. PINs are denoted as low-grade (LG) or high-grade (HG), and adenocarcinoma as AdCa. CF-101 supplierAll images were taken at 400x magnification. Bar, 50 .Figure 6. Assessment of -H2AX immunostaining in prostate tissue sections. (A) Representative view of -H2AX expression, indicated by arrows, in the prostate samples at 10 weeks from the conditional Pten knockout mice with single or double deletion of Survivin as compared to corresponding samples with intact Survivin. Images were taken at 400x magnification. Bar, 50 . (B) (C), and (D) show the results of quantitation of -H2AX positive cells in samples at 10, 21 and 37 weeks, respectively. P < 0.05 P <0.01 deleted tumor tissues, XIAP appeared to be down-regulated with the loss of survivin expression. This type of corollary expression, however, was not observed with Livin, another IAP family member. Livin protein expression did increase in the lesions of Pten- deleted prostates, but there was no significant alteration in its expression in the cases when Survivin was deleted (Figure 5A).In the cancer field, survivin stands as a unique member of the IAP family with essential roles in mitosis, cellular stress response and inhibition of cell death. It was, however, not known whether survivin plays a role in the development of the normal prostate and how this multi-functional protein might be relevant to its role in prostate carcinogenesis. For this purpose, we first determined if the organogenesis and growth of the prostate gland might be influenced by survivin. Mice with conditional inactivation of Survivin in prostate epithelium were generated by crossing mice of our PB-Cre4 line [23] with floxed Survivin mice [22]. Through breeding and analyses of these mice at various ages up to one year, we demonstrate that homozygous inactivation of Survivin alleles in the epithelial cells of the prostate does not interfere with the development of fertile males harboring prostate gland with generally normal gross and microscopic anatomy. The role of survivin in prostate cancer genesis and progression was then investigated using the conditional biallelic Pten deletion model [179] by crossing the tumor model with the floxed Survivin allelic mice. Our contention was that prostate epithelium-specific Survivin nullizygous condition would likely enhance cellular apoptosis to counter prostate tumorigenesis. Here, using this combined model we provide evidence that loss of survivin inhibits progression of premalignant lesions to adenocarcinoma, and that the premalignant lesions exhibiting decreased proliferation index are composed of atypical cells, many of which exhibit increased hypertrophy and senescence. Survivin expression was reported to be up-regulated in the early prostate tumor growth in both the conditional Pten knockout [16] and the TRAMP [31] mouse models. This implication of survivin in early pathogenesis is corroborated by our observation that deletion of either single or both alleles of Survivin can influence the phenotype of the PIN formed as early as 9 weeks of age. While all groups displayed low grade PIN formation, none of the four cPten-/-S-/- and only 1 of 4 cPten-/-S+/- mice was found to develop high grade PIN lesions at this age, compared to 3 out of 4 cPten-/-S+/+ mice. In the age group of 56 weeks, 80-100% of the mice with intact or singly deleted Survivin alleles harbored adenocarcinoma, while none (0/5) of the mice with the homozygous Survivin deletion developed adenocarcinoma, although high grade PINs were detected. In relation to the known influence of the genetic background and modifier genes on susceptibility to tumorigenesis, we believe that it is unlikely that the suppression of the progression to adenocarcinoma in our test model is due to variation in the mixed genetic background of the animals, because some of the cPten-/-S+/- mice developed adenocarcinoma at age as early as 21 weeks, whereas cPten-/-S-/- littermates with similar mixed background were free of cancer even at over one year age. Still, to increase the validity of the study, it will be necessary to breed all of the three pertinent strains, namely PBCre-4, floxed Pten, and the floxed Survivin, into a single genetic background that is also conducive for tumorigenesis. This major task, however, remains to be initiated. The question of whether the cPten-/-S-/mice might manifest cancer on further aging was examined very recently. Of the three cPten-/-S-/- 72 week-old males examined subsequently, only one was found to display development of adenocarcinoma in the prostate (data not shown). With respect to other histopathology parameters, hyperplasia was abundantly detected in the cPten-/-S-/- mice, but only rarely in cPten-/-S+/- or the control cPten-/-S+/+ group. Furthermore, hypertrophy or enlargement of cells, and polyploidy or hyperploidy were relatively more frequent in the prostate samples from cPten-/-S-/- compared to the other groups. Survivin deficient cells have been reported to exhibit multiple nuclei in vitro and in vivo, consistent with the known role of survivin in the regulation of cytokinesis and cell division [11,22]. Another striking difference between single and biallelic deletion of Survivin was the lack of desmoplasia observed in the prostate tissue of cPten-/-S-/- mice younger than the 56 weeks age group that was prominently present in cPten-/-S+/+ and cPten-/-S+/- samples from all four age groups tested. A reduction in the rate of cell proliferation was evident from the significant down-regulation of Ki67 expression in the specimens from the cPten-/-S-/- animals. This effect was the most striking with the 10 weeks age group, whether the samples were from the monoallelic or biallelic Survivin knockout animals. However, with time, the degree of proliferation became somewhat parallel with the extent of Survivin insufficiency. Another noteworthy point is that we found that the degree of Survivin deletion in the tumors differentially affected the detectable levels of expression of some other IAP family members. For example, the expression of XIAP, but not Livin was reduced. This phenomenon could probably be attributed to the function of the complex known to form between survivin and XIAP that stabilizes XIAP and protects it from ubiquitindependent degradation [3]. The survivin-XIAP complex was also described to enhance XIAP's capacity to inhibit caspases and to facilitate tumor growth in vivo [3,32]. The reduction of XIAP in the setting of Survivin deficiency may therefore further protect against tumorigenesis. Consistent with previous findings in various other study systems [335], we observed an increase in apoptosis as assessed by the expression level of activated caspase-3 in the prostatic lesions of the conditional Pten knockout mice lacking both alleles of Survivin. This effect was most pronounced and significant at the low grade PIN stage. We also detected a correlation between loss of survivin expression and senescence in Pten-deleted prostate tumor tissues. By using the simplified method of staining for senescence-associated galactosidase in the tissue sections [36], we attempted to measure the extent of senescence induction in the prostate of the various groups of mice. Presence of senescent cells was readily detected in conditional Pten deleted mice with intact Survivin at low grade and high grade PIN stages but minimally when the tumor had advanced to adenocarcinoma. This finding is consistent with previous reports that senescence may be an initial barrier in cancer development [379], and that senescent cells exist in premalignant tumors but not in malignant ones [39]. The pattern of senescence observed in the group with monoallelic and biallelic Survivin loss was not detectably different from the control tumor group at the low grade PIN stage. However, at the high grade PIN stage, lesions of the group with single and especially double deletion of Survivin loss exhibited a higher proportion of senescent cells and greater intensity of staining. Since -H2AX, whose expression is induced in cells following initial DNA fragmentation, is considered as a cellular senescence marker [402] our data on -galactosidase criterion appears to be supported by the results of -H2AX staining. Clearly, the increase in the detection of -H2AX-positive cells is found to be associated with the degree of Survivin deletion in the conditional Pten knockout mice. However, it remains unknown to what extent this increased staining may be subscribed to senescence vs. DNA double strand breaks and induced H2AX that occurs during apoptosis [43]. Although an association of survivin loss with senescence in the lesions is indicated, not clear is whether this relationship is direct or secondary to the loss of the multi-functionality of the survivin protein that may be critical for the progression of the preneoplastic lesions to cancer. It is also possible that the defects in microtubule assembly, loss of mitotic spindles, and formation of multinucleated cells, the abnormalities that are triggered by the loss of survivin [2,9,11] could make the cells prone for senescence. Further studies into the effect of survivin on cellular senescence would be important. In summary, the results of our investigation on the role of survivin in prostate cancer progression using a double conditional Pten and Survivin mouse model is particularly significant because it led to insights of the direct impact of Survivin deletion occurring simultaneously with that of Pten deletion in the process of tumorigenesis. Evidence is obtained to link a supporting role of survivin in the progression of PIN lesions to adenocarcinoma of the prostate in the model system. Additionally, it is apparent that lesions formed in the absence of survivin are variant in microscopic phenotypes with hallmarks of hypertrophy, exfoliation, apoptosis and senescence. These findings offer a potential for future pre-clinical or clinical investigation for the control of PIN lesions. It is projected from the findings of this study that inhibition of survivin activity may retard or block progression of the PIN lesions, and, thereby extending the therapeutic window for prostate cancer.Deep sequencing and transcriptome analysis revealed the existence of non-coding RNAs (ncRNAs) in mammalian cells [1]. MicroRNAs (miRNAs) are single-stranded 19- to 25nucleotide ncRNAs that play a critical role in posttranscriptional gene regulation. The miRNA-mediated gene silencing is regulated by complementarity between nucleotides at positions 2 of the miRNAs (i.e., the seed sequences) and the 39-untranslated regions (39-UTR) of their target genes [4,5]. Dysregulated expression of miRNA is involved in a variety of human diseases, including cancer and liver disease [6,7]. Recent studies show that expression levels of various miRNAs are downregulated in cancer, indicating that they may function as tumor suppressors [80]. For example, downregulation of miR-15 and miR-16 in most chronic lymphocytic leukemia cells leads to upregulation of anti-apoptotic B cell lymphoma 2 (Bcl-2) protein [8]. The upregulated Bcl-2 averts apoptotic cell death of leukemia cells and thereby promotes their survival. Let-7, which is downregulated in lung cancers relative to normal lung tissue, targets ras oncogenes [10]. The increased levels of Ras protein in lung cancer cells leads to upregulated cell growth. The miR-200 family and miR-205 target zinc finger homeodomain enhancer-binding protein (ZEB) transcription factors, which are known to be inducers of the epithelial-mesenchymal transition in breast cancer [9]. Downregulation of these miRNAs are likely to be an essential early step in breast cancer metastasis. Cholangiocarcinoma (CC) is a bile duct cancer, and is classified as intrahepatic or extrahepatic [113]. Intrahepatic CC (ICC) is derived from epithelial cells of the bile ducts. Although ICCs comprise only 50% of all cases of liver cancer, they are the second most common liver malignancy [14]. The incidence and mortality rate of ICC are increasing worldwide. Despite advances in surgical techniques, chemotherapies and radiotherapies, longterm survival remains low because of the late presentation of the disease [14,15]. Even after resection, the prognosis for patients with advanced ICC is extremely poor [14,16,17]. Some researchers have examined the miRNA expression profiles in ICC, to understand the molecular and clinical basis of carcinogenesis and the progression of this disease [18,19]. We reported previously that miR-376c (formerly designated as miR-368 in miRBase Release 12 currently designated as miR-376c3p in miRBase Release 19) was expressed in a normal intrahepatic biliary epithelial cell line (HIBEpiC), but was significantly suppressed in an ICC cell line (HuCCT1) [18]. However, the biological significance of the downregulation of miR-376c in HuCCT1 cells was unknown. We hypothesized that miR-376c could function as a tumor suppressor in these cells. 23316025To test this hypothesis, we sought the targets of this miRNA, and characterized the effect of miR-376c down-regulation in HuCCT1 cells. We identified a direct target mRNA of miR-376c by proteomic analysis. Enforced expression of miR-376c significantly impaired migration of HuCCT1 cells by reducing levels of the targeted mRNA, indicating that downregulation of miR-376c is critical for this response. Finally, we investigated the DNA methylation and histone modification status of the putative promoter regions of miR-376c gene in HuCCT1 cells.These criteria for spot selection were determined based on previous studies, in which the repression of cellular protein synthesis by miRNA overexpression was typically mild [21,22]. Proteins in the spots were subjected to digestion with 10 ng/ml trypsin (Sigma, St. Louis, MO, USA) at 37uC, followed by elution in an acetonitrile buffer. The eluted proteins were then identified using a liquid chromatography-tandem mass spectrometry mass spectrometer (LCQ DECA XP Plus Thermo-Finnigan, San Jose, CA, USA). Mass spectrometry data were analyzed using the MASCOT software (Matrix Science, London, England).