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PDF file 1687K, The supplementary data contains immunostains, mRNA and DNA qPCR, ELISA analyses and descriptive cartoons including a model highlighting the manuscript

PDF file 145K, Tables containing information about genes residing on the amplicon and compiled data

Table S1: List of primers oligos and siRNAs

Table S2. Full list of Up- and Down regulated genes in the RNA seq analysis

Several long noncoding RNAs (lncRNA) are abrogated in cancer but their precise contributions to oncogenesis are still emerging. Here we report that the lncRNA MALAT1 is upregulated in hepatocellular carcinoma and acts as a proto-oncogene through Wnt pathway activation and induction of the oncogenic splicing factor SRSF1. Induction of SRSF1 by MALAT1 modulates SRSF1 splicing targets, enhancing the production of antiapoptotic splicing isoforms and activating the mTOR pathway by modulating the alternative splicing of S6K1. Inhibition of SRSF1 expression or mTOR activity abolishes the oncogenic properties of MALAT1, suggesting that SRSF1 induction and mTOR activation are essential for MALAT1-induced transformation. Our results reveal a mechanism by which lncRNA MALAT1 acts as a proto-oncogene in hepatocellular carcinoma, modulating oncogenic alternative splicing through SRSF1 upregulation. Cancer Res; 77(5); 1155–67. ©2016 AACR.

Table S3. IPA enriched pathways based on RNA-seq analysis

Supplementary information text: Methods and figure legends

Reprogrammed glucose metabolism of enhanced aerobic glycolysis (or the Warburg effect) is known as a hallmark of cancer. The roles of long noncoding RNAs (lncRNA) in regulating cancer metabolism at the level of both glycolysis and gluconeogenesis are mostly unknown. We previously showed that lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) acts as a proto-oncogene in hepatocellular carcinoma (HCC). Here, we investigated the role of MALAT1 in regulating cancer glucose metabolism. MALAT1 upregulated the expression of glycolytic genes and downregulated gluconeogenic enzymes by enhancing the translation of the metabolic transcription factor TCF7L2. MALAT1-enhanced TCF7L2 translation was mediated by upregulation of SRSF1 and activation of the mTORC1–4EBP1 axis. Pharmacological or genetic inhibition of mTOR and Raptor or expression of a hypophosphorylated mutant version of eIF4E-binding protein (4EBP1) resulted in decreased expression of TCF7L2. MALAT1 expression regulated TCF7L2 mRNA association with heavy polysomes, probably through the TCF7L2 5′-untranslated region (UTR), as determined by polysome fractionation and 5′UTR-reporter assays. Knockdown of TCF7L2 in MALAT1-overexpressing cells and HCC cell lines affected their metabolism and abolished their tumorigenic potential, suggesting that the effects of MALAT1 on glucose metabolism are essential for its oncogenic activity. Taken together, our findings suggest that MALAT1 contributes to HCC development and tumor progression by reprogramming tumor glucose metabolism.Significance:These findings show that lncRNA MALAT1 contributes to HCC development by regulating cancer glucose metabolism, enhancing glycolysis, and inhibiting gluconeogenesis via elevated translation of the transcription factor TCF7L2.

Supplementary Information: Figure S1: MALAT1 affects cancer glucose metabolism Figure S2: Glucose metabolism in HCC cell lines with MALAT1 knockdown. Figure S3: Regulation of TCF7L2 protein expression by MALAT1 in HCC cell lines. Figure S4: A non-phosphorylatable mutant of 4EBP1 inhibits TCF7L2 protein expression and expression of glycolytic genes. Figure S5: SRSF1 regulates TCF7L2 levels post-transcriptionally. Figure S6: TCF7L2 modulates glucose metabolism in a HCC cell line. Figure S7: MALAT1 and TCF7L2 regulate gluconeogenesis through the same pathway. Figure S8: Oncogenic properties of HCC cell lines with TCF7L2 knockdown. Figure S9: TCF7L2 protein, Gluconeogenesis and Glycolytic enzyme expression in livers from mouse HCC model Mdr2-/-. Table S1: List and sequences of shRNAs, siRNAs and PCR primers used in the paper

Tertiary lymphoid structures (TLSs) are formed in many cancer types and have been correlated with better prognosis and response to immunotherapy. In liver cancer, TLSs have been reported to be pro-tumorigenic as they harbor tumor progenitor cells and nurture their growth. The processes involved in TLS development and acquisition of pro- or anti-tumorigenic phenotype in cancer are largely unknown. RORc expressing immune cells have been previously implicated in TLS formation, however we find that they are not necessary for TLS neogenesis in the liver under chronic inflammation conditions. On the contrary, RORc expressing cells rather negatively regulate TLS formation, since in their absence TLSs form in excess. Importantly, in chronically inflamed livers lacking RORc expressing cells, pro-tumorigenic TLSs become anti-tumorigenic, resulting in reduction of tumor load. Comparing liver pro- and anti-tumorigenic TLSs by transcriptional, proteomic and immunohistochemical analyses, revealed an enrichment of exhausted CD8 cells that retained effector functions and had a progenitor-like proliferative phenotype in anti-tumorigenic TLSs. Similar observations were found when comparing pro- and anti-tumorigenic TLSs in human tissues. Thus, RORc expressing cells negatively regulate CD8 cell abundance, and facilitate the pro-tumorigenic functions of hepatic TLSs.

2-hydroxyglutarate (2HG) is recognized as an epigenetic regulator in cancer and some transient biological processes. Of all organs, the testis harbors the highest baseline physiological levels of 2HG, yet it’s putative functions in germ cell biology are unknown. Here we show that 2HG is generated in specific stages of spermatogenesis by the testis specific lactate dehydrogenase C (LDHC), beginning at the last stages of prophase I. Unexpectedly LDHC enters nuclei and concentrates in centromeres. LDHC-generated L-2HG controls centromere condensation and pericentromeric heterochromatin organization through multiple effects including clustering of chromocenters, centromere and chromocenter condensation and expression of satellite RNAs. These effects are rapid and specific to L but not D-2HG.In vivodepletion of L-2HG causes centromere malfunction and activation of the spindle assembly checkpoint. Our findings reveal that 2HG can directly affect centromere and pericentromeric heterochromatin conformation and function and is necessary for licensing chromosome segregation.

An analytical platform for the selective miRNA-21-guided imaging of breast cancer cells and miRNA-221-guided imaging of ovarian cancer cells and the selective photodynamic therapy (PDT) of these cancer cells is introduced. The method is based on Zn(II)-protoporphyrin IX, Zn(II)-PPIX-loaded UiO-66 metal-organic framework nanoparticles, NMOFs, gated by two hairpins H

Reprogrammed glucose metabolism of enhanced aerobic glycolysis (or the Warburg effect) is known as a hallmark of cancer. The roles of long noncoding RNAs (lncRNA) in regulating cancer metabolism at the level of both glycolysis and gluconeogenesis are mostly unknown. We previously showed that lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) acts as a proto-oncogene in hepatocellular carcinoma (HCC). Here, we investigated the role of MALAT1 in regulating cancer glucose metabolism. MALAT1 upregulated the expression of glycolytic genes and downregulated gluconeogenic enzymes by enhancing the translation of the metabolic transcription factor TCF7L2. MALAT1-enhanced TCF7L2 translation was mediated by upregulation of SRSF1 and activation of the mTORC1–4EBP1 axis. Pharmacological or genetic inhibition of mTOR and Raptor or expression of a hypophosphorylated mutant version of eIF4E-binding protein (4EBP1) resulted in decreased expression of TCF7L2. MALAT1 expression regulated TCF7L2 mRNA association with heavy polysomes, probably through the TCF7L2 5′-untranslated region (UTR), as determined by polysome fractionation and 5′UTR-reporter assays. Knockdown of TCF7L2 in MALAT1-overexpressing cells and HCC cell lines affected their metabolism and abolished their tumorigenic potential, suggesting that the effects of MALAT1 on glucose metabolism are essential for its oncogenic activity. Taken together, our findings suggest that MALAT1 contributes to HCC development and tumor progression by reprogramming tumor glucose metabolism. Significance: These findings show that lncRNA MALAT1 contributes to HCC development by regulating cancer glucose metabolism, enhancing glycolysis, and inhibiting gluconeogenesis via elevated translation of the transcription factor TCF7L2.

Summary: The kinase Mnk2 is a substrate of the MAPK pathway and phosphorylates the translation initiation factor eIF4E. In humans, MKNK2, the gene encoding for Mnk2, is alternatively spliced yielding two splicing isoforms with differing last exons: Mnk2a, which contains a MAPK-binding domain, and Mnk2b, which lacks it. We found that the Mnk2a isoform is downregulated in breast, lung, and colon tumors and is tumor suppressive. Mnk2a directly interacts with, phosphorylates, activates, and translocates p38α-MAPK into the nucleus, leading to activation of its target genes, increasing cell death and suppression of Ras-induced transformation. Alternatively, Mnk2b is pro-oncogenic and does not activate p38-MAPK, while still enhancing eIF4E phosphorylation. We further show that Mnk2a colocalization with p38α-MAPK in the nucleus is both required and sufficient for its tumor-suppressive activity. Thus, Mnk2a downregulation by alternative splicing is a tumor suppressor mechanism that is lost in some breast, lung, and colon tumors. : The Mnk2 kinase is a MAPK pathway substrate and phosphorylates the translation initiation factor eIF4E. Mnk2 is alternatively spliced yielding two isoforms: Mnk2a and Mnk2b. Maimon et al. now report that Mnk2a is downregulated in many tumors and behaves like a tumor suppressor. They show that whereas Mnk2b is pro-oncogenic and does not activate p38α-MAPK, Mnk2a phosphorylates and activates p38α-MAPK leading to induction of its target genes, cell death, and suppression of Ras-induced transformation.

In recent years, it has become clear that splicing factors play a direct role in cancer development. We showed previously that splicing factors SRSF1, SRSF6, and hnRNP A2/B1 are up-regulated in several cancers and can act as oncogenes when up-regulated. Here we examined the role of splicing factors hnRNP A1/A1b and hnRNP A2/B1 in hepatocellular carcinoma (HCC). We show that the splicing factors hnRNP A1 and hnRNP A2 are up-regulated in HCC tumors derived from inflammation-induced liver cancer mouse model. Overexpression of hnRNP A1 or hnRNP A2, but not the splicing isoform hnRNP B1, induced tumor formation of immortalized liver progenitor cells, while knockdown of these proteins inhibited anchorage-independent growth and tumor growth of human liver cancer cell lines. In addition, we found that cells overexpressing hnRNP A2 showed constitutive activation of the Ras-MAPK-ERK pathway. In contrast, knockdown of hnRNP A2 inhibited the Ras-MAPK-ERK pathway and prevented ERK1/2 activation by EGF. Moreover, we found that hnRNP A2 regulates the splicing of A-Raf, reducing the production of a short dominant-negative isoform of A-Raf and elevating the full-length A-Raf transcript. Taken together, our data suggest that hnRNP A2 up-regulation in HCC induces an alternative splicing switch that down-regulates a dominant-negative isoform of A-Raf, leading to activation of the Raf-MEK-ERK pathway and cellular transformation. Science Foundation Ireland Isreali Science Foundation (ISF) MINERVA stiftung ARCHES award

Ectopic lymphoid-like structures (ELSs) are often observed in cancer, yet their function is obscure. Although ELSs signify good prognosis in certain malignancies, we found that hepatic ELSs indicated poor prognosis for hepatocellular carcinoma (HCC). We studied an HCC mouse model that displayed abundant ELSs and found that they constituted immunopathological microniches wherein malignant hepatocyte progenitor cells appeared and thrived in a complex cellular and cytokine milieu until gaining self-sufficiency. The egress of progenitor cells and tumor formation were associated with the autocrine production of cytokines previously provided by the niche. ELSs developed via cooperation between the innate immune system and adaptive immune system, an event facilitated by activation of the transcription factor NF-κB and abolished by depletion of T cells. Such aberrant immunological foci might represent new targets for cancer therapy.

In contrast to the accepted notion that tumor-derived signals polarize macrophages toward a protumorigenic M2 phenotype during tumor progression, we recently discovered that the inflammatory microenvironment is capable of driving macrophages toward an M2 phenotype. Moreover, our data suggests that inflammatory education is prominent during the early phases of hepatocellular carcinoma (HCC) suggesting that inflammatory modulation might effectively prevent HCC.

The causes of sporadic human cancer are seldom recognized, but it is estimated that carcinogen exposure and chronic inflammation are two important underlying conditions for tumour development, the latter accounting for approximately 20% of human cancer. Whereas the causal relationship between carcinogen exposure and cancer has been intensely investigated, the molecular and cellular mechanisms linking chronic inflammation to tumorigenesis remain largely unresolved. We proposed that activation of the nuclear factor kappaB (NF-kappaB), a hallmark of inflammatory responses that is frequently detected in tumours, may constitute a missing link between inflammation and cancer. To test this hypothesis, we studied the Mdr2-knockout mouse strain, which spontaneously develops cholestatic hepatitis followed by hepatocellular carcinoma, a prototype of inflammation-associated cancer. We monitored hepatitis and cancer progression in Mdr2-knockout mice, and here we show that the inflammatory process triggers hepatocyte NF-kappaB through upregulation of tumour-necrosis factor-alpha (TNFalpha) in adjacent endothelial and inflammatory cells. Switching off NF-kappaB in mice from birth to seven months of age, using a hepatocyte-specific inducible IkappaB-super-repressor transgene, had no effect on the course of hepatitis, nor did it affect early phases of hepatocyte transformation. By contrast, suppressing NF-kappaB inhibition through anti-TNFalpha treatment or induction of IkappaB-super-repressor in later stages of tumour development resulted in apoptosis of transformed hepatocytes and failure to progress to hepatocellular carcinoma. Our studies thus indicate that NF-kappaB is essential for promoting inflammation-associated cancer, and is therefore a potential target for cancer prevention in chronic inflammatory diseases.

The Src-related protein kinase Lyn plays an important role in B-cell activation. However, several lines of evidence suggest that it is also involved in the control of cellular proliferation and the inhibition of apoptosis. We have discovered that Lyn is expressed in normal prostate epithelia, in 95% of primary human prostate cancer (PC) specimens examined, and in all of the PC cell lines that we assayed. Moreover, Lyn knockout mice display abnormal prostate gland morphogenesis, which suggests that Lyn plays an important role in prostate epithelium development and implies that Lyn is a candidate target for specific therapy for PC. Using a drug-design strategy to construct sequence-based peptide inhibitors, a Lyn-specific inhibitor, KRX-123, targeting a unique interaction site within Lyn, was synthesized. KRX-123 was found to inhibit cellular proliferation in three hormone-refractory PC cell lines, DU145, PC3, and TSU-Pr1 with IC50 values of 2–4 μm. In vivo, tumor volume of DU145 explants in nude mice was significantly reduced after once-a-week injections of KRX-123, at a dose of 10 mg/kg, for a period of 5 weeks. Histological analyses of the treated tumors indicated extensive apoptosis. Thus, we suggest that Lyn inhibition may serve as a prime target for the treatment of hormone-refractory PC.

In contrast to common genomic amplifications that support cancer cell growth by rewiring intracellular signaling, VEGFA amplification drives tumor cell proliferation via the tumor microenvironment. VEGFA amplification is present in a subset of mouse and human hepatocellular carcinomas (HCCs) that appear to be particularly sensitive to sorafenib treatment, indicating its potential value as a biomarker for HCC treatment.

Death rates from hepatocellular carcinoma (HCC) are steadily increasing, yet therapeutic options for advanced HCC are limited. We identify a subset of mouse and human HCCs harboring VEGFA genomic amplification, displaying distinct biologic characteristics. Unlike common tumor amplifications, this one seems to work via heterotypic paracrine interactions; stromal VEGF receptors (VEGFR), responding to tumor VEGF-A, produce hepatocyte growth factor (HGF) that reciprocally affects tumor cells. VEGF-A inhibition results in HGF downregulation and reduced proliferation, specifically in amplicon-positive mouse HCCs. Sorafenib—the first-line drug in advanced HCC—targets multiple kinases, including VEGFRs, but has only an overall mild beneficial effect. We found that VEGFA amplification specifies mouse and human HCCs that are distinctly sensitive to sorafenib. FISH analysis of a retrospective patient cohort showed markedly improved survival of sorafenib-treated patients with VEGFA-amplified HCCs, suggesting that VEGFA amplification is a potential biomarker for HCC response to VEGF-A–blocking drugs. Significance: Using a mouse model of inflammation-driven cancer, we identified a subclass of HCC carrying VEGFA amplification, which is particularly sensitive to VEGF-A inhibition. We found that a similar amplification in human HCC identifies patients who favorably responded to sorafenib—the first-line treatment of advanced HCC—which has an overall moderate therapeutic efficacy. Cancer Discov; 4(6); 730–43. ©2014 AACR. See related commentary by Luo and Feng, p. 640 This article is highlighted in the In This Issue feature, p. 621

Expression of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen and a potent angiogenic factor, is upregulated in response to a hypoxic or hypoglycemic stress. Here we show that the increase in steady-state levels of VEGF mRNA is partly due to transcriptional activation but mostly due to increase in mRNA stability. Both oxygen and glucose deficiencies result in extension of the VEGF mRNA half-life in a protein synthesis-dependent manner. Viewing VEGF as a stress-induced gene, we compared its mode of regulation with that of other stress-induced genes. Results showed that under nonstressed conditions, VEGF shares with the glucose transporter GLUT-1 a relatively short half-life (0.64 and 0.52 h, respectively), which is extended fourfold and more than eightfold, respectively, when cells are deprived of either oxygen or glucose. In contrast, the mRNAs of another hypoxia-inducible and hypoglycemia-inducible gene, grp78, as well as that of HSP70, were not stabilized by these metabolic insults. To show that VEGF and GLUT-1 are coinduced in differentially stressed microenvironments, multicell spheroids representing a clonal population of glioma cells in which each cell layer is differentially stressed were analyzed by in situ hybridization. Cellular microenvironments conducive to induction of VEGF and GLUT-1 were completely coincidental. These findings show that two different consequences of tissue ischemia, namely, hypoxia and glucose deprivation, induce VEGF and GLUT-1 expression by similar mechanisms. These proteins function, in turn, to satisfy the tissue needs through expanding its vasculature and improving its glucose utilization, respectively.

The extension in human lifespan in the last century results in a significant increase in incidence of age related diseases. It is therefore crucial to identify key factors that control elderly healthspan. Similar to dietary restriction, mice overexpressing the NAD+ dependent protein deacylase SIRT6 (MOSES) live longer and have reduced IGF-1 levels. However, it is as yet unknown whether SIRT6 also affects various healthspan parameters. Here, a range of age related phenotypes was evaluated in MOSES mice. In comparison to their wild-type (WT) littermates, old MOSES mice showed amelioration of a variety of age-related disorders, including: improved glucose tolerance, younger hormonal profile, reduced age-related adipose inflammation and increased physical activity. The increased activity was accompanied with increased muscle AMP-activated protein kinase (AMPK) activity. Altogether, these results indicate that overexpression of SIRT6 in mice retards important aspects of the aging process and suggest SIRT6 to be a potential therapeutic target for the treatment of a set of age-related disorders.

The receptor for advanced glycation endproducts (RAGE) is a multiligand receptor and member of the immunoglobulin superfamily. RAGE is mainly involved in tissue damage and chronic inflammatory disorders, sustaining the inflammatory response upon engagement with damage-associated molecular pattern molecules (DAMPs) such as S100 proteins and high-mobility group box 1 (HMGB1). Enhanced expression of RAGE and its ligands has been demonstrated in distinct tumors and several studies support its crucial role in tumor progression and metastasis by still unknown mechanisms. Here we show that RAGE supports hepatocellular carcinoma (HCC) formation in the Mdr2−/− mouse model, a prototype model of inflammation-driven HCC formation, which mimics the human pathology. Mdr2−/− Rage−/− (dKO) mice developed smaller and fewer HCCs than Mdr2−/− mice. Interestingly, although in preneoplastic Mdr2−/− livers RAGE ablation did not affect the onset of inflammation, premalignant dKO livers showed reduced liver damage and fibrosis, in association with decreased oval cell activation. Oval cells expressed high RAGE levels and displayed reduced proliferation upon RAGE silencing. Moreover, stimulation of oval cells with HMGB1 promoted an ERK1/2-Cyclin D1-dependent oval cell proliferation in vitro. Finally, genetic and pharmacologic blockade of RAGE signaling impaired oval cell activation in an independent mouse model of oval cell activation, the choline deficient ethionine-supplemented dietary regime. Conclusion: Our data identified a novel function of RAGE in regulating oval cell activation and tumor development in inflammation-associated liver carcinogenesis. (Hepatology 2013)

The nuclear factor-kappaB (NF-κB) signaling pathway has been recently shown to participate in inflammation-induced cancer progression. Here, we describe a detailed analysis of the NF-κB–dependent gene regulatory network in the well-established Mdr2 knockout mouse model of inflammation-associated liver carcinogenesis. Expression profiling of NF-κB–deficient and NF-κB–proficient hepatocellular carcinoma (HCC) revealed a comprehensive list of known and novel putative NF-κB target genes, including S100a8 and S100a9. We detected increased co-expression of S100A8 and S100A9 proteins in mouse HCC cells, in human HCC tissue, and in the HCC cell line Hep3B on ectopic RelA expression. Finally, we found a synergistic function for S100A8 and S100A9 in Hep3B cells resulting in a significant induction of reactive oxygen species (ROS), accompanied by enhanced cell survival. Conclusion: We identified S100A8 and S100A9 as novel NF-κB target genes in HCC cells during inflammation-associated liver carcinogenesis and provide experimental evidence that increased co-expression of both proteins supports malignant progression by activation of ROS-dependent signaling pathways and protection from cell death. (HEPATOLOGY 2009.)

Clinical observations and mouse models have suggested that inflammation can be pro-tumorigenic. Since chemokines are critical in leukocyte trafficking, we hypothesized that chemokines play essential roles in inflammation-associated cancers. Screening for 37 chemokines in prostate cancer cell lines and xenografts revealed CXCL16, the ligand for the receptor CXCR6, as the most consistently expressed chemokine. Immunohistochemistry and/or immunofluorescence and confocal imaging of 121 human prostate specimens showed that CXCL16 and CXCR6 were co-expressed, both on prostate cancer cells and adjacent T cells. Expression levels of CXCL16 and CXCR6 on cancer cells correlated with poor prognostic features including high-stage and high-grade, and expression also correlated with post-inflammatory changes in the cancer stroma as revealed by loss of alpha-smooth muscle actin. Moreover, CXCL16 enhanced the growth of CXCR6-expressing cancer and primary CD4 T cells. We studied expression of CXCL16 in an additional 461 specimens covering 12 tumor types, and found that CXCL16 was expressed in multiple human cancers associated with inflammation. Our study is the first to describe the expression of CXCL16/CXCR6 on both cancer cells and adjacent T cells in humans, and to demonstrate correlations between CXCL16 and CXCR6 vs. poor both prognostic features and reactive changes in cancer stoma. Taken together, our data suggest that CXCL16 and CXCR6 may mark cancers arising in an inflammatory milieu and mediate pro-tumorigenic effects of inflammation through direct effects on cancer cell growth and by inducing the migration and proliferation of tumor-associated leukocytes.

Far upstream element-binding proteins (FBPs) represent a family of transcription factors, which are highly overexpressed in the majority human liver cancer (hepatocellular carcinoma; HCC). Nuclear accumulation of FBP-1, -2, and -3 in HCC cells support cell proliferation and migration. However, the mode of FBP dysregulation in HCC cells has not been defined so far. Genomic alterations of the fubp (FBP-1; chr. 1p31.1), khsrp (FBP-2; chr. 19.p13.3), and fubp3 (FBP-3; 9p34.11) gene loci in primary human HCCs were determined by matrix-comparative genomic hybridization (CGH). In order to identify specific stimuli of FBP enrichment, different human HCC cell lines were cultured under different cell density and hypoxic conditions or treated with growth factors (e.g., TGFß, IGF-II), chemical inhibitors (e.g., AG1478, SB203580, MEK1/2, sorafenib, and PI3K inhibitors), and gene-specific siRNAs (e.g., Akt1-3, rictor, raptor). The in vivo relevance of the findings was confirmed using HCC mouse models (e.g., Mdr2-/- animals) and human HCC tissue specimens (immunohistochemistry and western blotting). Only few genomic gains were found for all FBP family members in human HCC samples (e.g., 6% for fubp). In vitro, inhibition of EGFR (by AG1478) or administration of the multi-kinase inhibitor sorafenib reduced FBP protein levels. Furthermore, inhibition of the PI3K/Akt/mTOR pathway revealed strong effects on FBP protein half-live without significant impact on FBP transcription. Other pathways or stimuli only caused minor effects on FBP expression (e.g., p38 or Raf1 inhibition). These findings were confirmed in mouse HCC models with high-level expression of FBPs, where injection of sorafenib or rapamycin significantly diminished FBP amounts. Reduction of FBP concentrations after inhibition of the PI3K/Akt/mTOR pathway was rescued after inhibition of caspase-3/-7 activity. Lastly, a significant correlation between pAkt and nuclear FBP expression was detected in human HCC tissues. These results demonstrate that activation of the PI3K/Akt/mTOR signalling axis is responsible for increased stabilisation of FBPs in human HCC cells. Elevated FBP half-live is at least partly mediated through crosstalk between the PI3K/Akt/mTOR pathway and caspase activity. These data are important for a deeper understanding of how druggable upstream regulators may affect FBP bioavailability in hepatocarcinogenesis. Citation Format: Jana Samarin, Ilan Stein, Elad Horwitz, Xin Chen, Mona Malz, Eli Pikarsky, Diego Calvisi, Peter Schirmacher, Kai Breuhahn. PI3K/AKT-induced stabilization of FUSE binding proteins (FBPs) in liver cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2157. doi:10.1158/1538-7445.AM2015-2157

The Src-related protein kinase Lyn plays an important role in B-cell activation. However, several lines of evidence suggest that it is also involved in the control of cellular proliferation and the inhibition of apoptosis. We have discovered that Lyn is expressed in normal prostate epithelia, in 95% of primary human prostate cancer (PC) specimens examined, and in all of the PC cell lines that we assayed. Moreover, Lyn knockout mice display abnormal prostate gland morphogenesis, which suggests that Lyn plays an important role in prostate epithelium development and implies that Lyn is a candidate target for specific therapy for PC. Using a drug-design strategy to construct sequence-based peptide inhibitors, a Lyn-specific inhibitor, KRX-123, targeting a unique interaction site within Lyn, was synthesized. KRX-123 was found to inhibit cellular proliferation in three hormone-refractory PC cell lines, DU145, PC3, and TSU-Pr1 with IC(50) values of 2-4 micro M. In vivo, tumor volume of DU145 explants in nude mice was significantly reduced after once-a-week injections of KRX-123, at a dose of 10 mg/kg, for a period of 5 weeks. Histological analyses of the treated tumors indicated extensive apoptosis. Thus, we suggest that Lyn inhibition may serve as a prime target for the treatment of hormone-refractory PC.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent mitogen and migration factor for vascular smooth muscle cells (SMC), promoted neovascularization in vivo in the rabbit cornea. MRI demonstrated quantitatively the angiogenic effect of HB-EGF when introduced subcutaneously into nude mice. HB-EGF is not directly mitogenic to endothelial cells but it induced the migration of bovine endothelial cells and release of endothelial cell mitogenic activity from bovine vascular SMC. This mitogenic activity was specifically blocked by neutralizing anti-vascular endothelial growth factor (VEGF) antibodies. In contrast, EGF or transforming growth factor-α (TGF-α) had almost no effect on release of endothelial mitogenicity from SMC. In addition, RT-PCR analysis demonstrated that VEGF165 mRNA levels were increased in vascular SMC 4–10-fold by 0.35–2 nM of HB-EGF, respectively. Our data suggest that HB-EGF, as a mediator of intercellular communication, may play a new important role in supporting wound healing, tumor progression and atherosclerosis by stimulating angiogenesis.

Androgen activity plays a key role in prostate cancer progression. Androgen receptor (AR) is the main mediator of androgen activity in the prostate, through its ability to act as a transcription mediator. Here we performed a genome-wide analysis of human AR binding to promoters in the presence of an agonist or antagonist in an androgen dependent prostate cancer cell line. Many of the AR bound promoters are bound in all examined conditions while others are bound only in the presence of an agonist or antagonist. Several motifs are enriched in AR bound promoters, including the AR Response Element (ARE) half-site and recognition elements for the transcription factors OCT1 and SOX9. This suggests that these 3 factors could define a module of co-operating transcription factors in the prostate. Interestingly, AR bound promoters are preferentially located in AT rich genomic regions. Analysis of mRNA expression identified chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF1) as a direct AR target gene that is downregulated upon binding by the agonist liganded AR. COUP-TF1 immunostaining revealed nucleolar localization of COUP-TF1 in epithelium of human androgen dependent prostate cancer, but not in adjacent benign prostate epithelium. Stromal cells both in human and mouse prostate show nuclear COUP-TF1 staining. We further show that there is an inverse correlation between COUP-TF1 expression in prostate stromal cells and the rising levels of androgen with advancing puberty. This study extends the pool of recognized putative AR targets and identifies a negatively regulated target of AR – COUP-TF1 – which could possibly play a role in human prostate cancer.

in this study we tested the hypothesis that due to its growth promoting function, SIRT1 expression may be a positive factor in the growth of HCC and therefore inhibiting its activity may be a means to limit the growth of HCC. Methods: HCC cell lines (Hep3B, HepG2, HuH7, HLE, HLF, HepKK1, skHep1) were screened for mRNA expression of SIRT1–7. To inhibit SIRT1, cells were either transduced with lentiviruses expressing shRNA sequences targeting SIRT1 or treated with small molecule inhibitors, sirtinol or cambinol. Genetic deletion was confirmed by RT-PCR and Western-blot. Effect of SIRT1 inhibition was monitored by changes in morphology, proliferation and cell cycle in vitro. Half a million wild type and SIRT1 knock-down HCC cells were transplanted under the liver capsule of Rag2gc−/− immunodeficient mice and tumor growth was monitored by bioluminescence. Results: SIRT1 mRNA and protein was significantly higher expressed in HCC cell lines compared to normal liver cells, whereas other family members, SIRT2–7, were expressed at equal or lower levels. Inhibition of SIRT1 in HepG2 and Hep3B cells either by shRNA or with sirtinol or cambinol altered cell morphology, impaired proliferation and in HepG2 resulted in a decrease of dedifferentiation markers AFP and glypican. SIRT1 inhibition in Hep3B cells resulted a senescent morphology and expression of SA b-gal. Intrahepatic tumors developed in 84% of mice injected with control cells, while only 33% of the animals developed tumors with SIRT1 knock-down cells. In addition, cambinol reduced xenograft growth in mice compared to vehicle treated controls. Conclusion: Targeted deletion of SIRT1 expression impairs the growth of HCC cells in vivo and vitro. Activation of SIRT1 may have a beneficial role in non-malignant liver tissue, however in malignant HCCs, inhibiting SIRT1 activity may be a novel therapeutic option.

The yeast mitochondrial and cytosolic isoenzymes of fumarase, which are encoded by a single nuclear gene (FUM1), follow a unique mechanism of protein subcellular localization and distribution. Translation of all FUM1 messages initiates only from the 5'-proximal AUG codon and results in a single translation product that contains the targeting sequence located within the first 32 amino acids of the precursor. All fumarase molecules synthesized in the cell are processed by the mitochondrial matrix signal peptidase; nevertheless, most of the enzyme (80 to 90%) ends up in the cytosol. The translocation and processing of fumarase are cotranslational. We suggest that in Saccharomyces cerevisiae, the single type of initial translation product of the FUM1 gene is first partially translocated, and then a subset of these molecules continues to be fully translocated into the organelle, whereas the rest are folded into an import-incompetent state and are released by the retrograde movement of fumarase into the cytosol.