NAMPT Overexpression Drives Cell Growth in Polycystic Liver Disease through Mitochondrial Metabolism Regulation.

A group of genetic diseases known as polycystic liver disease (PLD) are distinguished by the gradual development of fluid-filled hepatic cysts formed from cholangiocytes and commonly related to primary cilia defects. The NAD salvage pathway, which sustains cellular bioenergetics and supplies a required substrate for tasks important to rapidly multiplying cells, has a rate-limiting phase that is mediated by nicotinamide phosphoribosyltransferase (NAMPT). In this study, the efficacy and mechanisms of action of FK866, a novel, high-potency NAMPT inhibitor with a good toxicity profile, were assessed. NAMPT-siRNA and FK866 reduced NAD levels and inhibited the proliferation of PLD cells in a dose-dependent manner. Notably, this pharmacologic and siRNA-mediated suppression of NAMPT was less effective in normal cells at the same concentrations. The addition of nicotinamide mononucleotide (NMN), a byproduct of NAMPT that restores NAD concentration, rescued the cellular viability of PLD cells and verified the on-target action of FK866. In FK866-treated PLD cells, mitochondrial respiration and ATP production were impaired and reactive oxygen species production was induced. Importantly, FK866 treatment was associated with improved effects of octreotide, a drug used for PLD treatment. As a result, the use of NAMPT inhibitors, including FK866 therapy, offers the possibility of a further targeted strategy for the therapeutic treatment of PLD.

Cholangiocyte ciliary defects induce sustained epidermal growth factor receptor signaling.

BACKGROUND AND AIMS: The primary cilium, an organelle that protrudes from cell surfaces, is essential for sensing extracellular signals. With disturbed cellular communication and chronic liver pathologies, this organelle's dysfunctions have been linked to disorders, including polycystic liver disease and cholangiocarcinoma. The goal of this study was to elucidate the relationship between primary cilia and the crucial regulator of cellular proliferation, the epidermal growth factor receptor (EGFR) signaling pathway, which has been associated with various clinical conditions. APPROACH AND RESULTS: The study identified aberrant EGFR signaling pathways in cholangiocytes lacking functional primary cilia using liver-specific intraflagellar transport 88 knockout mice, a Pkhd1 mutant rat model, and human cell lines that did not have functional cilia. Cilia-deficient cholangiocytes showed persistent EGFR activation because of impaired receptor degradation, in contrast to their normal counterparts, where EGFR localization to the cilia promotes appropriate signaling. Using histone deacetylase 6 inhibitors to restore primary cilia accelerates EGFR degradation, thereby reducing maladaptive signaling. Importantly, experimental intervention with the histone deacetylase 6 inhibitor tubastatin A in an orthotopic rat model moved EGFR to cilia and reduced ERK phosphorylation. Concurrent administration of EGFR and histone deacetylase 6 inhibitors in cholangiocarcinoma and polycystic liver disease cells demonstrated synergistic antiproliferative effects, which were associated with the restoration of functioning primary cilia. CONCLUSIONS: This study's findings shed light on ciliary function and robust EGFR signaling with slower receptor turnover. We could use therapies that restore the function of primary cilia to treat EGFR-driven diseases in polycystic liver disease and cholangiocarcinoma.

The Role of Gut Microbiome-Derived Short-Chain Fatty Acid Butyrate in Hepatobiliary Diseases.

The short-chain fatty acid butyrate, produced from fermentable carbohydrates by gut microbiota in the colon, has multiple beneficial effects on human health. At the intestinal level, butyrate regulates metabolism, helps in the transepithelial transport of fluids, inhibits inflammation, and induces the epithelial defense barrier. The liver receives a large amount of short-chain fatty acids via the blood flowing from the gut via the portal vein. Butyrate helps prevent nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, inflammation, cancer, and liver injuries. It ameliorates metabolic diseases, including insulin resistance and obesity, and plays a direct role in preventing fatty liver diseases. Butyrate has different mechanisms of action, including strong regulatory effects on the expression of many genes by inhibiting the histone deacetylases and modulating cellular metabolism. The present review highlights the wide range of beneficial therapeutic and unfavorable adverse effects of butyrate, with a high potential for clinically important uses in several liver diseases.

Histone Deacetylase Sirtuin 1 Promotes Loss of Primary Cilia in Cholangiocarcinoma.

BACKGROUND AND AIMS: Sirtuin 1 (SIRT1) is a complex NAD+ -dependent protein deacetylase known to act as a tumor promoter or suppressor in different cancers. Here, we describe a mechanism of SIRT1-induced destabilization of primary cilia in cholangiocarcinoma (CCA). APPROACH AND RESULTS: A significant overexpression of SIRT1 was detected in human CCA specimens and CCA cells including HuCCT1, KMCH, and WITT1 as compared with normal cholangiocytes (H69 and NHC). Small interfering RNA (siRNA)-mediated knockdown of SIRT1 in HuCCT1 cells induced cilia formation, whereas overexpression of SIRT1 in normal cholangiocytes suppressed ciliary expression. Activity of SIRT1 was regulated by presence of NAD+ in CCA cells. Inhibition of NAD -producing enzyme nicotinamide phosphoribosyl transferase increased ciliary length and frequency in CCA cells and in SIRT1-overexpressed H69 cells. Furthermore, we also noted that SIRT1 induces the proteasomal mediated degradation of ciliary proteins, including α-tubulin, ARL13B, and KIF3A. Moreover, overexpression of SIRT1 in H69 and NHC cells significantly induced cell proliferation and, conversely, SIRT1 inhibition in HuCCT1 and KMCH cells using siRNA or sirtinol reduced cell proliferation. In an orthotopic transplantation rat CCA model, the SIRT1 inhibitor sirtinol reduced tumor size and tumorigenic proteins (glioma-associated oncogene 1, phosphorylated extracellular signal-regulated kinase, and IL-6) expression. CONCLUSIONS: In conclusion, these results reveal the tumorigenic role of SIRT1 through modulation of primary cilia formation and provide the rationale for developing therapeutic approaches for CCA using SIRT1 as a target.

Synthetic Conjugates of Ursodeoxycholic Acid Inhibit Cystogenesis in Experimental Models of Polycystic Liver Disease.

BACKGROUND AND AIMS: Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive development of symptomatic biliary cysts. Current surgical and pharmacological approaches are ineffective, and liver transplantation represents the only curative option. Ursodeoxycholic acid (UDCA) and histone deacetylase 6 inhibitors (HDAC6is) have arisen as promising therapeutic strategies, but with partial benefits. APPROACH AND RESULTS: Here, we tested an approach based on the design, synthesis, and validation of a family of UDCA synthetic conjugates with selective HDAC6i capacity (UDCA-HDAC6i). Four UDCA-HDAC6i conjugates presented selective HDAC6i activity, UDCA-HDAC6i #1 being the most promising candidate. UDCA orientation within the UDCA-HDAC6i structure was determinant for HDAC6i activity and selectivity. Treatment of polycystic rats with UDCA-HDAC6i #1 reduced their hepatomegaly and cystogenesis, increased UDCA concentration, and inhibited HDAC6 activity in liver. In cystic cholangiocytes UDCA-HDAC6i #1 restored primary cilium length and exhibited potent antiproliferative activity. UDCA-HDAC6i #1 was actively transported into cells through BA and organic cation transporters. CONCLUSIONS: These UDCA-HDAC6i conjugates open a therapeutic avenue for PLDs.

HDAC6-dependent ciliophagy is involved in ciliary loss and cholangiocarcinoma growth in human cells and murine models.

Reduced ciliary expression is reported in several tumors, including cholangiocarcinoma (CCA). We previously showed primary cilia have tumor suppressor characteristics, and HDAC6 is involved in ciliary loss. However, mechanisms of ciliary disassembly are unknown. Herein, we tested the hypothesis that HDAC6-dependent autophagy of primary cilia, i.e., ciliophagy, is the main mechanism driving ciliary disassembly in CCA. Using the cancer genome atlas database, human CCA cells, and a rat orthotopic CCA model, we assessed basal and HDAC6-regulated autophagy levels. The effects of RNA-silencing or pharmacological manipulations of ciliophagy on ciliary expression were assessed. Interactions of ciliary proteins with autophagy machinery was assessed by immunoprecipitations. Cell proliferation was assessed by MTS and IncuCyte. A CCA rat model was used to assess the effects of pharmacological inhibition of ciliophagy in vivo. Autophagy is increased in human CCA, as well as in a rat orthotopic CCA model and human CCA cell lines. Autophagic flux was decreased via inhibition of HDAC6, while it was increased by its overexpression. Inhibition of autophagy and HDAC6 restores cilia and decreases cell proliferation. LC3 interacts with HDAC6 and ciliary proteins, and the autophagy cargo receptor involved in targeting ciliary components to the autophagy machinery is primarily NBR1. Treatment with chloroquine, Ricolinostat (ACY-1215), or their combination decreased tumor growth in vivo. Mice that overexpress the autophagy transcription factor TFEB show a decrease of ciliary number. These results suggest that ciliary disassembly is mediated by HDAC6-regulated autophagy, i.e., ciliophagy. Inhibition of ciliophagy may decrease cholangiocarcinoma growth and warrant further investigations as a potential therapeutic approach.NEW & NOTEWORTHY This work identifies novel targets against primary ciliary disassembly that can lead to new cholangiocarcinoma therapeutic strategies. Furthermore, ciliary loss has been described in different tumors, increasing the significance of our research.

The Chemosensory Function of Primary Cilia Regulates Cholangiocyte Migration, Invasion, and Tumor Growth.

Cholangiocytes, the epithelial cells lining the biliary tree in the liver, express primary cilia that can detect several kinds of environmental signals and then transmit this information into the cell. We have reported that cilia are significantly reduced in cholangiocarcinoma (CCA) and that the experimental deciliation of normal cells induces a malignant-like phenotype with increased proliferation, anchorage-independent growth, invasion, and migration. Here, we tested the hypothesis that the chemosensory function of cholangiocyte primary cilia acts as a mechanism for tumor suppression. We found that in the presence of extracellular nucleotides cilia-dependent chemosensation of the nucleotides inhibited migration and invasion in normal ciliated cholangiocytes through a P2Y11 receptor and liver kinase B1 (LKB1)-phosphatase and tensin homolog-AKT-dependent mechanism. In contrast, in normal deciliated cholangiocytes and CCA cells, the nucleotides induced the opposite effects, i.e., increased migration and invasion. As activation of LKB1 through a cilia-dependent mechanism was required for the nucleotide-mediated inhibitory effects on migration and invasion, we attempted to activate LKB1 directly, independent of ciliary expression, using the compound hesperidin methyl chalcone (HMC). We found that HMC induced activation of LKB1 in both ciliated and deciliated cells in vitro, resulting in the inhibition of migration and proliferation. Furthermore, using a rat syngeneic orthotopic CCA model, we found that HMC inhibited tumor growth in vivo. Conclusion: These findings highlight the importance of the chemosensory function of primary cilia for the control of migration and invasion and suggest that, by directly activating LKB1 and bypassing the need for primary cilia, it is possible to emulate this chemosensory function in CCA cells; these data warrant further studies evaluating the possibility of using HMC as therapy for CCA.

Combination of a Histone Deacetylase 6 Inhibitor and a Somatostatin Receptor Agonist Synergistically Reduces Hepatorenal Cystogenesis in an Animal Model of Polycystic Liver Disease.

Hepatic cystogenesis in polycystic liver disease (PLD) is associated with abnormalities in multiple cellular processes, including elevated cAMP and overexpression of histone deacetylase 6 (HDAC6). Disease progression in polycystic kidney (PCK) rats (an animal model of PLD) is attenuated by inhibition of either cAMP production or HDAC6. Therefore, we hypothesized that concurrent targeting of HDAC6 and cAMP would synergistically reduce cyst growth. Changes in hepatorenal cystogenesis were examined in PCK rats treated with a pan-HDAC inhibitor, panobinostat; three specific HDAC6 inhibitors, ACY-1215, ACY-738, and ACY-241; and a combination of ACY-1215 and the somatostatin receptor analogue, pasireotide. We also assessed effects of ACY-1215 and pasireotide alone and in combination on cell proliferation, cAMP production, and expression of acetylated α-tubulin in vitro in cultured cholangiocytes and the length of primary cilia and the frequency of ciliated cholangiocytes in vivo in PCK rats. Panobinostat and all three HDAC6 inhibitors decreased hepatorenal cystogenesis in PCK rats. ACY-1215 was more effective than other HDAC inhibitors and was chosen for combinational treatment. ACY-1215 + pasireotide combination synergistically reduced cyst growth and increased length of primary cilia in PCK rats. In cultured cystic cholangiocytes, ACY-1215 + pasireotide combination concurrently decreased cell proliferation and inhibited cAMP levels. These data suggest that the combination of drugs that inhibit HDAC6 and cAMP may be an effective therapy for PLD.

MicroRNA (miR)-433 and miR-22 dysregulations induce histone-deacetylase-6 overexpression and ciliary loss in cholangiocarcinoma.

Cholangiocytes normally express primary cilia, a multisensory organelle that detects signals from the cellular environment. Cilia are significantly reduced in cholangiocarcinoma (CCA) by a mechanism involving overexpression of histone deacetylase 6 (HDAC6). Despite HDAC6 overexpression in CCA, we found no differences in its mRNA level, suggesting a posttranscriptional regulation, possibly involving microRNAs (miRNAs). Here, we describe that at least two HDAC6-targeting miRNAs, miR-433 and miR-22, are down-regulated in CCA both in vitro and in vivo. Experimental restoration of these miRNAs in CCA cells reduced HDAC6 expression, induced ciliary restoration, and decreased the malignant phenotype. Furthermore, in contrast to the mature forms, levels of precursor forms of these miRNAs were higher in CCA compared to normal cholangiocytes and accumulated in the nuclei, suggesting a defective nuclear export. We assessed the expression of Exportin-5, the protein responsible for transporting miRNA precursors out of the nucleus, and found it to be reduced by 50% in CCA compared to normal cholangiocytes. Experimental overexpression of Exportin-5 in CCA cells restored precursor and mature forms of these miRNAs to normal levels, inducing a decrease in the expression of HDAC6 and a decrease in the malignant phenotype. Conversely, short hairpin RNA (shRNA) depletion of Exportin-5 in normal cholangiocytes resulted in increased nuclear retention of precursor miRNAs, decreased mature miRNAs, increased cell proliferation, and shorter cilia. CONCLUSION: These data suggest that down-regulated Exportin-5 impairs the nuclear export of miR-433 and miR-22 precursor forms, causing a decrease in levels of mature miR-433 and miR-22 forms, and leading to overexpression of HDAC6 and ciliary loss in CCA. (Hepatology 2018).

The cholangiocyte primary cilium in health and disease.

Cholangiocytes, like most cells, express primary cilia extending from their membranes. These organelles function as antennae which detect stimuli from bile and transmit the information into cells regulating several signaling pathways involved in secretion, proliferation and apoptosis. The ability of primary cilia to detect different signals is provided by ciliary associated proteins which are expressed in its membrane. Defects in the structure and/or function of these organelles lead to cholangiociliopathies that result in cholangiocyte hyperproliferation, altered fluid secretion and absorption. Since primary cilia dysfunction has been observed in several epithelial tumors, including cholangiocarcinoma (CCA), primary cilia have been proposed as tumor suppressor organelles. In addition, the loss of cilia is associated with dysregulation of several molecular pathways resulting in CCA development and progression. Thus, restoration of the primary cilia may be a potential therapeutic approach for several ciliopathies and CCA.

SOX17 regulates cholangiocyte differentiation and acts as a tumor suppressor in cholangiocarcinoma.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA) is a biliary malignancy linked to genetic and epigenetic abnormalities, such as hypermethylation of SOX17 promoter. Here, the role of SOX17 in cholangiocyte differentiation and cholangiocarcinogenesis was studied. METHODS: SOX17 expression/function was evaluated along the differentiation of human induced pluripotent stem cells (iPSC) into cholangiocytes, in the dedifferentiation process of normal human cholangiocytes (NHC) in culture and in cholangiocarcinogenesis. Lentiviruses for SOX17 overexpression or knockdown were used. Gene expression and DNA methylation profiling were performed. RESULTS: SOX17 expression is induced in the last stage of cholangiocyte differentiation from iPSC and regulates the acquisition of biliary markers. SOX17 becomes downregulated in NHC undergoing dedifferentiation; experimental SOX17 knockdown in differentiated NHC downregulated biliary markers and promoted baseline and Wnt-dependent proliferation. SOX17 expression is lower in human CCA than in healthy tissue, which correlates with worse survival after tumor resection. In CCA cells, SOX17 overexpression decreased their tumorigenic capacity in murine xenograft models, which was related to increased oxidative stress and apoptosis. In contrast, SOX17 overexpression in NHC did not affect their survival but inhibited their baseline proliferation. In CCA cells, SOX17 inhibited migration, anchorage-independent growth and Wnt/ß-catenin-dependent proliferation, and restored the expression of biliary markers and primary cilium length. In human CCA, SOX17 promoter was found hypermethylated and its expression inversely correlates with the methylation grade. In NHC, Wnt3a decreased SOX17 expression in a DNMT-dependent manner, whereas in CCA, DNMT1 inhibition or silencing upregulated SOX17. CONCLUSIONS: SOX17 regulates the differentiation and maintenance of the biliary phenotype and functions as a tumor suppressor for CCA, being a potential prognostic marker and a promising therapeutic target. LAY SUMMARY: Understanding the molecular mechanisms involved in the pathogenesis of CCA is key in finding new valuable diagnostic and prognostic biomarkers, as well as therapeutic targets. This study provides evidence that SOX17 regulates the differentiation and maintenance of the biliary phenotype, and its downregulation promotes their tumorigenic transformation. SOX17 acts as a tumor suppressor in CCA and its genetic, molecular and/or pharmacological restoration may represent a new promising therapeutic strategy. Moreover, SOX17 expression correlates with the outcome of patients after tumor resection, being a potential prognostic biomarker.

Targeting IRES-Mediated p53 Synthesis for Cancer Diagnosis and Therapeutics.

While translational regulation of p53 by the internal ribosome entry site (IRES) at its 5'-untranslated region following DNA damage has been widely accepted, the detailed mechanism underlying the translational control of p53 by its IRES sequence is still poorly understood. In this review, we will focus on the latest progress in identifying novel regulatory proteins of the p53 IRES and in uncovering the functional connection between defective IRES-mediated p53 translation and tumorigenesis. We will also discuss how these findings may lead to a better understanding of the process of oncogenesis and open up new avenues for cancer diagnosis and therapeutics.

MicroRNAs and benign biliary tract diseases.

Cholangiocytes, the epithelial cells lining the biliary tree, represent only a small portion of the total liver cell population (3-5%), but they are responsible for the secretion of up to 40% of total daily bile volume. In addition, cholangiocytes are the target of a diverse group of liver diseases affecting the biliary tract, the cholangiopathies; for most of these conditions, the pathological mechanisms are unclear. MicroRNAs (miRNAs) are small, noncoding RNAs that posttranscriptionally regulate gene expression. Thus, it is not surprising that altered miRNA profiles underlie the dysregulation of several proteins involved in the pathobiology of the cholangiopathies, as well as showing promise as diagnostic and prognostic tools. Here the authors review recent work relevant to the role of miRNAs in the etiopathogenesis of several of the cholangiopathies (i.e., fibroinflammatory cholangiopathies and polycystic liver diseases), discuss their value as prognostic and diagnostic tools, and provide suggestions for further research.

HDAC6 is overexpressed in cystic cholangiocytes and its inhibition reduces cystogenesis.

Polycystic liver disease (PLD) is a member of the cholangiopathies, a group of liver diseases in which cholangiocytes, the epithelia lining of the biliary tree, are the target cells. PLDs are caused by mutations in genes involved in intracellular signaling pathways, cell cycle regulation, and ciliogenesis, among others. We previously showed that cystic cholangiocytes have abnormal cell cycle profiles and malfunctioning cilia. Because histone deacetylase 6 (HDAC6) plays an important role in both cell cycle regulation and ciliary disassembly, we examined the role of HDAC6 in hepatic cystogenesis. HDAC6 protein was increased sixfold in cystic liver tissue and in cultured cholangiocytes isolated from both PCK rats (an animal model of PLD) and humans with PLD. Furthermore, pharmacological inhibition of HDAC6 by Tubastatin-A, Tubacin, and ACY-1215 decreased proliferation of cystic cholangiocytes in a dose- and time-dependent manner, and inhibited cyst growth in three-dimensional cultures. Importantly, ACY-1215 administered to PCK rats diminished liver cyst development and fibrosis. In summary, we show that HDAC6 is overexpressed in cystic cholangiocytes both in vitro and in vivo, and its pharmacological inhibition reduces cholangiocyte proliferation and cyst growth. These data suggest that HDAC6 may represent a potential novel therapeutic target for cases of PLD.

Centrosomal abnormalities characterize human and rodent cystic cholangiocytes and are associated with Cdc25A overexpression.

Hepatic cystogenesis in polycystic liver diseases is associated with abnormalities of cholangiocyte cilia. Given the crucial association between cilia and centrosomes, we tested the hypothesis that centrosomal defects occur in cystic cholangiocytes of rodents (Pkd2(WS25/-) mice and PCK rats) and of patients with polycystic liver diseases, contributing to disturbed ciliogenesis and cyst formation. We examined centrosomal cytoarchitecture in control and cystic cholangiocytes, the effects of centrosomal abnormalities on ciliogenesis, and the role of the cell-cycle regulator Cdc25A in centrosomal defects by depleting cholangiocytes of Cdc25A in vitro and in vivo and evaluating centrosome morphology, cell-cycle progression, proliferation, ciliogenesis, and cystogenesis. The cystic cholangiocytes had atypical centrosome positioning, supernumerary centrosomes, multipolar spindles, and extra cilia. Structurally aberrant cilia were present in cystic cholangiocytes during ciliogenesis. Depletion of Cdc25A resulted in i) a decreased number of centrosomes and multiciliated cholangiocytes, ii) an increased fraction of ciliated cholangiocytes with longer cilia, iii) a decreased proportion of cholangiocytes in G1/G0 and S phases of the cell cycle, iv) decreased cell proliferation, and v) reduced cyst growth in vitro and in vivo. Our data support the hypothesis that centrosomal abnormalities in cholangiocytes are associated with aberrant ciliogenesis and that accelerated cystogenesis is likely due to overexpression of Cdc25A, providing additional evidence that pharmacological targeting of Cdc25A has therapeutic potential in polycystic liver diseases.

Non-canonical Hedgehog signaling contributes to chemotaxis in cholangiocarcinoma.

BACKGROUND & AIMS: The Hedgehog signaling pathway contributes to cholangiocarcinoma biology. However, canonical Hedgehog signaling requires cilia, and cholangiocarcinoma cells often do not express cilia. To resolve this paradox, we examined non-canonical (G-protein coupled, pertussis toxin sensitive) Hedgehog signaling in cholangiocarcinoma cells. METHODS: Human [non-malignant (H69), malignant (HuCC-T1 and Mz-ChA-1)] and rat [non-malignant (BDE1 and NRC), and malignant (BDEneu)] cell lines were employed for this study. A BDE(ΔLoop2) cell line with the dominant-negative receptor Patched-1 was generated with the Sleeping Beauty transposon transfection system. RESULTS: Cilia expression was readily identified in non-malignant, but not in malignant cholangiocarcinoma cell lines. Although the canonical Hh signaling pathway was markedly attenuated in cholangiocarcinoma cells, they were chemotactic to purmorphamine, a small-molecule direct Smoothened agonist. Purmorphamine also induced remodeling of the actin cytoskeleton with formation of filopodia and lamellipodia-like protrusions. All these biological features of cell migration were pertussis toxin sensitive, a feature of G-protein coupled (Gis) receptors. To further test the role of Hedgehog signaling in vivo, we employed a syngeneic orthotopic rat model of cholangiocarcinoma. In vivo, genetic inhibition of the Hedgehog signaling pathway employing BDE(ΔLoop2) cells or pharmacological inhibition with a small-molecule antagonist of Smoothened, vismodegib, was tumor and metastasis suppressive. CONCLUSIONS: Cholangiocarcinoma cells exhibit non-canonical Hedgehog signaling with chemotaxis despite impaired cilia expression. This non-canonical Hedgehog signaling pathway appears to contribute to cholangiocarcinoma progression, thereby, supporting a role for Hedgehog pathway inhibition in human cholangiocarcinoma.

Pasireotide is more effective than octreotide in reducing hepatorenal cystogenesis in rodents with polycystic kidney and liver diseases.

UNLABELLED: In polycystic liver (PLD) and kidney (PKD) diseases, increased cyclic adenosine monophosphate (cAMP) levels trigger hepatorenal cystogenesis. A reduction of the elevated cAMP by targeting somatostatin receptors (SSTRs) with octreotide (OCT; a somatostatin analog that preferentially binds to SSTR2) inhibits cyst growth. Here we compare the effects of OCT to pasireotide (PAS; a more potent somatostatin analog with broader receptor specificity) on: (1) cAMP levels, cell cycle, proliferation, and cyst expansion in vitro using cholangiocytes derived from control and PCK rats (a model of autosomal recessive PKD [ARPKD]), healthy human beings, and patients with autosomal dominant PKD (ADPKD); and (2) hepatorenal cystogenesis in vivo in PCK rats and Pkd2(WS25/-) mice (a model of ADPKD). Expression of SSTRs was assessed in control and cystic cholangiocytes of rodents and human beings. Concentrations of insulin-like growth factor 1 (IGF1) and vascular endothelial growth factor (VEGF) (both involved in indirect action of somatostatin analogs), and expression and localization of SSTRs after treatment were evaluated. We found that PAS was more potent (by 30%-45%) than OCT in reducing cAMP and cell proliferation, affecting cell cycle distribution, decreasing growth of cultured cysts in vitro, and inhibiting hepatorenal cystogenesis in vivo in PCK rats and Pkd2(WS25/-) mice. The levels of IGF1 (but not VEGF) were reduced only in response to PAS. Expression of SSTR1 and SSTR2 (but not SSTR3 and SSTR5) was decreased in cystic cholangiocytes compared to control. Although both OCT and PAS increased the immunoreactivity of SSTR2, only PAS up-regulated SSTR1; neither drug affected cellular localization of SSTRs. CONCLUSION: PAS is more effective than OCT in reducing hepatorenal cystogenesis in rodent models; therefore, it might be more beneficial for the treatment of PKD and PLD.

Myeloid differentiation factor-2/LY96, a potential predictive biomarker of metastasis and poor outcomes in prostate cancer: clinical implications as a potential therapeutic target.

Prostate cancer (CaP) is the most diagnosed cancer in males and the second leading cause of cancer deaths. Patients with localized tumors are generally curable. However, no curative treatment exists for patients with advanced and metastatic disease. Therefore, identifying critical proteins involved in the metastatic process would help to develop new therapeutic options for patients with advanced and aggressive CaP. We provide strong evidence that Myeloid differentiation factor-2 (MD2) plays a critical role in metastasis and CaP progression. Analysis of tumor genomic data showed that amplifications of MD2 and increased expression are associated with poor outcomes in patients. Immunohistochemistry analysis of tumor tissues showed a correlation between the expression of MD2 and cancer progression. The Decipher-genomic test validated the potential of MD2 in predicting metastasis. In vitro studies demonstrated that MD2 confers invasiveness by activating MAPK and NF-kB signaling pathways and inducing epithelial-mesenchymal transition. Furthermore, we show that metastatic cells release MD2 (sMD2). We measured serum-sMD2 in patients and found that the level is correlated to disease extent. We determined the significance of MD2 in metastasis in vivo and as a therapeutic target, showing that the molecular and pharmacological targeting of MD2 significantly inhibited metastasis in murine models. We conclude that MD2 predicts metastatic behavior, and serum-MD2 could be studied as a potential non-invasive biomarker for metastasis, whereas MD2 presence on prostate biopsy predicts adverse disease outcome. We suggest MD2-targeted therapies could be developed as potential treatments for aggressive metastatic disease.

Ciliary subcellular localization of TGR5 determines the cholangiocyte functional response to bile acid signaling.

TGR5, the G protein-coupled bile acid receptor that transmits bile acid signaling into a cell functional response via the intracellular cAMP signaling pathway, is expressed in human and rodent cholangiocytes. However, detailed information on the localization and function of cholangiocyte TGR5 is limited. We demonstrated that in human (H69 cells) and rat cholangiocytes, TGR5 is localized to multiple, diverse subcellular compartments, with its strongest expression on the apical plasma, ciliary, and nuclear membranes. To evaluate the relationship between ciliary TGR5 and the cholangiocyte functional response to bile acid signaling, we used a model of ciliated and nonciliated H69 cells and demonstrated that TGR5 agonists induce opposite changes in cAMP and ERK levels in cells with and without primary cilia. The cAMP level was increased in nonciliated cholangiocytes but decreased in ciliated cells. In contrast, ERK signaling was induced in ciliated cholangiocytes but suppressed in cells without cilia. TGR5 agonists inhibited proliferation of ciliated cholangiocytes but activated proliferation of nonciliated cells. The observed differential effects of TGR5 agonists were associated with the coupling of TGR5 to Gαi protein in ciliated cells and Gαs protein in nonciliated cholangiocytes. The functional responses of nonciliated and ciliated cholangiocytes to TGR5-mediated bile acid signaling may have important pathophysiological significance in cilia-related liver disorders (i.e., cholangiociliopathies), such as polycystic liver disease. In summary, TGR5 is expressed on diverse cholangiocyte compartments, including a primary cilium, and its ciliary localization determines the cholangiocyte functional response to bile acid signaling.