Annalisa Morgano

Down‐regulation of the LXR transcriptome provides the requisite cholesterol levels to proliferating hepatocytes

Cholesterol homeostasis is critical for cellular proliferation. Liver X receptor (LXR) α and β are the nuclear receptors responsible for regulation of cholesterol metabolism. In physiological conditions, high intracellular cholesterol levels cause increased synthesis of oxysterols, which activate LXR, thus triggering a transcriptional response for cholesterol secretion and catabolism. Here we employed a mouse model of partial hepatectomy (PH) to dissect the molecular pathways connecting cholesterol homeostasis, cellular proliferation, and LXR. First, we show that hepatic cholesterol content increases after PH, whereas the entire LXR transcriptome is down‐regulated. Although LXR messenger RNA (mRNA) levels are unmodified, LXR target genes are significantly down‐regulated on day 1 after PH and restored to control levels on day 7, when the liver reaches normal size. The inactivation of LXR following PH is related to the reduced oxysterol availability by way of decreased synthesis, and increased sulfation and secretion. On the contrary, cholesterol synthesis is up‐regulated, and extracellular matrix remodeling is enhanced. Second, we show that reactivation of LXR by way of a synthetic ligand determines a negative modulation of hepatocyte proliferation. This effect is sustained by the reactivation of hepatic cholesterol catabolic and secretory pathways, coupled with a significant reduction of cholesterol biosynthesis. Our data unveil a previously unrecognized and apparently paradoxical scenario of LXR modulation. During liver regeneration LXR activity is abated in spite of increasing intracellular cholesterol levels. Turning off LXR‐transcriptional pathways is crucial to guaranteeing the requisite intracellular cholesterol levels of regenerating hepatocytes. In line with this hypothesis, pharmacological LXR reactivation during PH significantly reduces liver regeneration capacity. (HEPATOLOGY 2010.)

Prevention of spontaneous hepatocarcinogenesis in farnesoid X receptor-null mice by intestinal-specific farnesoid X receptor reactivation

Farnesoid X receptor (FXR) is the master regulator of bile acid (BA) homeostasis because it controls BA synthesis, influx, efflux, and detoxification in the gut/liver axis. Deregulation of BA homeostasis has been linked to hepatocellular carcinoma (HCC), and spontaneous hepatocarcinogenesis has been observed in FXR‐null mice. This dreaded liver neoplasm has been associated with both FXR gene deletion and BA‐mediated metabolic abnormalities after inactivation of FXR transcriptional activity. In the present study, we addressed the hypothesis that intestinal selective FXR reactivation would be sufficient to restore the fibroblast growth factor 15 (FGF15)/cholesterol‐7alpha‐hydroxylase (Cyp7a1) enterohepatic axis and eventually provide protection against HCC. To this end, we generated FXR‐null mice with re‐expression of constitutively active FXR in enterocytes (FXR−/−iVP16FXR) and corresponding control mice (FXR−/−iVP16). In FXR‐null mice, intestinal selective FXR reactivation normalized BA enterohepatic circulation along with up‐regulation of intestinal FXR transcriptome and reduction of hepatic BA synthesis. At 16 months of age, intestinal FXR reactivation protected FXR‐null mice from spontaneous HCC development that occurred in otherwise FXR‐null mice. Activation of intestinal FXR conferred hepatoprotection by restoring hepatic homeostasis, limiting cellular proliferation through reduced cyclinD1 expression, decreasing hepatic inflammation and fibrosis (decreased signal transducer and activator of transcription 3 activation and curtailed collagen deposition). Conclusion: Intestinal FXR is sufficient to restore BA homeostasis through the FGF15 axis and prevent progression of liver damage to HCC even in the absence of hepatic FXR. Intestinal‐selective FXR modulators could stand as potential therapeutic intervention to prevent this devastating hepatic malignancy, even if carrying a somatic FXR mutation. (Hepatology 2015;61:161–170)

The Insulin Receptor Substrate 1 (Irs1) in Intestinal Epithelial Differentiation and in Colorectal Cancer

Colorectal cancer (CRC) is associated with lifestyle factors that affect insulin/IGF signaling, of which the insulin receptor substrate 1 (IRS1) is a key transducer. We investigated expression, localization and pathologic correlations of IRS1 in cancer-uninvolved colonic epithelium, primary CRCs with paired liver metastases and in vitro polarizing Caco2 and HT29 cells. IRS1 mRNA and protein resulted higher, relative to paired mucosa, in adenomas of familial adenomatous polyposis patients and in CRCs that overexpressed c-MYC, ß-catenin, InsRß, and IGF1R. Analysis of IRS1 immunostaining in 24 cases of primary CRC with paired colonic epithelium and hepatic metastasis showed that staining intensity was significantly higher in metastases relative to both primary CRC (P<0.01) and colonic epithelium (P<0.01). Primary and metastatic CRCs, compared to colonic epithelium, contained significantly higher numbers of IRS1-positive cells (P = 0.013 and P = 0.014, respectively). Pathologic correlations in 163 primary CRCs revealed that diffuse IRS1 staining was associated with tumors combining differentiated phenotype and aggressive markers (high Ki67, p53, and ß-catenin). In Caco 2 IRS1 and InsR were maximally expressed after polarization, while IGF1R was highest in pre-polarized cells. No nuclear IRS1 was detected, while, with polarization, phosphorylated IRS1 (pIRS1) shifted from the lateral to the apical plasma membrane and was expressed in surface cells only. In HT29, that carry mutations constitutively activating survival signaling, IRS1 and IGF1R decreased with polarization, while pIRS1 localized in nuclear spots throughout the course. Overall, these data provide evidence that IRS1 is modulated according to CRC differentiation, and support a role of IRS1 in CRC progression and liver metastatization.

Expression and localisation of insulin receptor substrate 2 in normal intestine and colorectal tumours. Regulation by intestine-specific transcription factor CDX2

Background and aims: Self-renewal and differentiation of intestinal epithelium is a tightly regulated process, whose perturbations are implicated in human colorectal tumourigenesis. The insulin/insulin-like growth factor (IGF) signalling pathway may play an important role in intestinal epithelium homeostasis. Insulin receptor substrate 2 (IRS2) is a poorly characterised component in this pathway. Methods: Using complementary in vitro and in vivo human and murine models, expression (mRNA and protein levels), localisation (immunohistochemistry) and regulation of IRS2 were investigated in the normal intestine and colorectal tumours. In silico analysis of the human IRS2 promoter was performed together with reporter and chromatin immunoprecipitation assays. Results: Significant IRS2 expression was detected in the intestine, with specific protein localisation in the villus region of the ileum and in the surface epithelium of the colon. In human HT29 and Caco2 cells, IRS2 mRNA levels increased with spontaneous and induced differentiation, together with CDX2 (caudal-related homeobox protein 2), P21 and KLF4 (Krüppel-like factor 4). Adenoviral infection with human CDX2 induced IRS2 expression in APC- (adenomatous polyposis coli) and β-catenin-mutated cells. On the other hand, IRS2 downregulation was observed in differentiated enterocytes after adenoviral infection with short hairpin CDX2 (shCDX2), in the intestine of CDX2 heterozygous mice and in colorectal tumours of ApcMin/+ and patients with familial adenomatous polyposis (FAP). The human IRS2 promoter region presents several CDX2-binding sites where CDX2 immunoprecipitated in vivo. IRS2 reporters were functionally activated via CDX2 and blocked via a dominant-negative CDX2 protein. Conclusions: Combining gain- and loss-of-function approaches, an intriguing scenario is presented whereby IRS2 is significantly expressed in the apical intestinal compartment and is directly controlled by CDX2 in normal intestine and tumours.

PGC-1β promotes enterocyte lifespan and tumorigenesis in the intestine

Significance The mucosa of the small intestine is renewed completely every 3–5 d during the entire lifetime through the continuous steps of proliferation, migration, and differentiation of the cells of the mucosa from the crypt site on the bottom to the villus site on the top of the mucosa. The factors that regulate enterocyte lifespan and aging are of special interest as related to colon cancer susceptibility. Here, using genetically modified gain- and loss-of-function models, we present the importance of the mitochondrial respiration chain and reactive oxygen species homeostasis in the gut and identify the protein peroxisome proliferator-activated receptor-γ coactivator-1β as a gene-expression modulator of enterocyte lifespan in both normal and tumoral conditions. The mucosa of the small intestine is renewed completely every 3–5 d throughout the entire lifetime by small populations of adult stem cells that are believed to reside in the bottom of the crypts and to migrate and differentiate into all the different populations of intestinal cells. When the cells reach the apex of the villi and are fully differentiated, they undergo cell death and are shed into the lumen. Reactive oxygen species (ROS) production is proportional to the electron transfer activity of the mitochondrial respiration chain. ROS homeostasis is maintained to control cell death and is finely tuned by an inducible antioxidant program. Here we show that peroxisome proliferator-activated receptor-γ coactivator-1β (PGC-1β) is highly expressed in the intestinal epithelium and possesses dual activity, stimulating mitochondrial biogenesis and oxygen consumption while inducing antioxidant enzymes. To study the role of PGC-1β gain and loss of function in the gut, we generated both intestinal-specific PGC-1β transgenic and PGC-1β knockout mice. Mice overexpressing PGC-1β present a peculiar intestinal morphology with very long villi resulting from increased enterocyte lifespan and also demonstrate greater tumor susceptibility, with increased tumor number and size when exposed to carcinogens. PGC-1β knockout mice are protected from carcinogenesis. We show that PGC-1β triggers mitochondrial respiration while protecting enterocytes from ROS-driven macromolecule damage and consequent apoptosis in both normal and dysplastic mucosa. Therefore, PGC-1β in the gut acts as an adaptive self-point regulator, capable of providing a balance between enhanced mitochondrial activity and protection from increased ROS production.

Synthesis and Biological Evaluation of 2-Heteroarylthioalkanoic Acid Analogues of Clofibric Acid as Peroxisome Proliferator-Activated Receptor α Agonists

A series of 2-heteroarylthioalkanoic acids were synthesized through systematic structural modifications of clofibric acid and evaluated for human peroxisome proliferator-activated receptor alpha (PPARalpha) transactivation activity, with the aim of obtaining new hypolipidemic compounds. Some thiophene and benzothiazole derivatives showing a good activation of the receptor alpha were screened for activity against the PPARgamma isoform. The gene induction of selected compounds was also investigated in the human hepatoma cell line.

Micellar lipid composition profoundly affects LXR-dependent cholesterol transport across CaCo2 cells

Intraluminal phospholipids affect micellar solubilization and absorption of cholesterol. We here study cholesterol transport from taurocholate–phospholipid–cholesterol micelles to CaCo2 cells, and associated effects on ABC‐A1 mediated cholesterol efflux. Micellar incorporation of egg‐yolk‐phosphatidylcholine markedly increased apical retention of the sterol with decreased expression of ABC‐A1, an effect that is prevented by synthetic liver X receptor (LXR) or retinoid X receptor (RXR) agonists. On the other hand, incorporation of lyso‐phosphatidylcholine (LysoPC) increased ABC‐A1–HDL‐dependent basolateral cholesterol efflux, an effect that is abated when LXR is silenced. Thus, the modulation of cholesterol metabolism via intraluminal phospholipids is related to the activity of the oxysterol nuclear receptor LXR.

Methods for routine diagnosis of genomic rearrangements: multiplex PCR-based methods and future perspectives.

Germline and somatic genomic rearrangement play a relevant role in the pathogenesis of genetic disorders, and their identification is a fundamental task in molecular diagnosis. However, screening for structural genomic abnormalities is often not included in routine mutational analyses and consequently the proportion of rearrangements playing a pathogenic role in several genetic disorders is likely to be underestimated. A wide range of molecular techniques for the detection of large genomic rearrangements has been developed: some methods have the power to screen the whole genome, others are designed to analyze one or few loci that are known to be involved in a specific disease; some may detect balanced rearrangements, while others only unbalanced rearrangements; some are suitable for detection of germline abnormalities, yet others also detect somatic abnormalities. This review provides a brief summary of principles, applications and limitations of the methods available for the screening of genomic rearrangements, focusing on multiplex PCR-based protocols that are currently employed in routine detection of extended germline genomic deletions or duplications. Future developments based on microarray platforms and high-throughput sequencing are also discussed.

Transcriptional Regulation of the Intestinal Nuclear Bile Acid Farnesoid X Receptor (FXR) by the caudal-related Homeobox 2 (CDX2)

Background: Farnesoid X receptor (FXR) regulates bile acid (BA) metabolism. High BA levels increase susceptibility to intestinal tumorigenesis. Results: caudal-related homeobox 2 (CDX2) directly binds the FXR promoter and regulates FXR expression. Conclusion: CDX2 transcription factor is a positive regulator of FXR in the gut. Significance: Manipulation of the APC-CDX2-FXR axis could reduce BA toxicity and intestinal tumor susceptibility. Farnesoid X receptor (FXR, NR1H4) is a bile acid-activated transcription factor that belongs to the nuclear receptor superfamily. It is highly expressed in the enterohepatic system, where it senses bile acid levels to consequently reduce their synthesis while inducing their detoxification. Bile acids are intestinal tumor promoters and their concentrations have to be tightly regulated. Indeed, reduced expression of FXR in the intestine increases colorectal cancer susceptibility in mice, whereas its activation can promote apoptosis in genetically modified cells. Notably, despite the broad knowledge of the FXR enterohepatic transcriptional activity, the molecular mechanisms regulating FXR expression in the intestine are still unknown. Herein, by combining both gain and loss of function approaches and FXR promoter activity studies, we identified caudal-related homeobox 2 (CDX2) transcription factor as a positive regulator of FXR expression in the enterocytes. Our results provide a putative novel tool for modulating FXR expression against bile acid-related colorectal cancer progression.

TRIM8 restores p53 tumour suppressor function by blunting N-MYC activity in chemo-resistant tumours

BackgroundTRIM8 plays a key role in controlling the p53 molecular switch that sustains the transcriptional activation of cell cycle arrest genes and response to chemotherapeutic drugs. The mechanisms that regulate TRIM8, especially in cancers like clear cell Renal Cell Carcinoma (ccRCC) and colorectal cancer (CRC) where it is low expressed, are still unknown. However, recent studies suggest the potential involvement of some microRNAs belonging to miR-17-92 and its paralogous clusters, which could include TRIM8 in a more complex pathway.MethodsWe used RCC and CRC cell models for in-vitro experiments, and ccRCC patients and xenograft transplanted mice for in vivo assessments. To measure microRNAs levels we performed RT-qPCR, while steady-states of TRIM8, p53, p21 and N-MYC were quantified at protein level by Western Blotting as well as at transcript level by RT-qPCR. Luciferase reporter assays were performed to assess the interaction between TRIM8 and specific miRNAs, and the potential effects of this interaction on TRIM8 expression. Moreover, we treated our cell models with conventional chemotherapeutic drugs or tyrosine kinase inhibitors, and measured their response in terms of cell proliferation by MTT and colony suppression assays.ResultsWe showed that TRIM8 is a target of miR-17-5p and miR-106b-5p, whose expression is promoted by N-MYC, and that alterations of their levels affect cell proliferation, acting on the TRIM8 transcripts stability, as confirmed in ccRCC patients and cell lines. In addition, reducing the levels of miR-17-5p/miR-106b-5p, we increased the chemo-sensitivity of RCC/CRC-derived cells to anti-tumour drugs used in the clinic. Intriguingly, this occurs, on one hand, by recovering the p53 tumour suppressor activity in a TRIM8-dependent fashion and, on the other hand, by promoting the transcription of miR-34a that turns off the oncogenic action of N-MYC. This ultimately leads to cell proliferation reduction or block, observed also in colon cancer xenografts overexpressing TRIM8.ConclusionsIn this paper we provided evidence that TRIM8 and its regulators miR-17-5p and miR-106b-5 participate to a feedback loop controlling cell proliferation through the reciprocal modulation of p53, miR-34a and N-MYC. Our experiments pointed out that this axis is pivotal in defining drug responsiveness of cancers such ccRCC and CRC.