Lina Sabatino

Fenofibrate prevents and reduces body weight gain and adiposity in diet-induced obese rats

Fibrates are hypolipidemic drugs that activate the peroxisome proliferator‐activated receptors. Since fibrates may also increase energy expenditure, we investigated whether fenofibrate (FF) had this effect in diet‐induced obese rats. A 2‐month administration of a high‐fat palatable diet to adult rats increased body weight by 25% and white adipose mass by 163% compared with a standard diet. These effects were prevented by FF, both when administered for the 2 months of high‐fat feeding and when given for only the second month. Consequently, FF‐treated rats had a final body weight and white adipose tissue mass similar to untreated animals on the standard diet. FF also increased resting metabolic rate, hepatic peroxisomal and mitochondrial palmitoyl‐dependent oxygen uptake and mRNA levels of acyl‐CoA oxidase and lipoprotein lipase. Finally, FF lowered mRNA levels of uncoupling protein‐2 and did not affect mitochondrial respiration in skeletal muscle. Therefore, FF seems to act as a weight‐stabilizer mainly through its effect on liver metabolism.

Comparative genome-wide DNA methylation analysis of colorectal tumor and matched normal tissues

Aberrant DNA methylation often occurs in colorectal cancer (CRC). In our study we applied a genome-wide DNA methylation analysis approach, MethylCap-seq, to map the differentially methylated regions (DMRs) in 24 tumors and matched normal colon samples. In total, 2687 frequently hypermethylated and 468 frequently hypomethylated regions were identified, which include potential biomarkers for CRC diagnosis. Hypermethylation in the tumor samples was enriched at CpG islands and gene promoters, while hypomethylation was distributed throughout the genome. Using epigenetic data from human embryonic stem cells, we show that frequently hypermethylated regions coincide with bivalent loci in human embryonic stem cells. DNA methylation is commonly thought to lead to gene silencing; however, integration of publically available gene expression data indicates that 75% of the frequently hypermethylated genes were most likely already lowly or not expressed in normal tissue. Collectively, our study provides genome-wide DNA methylation maps of CRC, comprehensive lists of DMRs, and gives insights into the role of aberrant DNA methylation in CRC formation.

Reduced β-catenin and peroxisome proliferator–activated receptor–γ expression levels are associated with colorectal cancer metastatic progression: correlation with tumor-associated macrophages, cyclooxygenase 2, and patient outcome

Recent studies have reported cross talk between beta-catenin, peroxisome proliferator-activated receptor-gamma, and cyclooxygenase 2 signaling pathways. We examined whether molecular changes of these pathways could be related to colorectal cancer metastatic progression. Seventy-two sporadic colorectal cancers and the distant nonneoplastic mucosa were analyzed for beta-catenin, peroxisome proliferator-activated receptor-gamma, cyclooxygenase 2, and nuclear factor kappaB levels by immunohistochemistry and Western blot. The expression profiles were correlated with patient outcome and 5-year survival. Nuclear beta-catenin staining was detected in only 18.1% of tumors and correlated with poor survival as compared with cases showing cytosolic/membrane accumulation (59.7%, P < .05). This latter group and tumor samples showing cytosolic/nuclear peroxisome proliferator-activated receptor-gamma expression (70.8%) were significantly associated with a favorable prognosis (P < .001). Remarkably, reduction or loss of beta-catenin (22.2%) and peroxisome proliferator-activated receptor-gamma (29.2%) expression was strongly correlated with marked infiltration of tumor-associated macrophages (P < .01), presence of liver metastases, and very short survival (P = .0001). Moreover, beta-catenin and peroxisome proliferator-activated receptor-gamma levels were inversely correlated with cyclooxygenase 2 (P < .01) and nuclear factor kappaB expression (P < .05). Our results suggest that reduced expression of beta-catenin and peroxisome proliferator-activated receptor-gamma could play a key role in aggressive colorectal cancer behavior. This finding may provide a relevant prognostic tool and contribute to early identification of patients at high risk of mortality.

UHRF1 coordinates peroxisome proliferator activated receptor gamma (PPARG) epigenetic silencing and mediates colorectal cancer progression

Peroxisome proliferator-activated receptor gamma (PPARG) inactivation has been identified as an important step in colorectal cancer (CRC) progression, although the events involved have been partially clarified. UHRF1 is emerging as a cofactor that coordinates the epigenetic silencing of tumor suppressor genes, but its role in CRC remains elusive. Here, we report that UHRF1 negatively regulates PPARG and is associated with a higher proliferative, clonogenic and migration potential. Consistently, UHRF1 ectopic expression induces PPARG repression through its recruitment on the PPARG promoter fostering DNA methylation and histone repressive modifications. In agreement, UHRF1 knockdown elicits PPARG re-activation, accompanied by positive histone marks and DNA demethylation, corroborating its role in PPARG silencing. UHRF1 overexpression, as well as PPARG-silencing, imparts higher growth rate and phenotypic features resembling those occurring in the epithelial-mesenchymal transition. In our series of 110 sporadic CRCs, high UHRF1-expressing tumors are characterized by an undifferentiated phenotype, higher proliferation rate and poor clinical outcome only in advanced stages III–IV. In addition, the inverse relationship with PPARG found in vitro is detected in vivo and UHRF1 prognostic significance appears closely related to PPARG low expression, as remarkably validated in an independent dataset. The results demonstrate that UHRF1 regulates PPARG silencing and both genes appear to be part of a complex regulatory network. These findings suggest that the relationship between UHRF1 and PPARG may have a relevant role in CRC progression.

De novo expression of uncoupling protein 3 is associated to enhanced mitochondrial thioesterase‐1 expression and fatty acid metabolism in liver of fenofibrate‐treated rats

Uncoupling protein 3 (UCP3) is a member of the mitochondrial carrier superfamily, preferentially expressed in skeletal muscle. Its function is not fully understood and it is debated whether it uncouples oxidative phosphorylation as does UCP1 in brown adipose tissue. Recent evidences suggest a role for UCP3 in the flux of fatty acids in and out mitochondria and their utilization in concert with mitochondrial thioesterase‐1 (MTE‐1). In fact, mice overexpressing muscle UCP3 also show high levels of MTE‐1. Fenofibrate is a hypolipidemic drug that prevents body weight gain in diet‐induced obese rats and enhances lipid metabolism by activating peroxisome proliferator‐activated receptors (PPARs). Because fatty acids and fenofibrate stimulate PPARs and in turn UCP3, we investigated whether UCP3 expression might be induced ‘de novo’ in situations of increased hepatic mitochondrial fatty acid utilization caused by a combined effect of a high‐fat diet and fenofibrate treatment. We also investigated whether Mte‐1 expression and β‐oxidation were affected. We show here that Ucp3 is induced in liver of fenofibrate‐treated rats at the mRNA and protein level. Expression was restricted to hepatocytes and was unevenly distributed in the liver. No increase in cell proliferation, inflammatory or fibrotic responses was found. Mte‐1 expression and mitochondrial β‐oxidation were upregulated. Thus, Ucp3 can be transactivated in tissues where it is normally silent and fenofibrate can attain this effect in liver. The data demonstrate that UCP3 is involved in fatty acid utilization and support the notion that UCP3 and MTE‐1 are linked within the same metabolic pathway.

Fasting plasma free fatty acid concentrations and Pro12Ala polymorphism of the peroxisome proliferator‐activated receptor (PPAR) γ2 gene in healthy individuals

background The Pro12Ala polymorphism of the peroxisome proliferator‐activated receptor (PPAR) γ gene has been associated in some, but not all, studies with lower body mass index (BMI) and improved insulin sensitivity; how an altered transcriptional activity of PPARγ2 could influence insulin sensitivity is currently unclear. The free fatty acids (FFAs) released from adipose tissue triglycerides via lipolysis are key mediators of impaired insulin sensitivity; however, no study has described the relationship of the Pro12Ala mutation with circulating levels of FFAs under physiological conditions.

Lessons to be learned from the clinical management of a MEN 2A patient bearing a novel 634/640/700 mutation of the RET proto‐oncogene

A cluster of germline gain-of-function mutations of the RET proto-oncogene are responsible for Multiple Endocrine Neoplasia type 2A (MEN 2A), an autosomal dominant, inherited disorder characterized by medullary thyroid carcinoma (MTC), phaeochromocytoma (Phaeo) and hyperparathyroidism. Genetic screening of MEN 2A patients has been available for the past decade and useful genotype–phenotype correlations have been established: specific RET mutations are associated with age at first diagnosis and tumour aggressiveness. Accordingly, MEN 2A patients can be stratified into three risk groups depending on the RET mutation. Management uncertainties remain regarding patients bearing uncommon RET mutations or genetic variations for which mutation-specific risk profiles and treatment recommendations are unavailable. Here we report the thirteen-year clinical and surgical follow-up of a patient with MEN 2A bearing three de novo RET mutations at codons 634, 640 and 700 (p. C634R, p.A640G and p.M700L) in exon 11: a combination of mutations which has not previously been described. In May 1998, a 26-year-old female patient was admitted to our unit because of recurrent episodes of hypertension, tachycardia and headache. She had recently been diagnosed with a MTC which had been removed surgically, with associated cervical central lymphectomy. Elevated urinary catecholamine levels and imaging examinations showing a left adrenal mass were suggestive of a Phaeo. Hyperplasia of the contralateral gland was also detected. The patient underwent left “open” adrenalectomy 3 months later; post-operative catecholamine levels decreased to normal values, while the serum calcitonin remained elevated (Fig. 1). The diagnosis of MEN 2A was confirmed by testing the patient’s DNA for RET mutations. Two heterozygous germline mutations were identified: a transition at position c.1900 replaced a T with a C and a transversion at position c.1919 replaced a C with a G, resulting in the substitution of a cysteine with an arginine and an alanine with a glycine at positions p.634 and p.640, respectively. The patient presented with MTC and Phaeo without parathyroid gland involvement, so we speculated that this clinical picture could be correlated with the two RET mutations identified and the unusual feature of calcitonin production by the adrenal tissue. No clinical symptoms suggestive of MEN 2A and no RET mutations were found in either parent or any of the available relatives tested (three sisters and one brother). In 2003, at

Immune escape mechanisms in colorectal cancer pathogenesis and liver metastasis.

Over the past decade, growing evidence indicates that the tumor microenvironment (TME) contributes with genomic/epigenomic aberrations of malignant cells to enhance cancer cells survival, invasion, and dissemination. Many factors, produced or de novo synthesized by immune, stromal, or malignant cells, acting in a paracrine and autocrine fashion, remodel TME and the adaptive immune response culminating in metastasis. Taking into account the recent accomplishments in the field of immune oncology and using metastatic colorectal cancer (mCRC) as a model, we propose that the evasion of the immune surveillance and metastatic spread can be achieved through a number of mechanisms that include (a) intrinsic plasticity and adaptability of immune and malignant cells to paracrine and autocrine stimuli or genotoxic stresses; (b) alteration of positional schemes of myeloid-lineage cells, produced by factors controlling the balance between tumour-suppressing and tumour-promoting activities; (c) acquisition by cancer cells of aberrant immune-phenotypic traits (NT5E/CD73, CD68, and CD163) that enhance the interactions among TME components through the production of immune-suppressive mediators. These properties may represent the driving force of metastatic progression and thus clinically exploitable for cancer prevention and therapy. In this review we summarize results and suggest new hypotheses that favour the growing impact of tumor-infiltrating immune cells on tumour progression, metastasis, and therapy resistance.

Transcriptional activity of the murine retinol binding protein gene is regulated by a multiprotein complex containing HMGA1, p54nrb/NonO, protein-associated splicing factor (PSF) and steroidogenic factor 1 (SF1)/liver receptor homologue 1 (LRH-1)

Retinol-binding protein (RBP4) transports retinol in the circulation from hepatic stores to peripheral tissues. Since little is known regarding the regulation of this gene, we analysed the cis-regulatory sequences of the mouse RBP4 gene. Our data show that transcription of the gene is regulated through a bipartite promoter: a proximal region necessary for basal expression and a distal segment responsible for cAMP-induction. This latter region contains several binding sites for the structural HMGA1 proteins, which are important to promoter regulation. We further demonstrate that HMGA1s play a key role in basal and cAMP-induction of Rbp4 transcription and the RBP4 and HMGA1 genes are coordinately regulated in vitro and in vivo. HMGA1 acts to recruit transcription factors to the RBP4 promoter and we specifically identified p54(nrb)/NonO and protein-associated splicing factor (PSF) as components that interact with this complex. Steroidogenic factor 1 (SF1) or the related liver receptor homologue 1 (LRH-1) are also associated with this complex upon cAMP-induction. Depletion of SF1/LRH-1 by RNA interference resulted in a dramatic loss of cAMP-induction. Collectively, our results demonstrate that basal and cAMP-induced Rbp4 transcription is regulated by a multiprotein complex that is similar to ones that modulate expression of genes of steroid hormone biosynthesis. Since genes related to glucose metabolism are regulated in a similar fashion, this suggests that Rbp4 expression may be regulated as part of a network of pathways relevant to the onset of type 2 diabetes.

Emerging role of the β-catenin-PPARγ axis in the pathogenesis of colorectal cancer.

Multiple lines of evidence indicate that Wnt/β-catenin signaling plays a fundamental role in colorectal cancer (CRC) initiation and progression. Recent genome-wide data have confirmed that in CRC this pathway is one of the most frequently modified by genetic or epigenetic alterations affecting almost 90% of Wnt/β-catenin gene members. A major challenge is thus learning how the corrupted coordination of this pathway is tied to other signalings to enhance cell growth. Peroxisome proliferator activated receptor γ (PPARγ) is emerging as a growth-limiting and differentiation-promoting factor. In tumorigenesis it exerts a tumor suppressor role and is potentially linked with the Wnt/β-catenin pathway. Based on these results, the identification of new selective PPARγ modulators with inhibitory effects on the Wnt/β-catenin pathway is becoming an interesting perspective. Should, in fact, these molecules display such properties, new research avenues would be opened aimed at developing new molecular targeted drugs. Herein, we review the basic principles and present new hypotheses underlying the crosstalk between Wnt/β-catenin and PPARγ signaling. Furthermore, we discuss the advances in our understanding as to how their altered regulation can culminate in colon cancer and the efforts aimed at designing novel PPARγ agonists endowed with Wnt/β-catenin inhibitory effects to be used as therapeutic and/or preventive agents.