Oncogenic Biogenesis of pri-miR-17∼92 Reveals Hierarchy and Competition among Polycistronic MicroRNAs.

The microRNAs encoded by the miR-17∼92 polycistron are commonly overexpressed in cancer and orchestrate a wide range of oncogenic functions. Here, we identify a mechanism for miR-17∼92 oncogenic function through the disruption of endogenous microRNA (miRNA) processing. We show that, upon oncogenic overexpression of the miR-17∼92 primary transcript (pri-miR-17∼92), the microprocessor complex remains associated with partially processed intermediates that aberrantly accumulate. These intermediates reflect a series of hierarchical and conserved steps in the early processing of the pri-miR-17∼92 transcript. Encumbrance of the microprocessor by miR-17∼92 intermediates leads to the broad but selective downregulation of co-expressed polycistronic miRNAs, including miRNAs derived from tumor-suppressive miR-34b/c and from the Dlk1-Dio3 polycistrons. We propose that the identified steps of polycistronic miR-17∼92 biogenesis contribute to the oncogenic re-wiring of gene regulation networks. Our results reveal previously unappreciated functional paradigms for polycistronic miRNAs in cancer.

Alternative polyadenylation confers Pten mRNAs stability and resistance to microRNAs.

Fine regulation of the phosphatase and tensin homologue (PTEN) phosphatase dosage is critical for homeostasis and tumour suppression. The 3'-untranslated region (3'-UTR) of Pten mRNA was extensively linked to post-transcriptional regulation by microRNAs (miRNAs). In spite of this critical regulatory role, alternative 3'-UTRs of Pten have not been systematically characterized. Here, we reveal an important diversity of Pten mRNA isoforms generated by alternative polyadenylation sites. Several 3'-UTRs are co-expressed and their relative expression is dynamically regulated. In spite of encoding multiple validated miRNA-binding sites, longer isoforms are largely refractory to miRNA-mediated silencing, are more stable and contribute to the bulk of PTEN protein and signalling functions. Taken together, our results warrant a mechanistic re-interpretation of the post-transcriptional mechanisms involving Pten mRNAs and raise concerns on how miRNA-binding sites are being validated.

The miR-17∼92 microRNA Cluster Is a Global Regulator of Tumor Metabolism.

A central hallmark of cancer cells is the reprogramming of cellular metabolism to meet the bioenergetic and biosynthetic demands of malignant growth. Here, we report that the miR-17∼92 microRNA (miRNA) cluster is an oncogenic driver of tumor metabolic reprogramming. Loss of miR-17∼92 in Myc(+) tumor cells leads to a global decrease in tumor cell metabolism, affecting both glycolytic and mitochondrial metabolism, whereas increased miR-17∼92 expression is sufficient to drive increased nutrient usage by tumor cells. We mapped the metabolic control element of miR-17∼92 to the miR-17 seed family, which influences cellular metabolism and mammalian target of rapamycin complex 1 (mTORC1) signaling through negative regulation of the LKB1 tumor suppressor. miR-17-dependent tuning of LKB1 levels regulates both the metabolic potential of Myc(+) lymphomas and tumor growth in vivo. Our results establish metabolic reprogramming as a central function of the oncogenic miR-17∼92 miRNA cluster that drives the progression of MYC-dependent tumors.

Identification of GATA-4 as a novel transcriptional regulatory component of regenerating islet-derived family members.

Intestinal epithelial cells are exposed to luminal bacterial threat and require adequate defense mechanisms to ensure host protection and epithelium regeneration against possible deleterious damage. Differentiated intestinal epithelial cells produce antimicrobial and regenerative components that protect against such challenges. Few intestinal specific transcription factors have been identified to control the switching from repression to activation of this class of gene. Herein, we show that gene transcription of some regenerating islet-derived (REG) family members is dependent on the transcription factor GATA-4. Silencing of GATA-4 expression in cultured intestinal epithelial cells identified Reg3ß as a target gene using an unbiased approach of gene expression profiling. Co-transfection and RNA interference assays identified complex GATA-4-interactive transcriptional components required for the activation or repression of Reg3ß gene activity. Conditional deletion of Gata4 in the mouse intestinal epithelium supported its regulatory role for Reg1, Reg3α, Reg3ß and Reg3γ genes. Reg1 dramatic down-modulation of expression in Gata4 conditional null mice was associated with a significant decrease in intestinal epithelial cell migration. Altogether, these results identify a novel and complex role for GATA-4 in the regulation of REG family members gene expression.

Ghrelin Inhibition Restores Glucose Homeostasis in Hepatocyte Nuclear Factor-1α (MODY3)-Deficient Mice.

Hepatocyte nuclear factor-1α (HNF1α) is a transcription factor expressed in tissues of endoderm origin. Mutations in HNF1A are associated with maturity-onset diabetes of the young 3 (MODY3). Mice deficient for Hnf1α are hyperglycemic, with their pancreatic ß-cells being defective in glucose-sensing insulin secretion. The specific mechanisms involved in this defect are unclear. Gut hormones control glucose homeostasis. Our objective was to explore whether changes in these hormones play a role in glucose homeostasis in the absence of Hnf1α. An increase in ghrelin gene transcript and a decrease in glucose-dependent insulinotropic polypeptide (GIP) gene transcripts were observed in the gut of Hnf1α-null mice. These changes correlated with an increase of ghrelin and a decrease of GIP-labeled cells. Ghrelin serological levels were significantly induced in Hnf1α-null mice. Paradoxically, GIP levels were also induced in these mice. Treatment of Hnf1α-null mice with a ghrelin antagonist led to a recovery of the diabetic symptoms. We conclude that upregulation of ghrelin in the absence of Hnf1α impairs insulin secretion and can be reversed by pharmacological inhibition of ghrelin/GHS-R interaction. These observations open up on future strategies to counteract ghrelin action in a program that could become beneficial in controlling non-insulin-dependent diabetes.

Loss of hepatocyte-nuclear-factor-1alpha impacts on adult mouse intestinal epithelial cell growth and cell lineages differentiation.

BACKGROUND AND AIMS: Although Hnf1alpha is crucial for pancreas and liver functions, it is believed to play a limited functional role for intestinal epithelial functions. The aim of this study was to assess the consequences of abrogating Hnf1alpha on the maintenance of adult small intestinal epithelial functions. METHODOLOGY/PRINCIPAL FINDINGS: An Hnf1alpha knockout mouse model was used. Assessment of histological abnormalities, crypt epithelial cell proliferation, epithelial barrier, glucose transport and signalling pathways were measured in these animals. Changes in global gene expression were also analyzed. Mice lacking Hnf1alpha displayed increased crypt proliferation and intestinalomegaly as well as a disturbance of intestinal epithelial cell lineages production during adult life. This phenotype was associated with a decrease of the mucosal barrier function and lumen-to-blood glucose delivery. The mammalian target of rapamycin (mTOR) signalling pathway was found to be overly activated in the small intestine of adult Hnf1alpha mutant mice. The intestinal epithelium of Hnf1alpha null mice displayed a reduction of the enteroendocrine cell population. An impact was also observed on proper Paneth cell differentiation with abnormalities in the granule exocytosis pathway. CONCLUSIONS/SIGNIFICANCE: Together, these results unravel a functional role for Hnf1alpha in regulating adult intestinal growth and sustaining the functions of intestinal epithelial cell lineages.

Integrin alpha8beta1 regulates adhesion, migration and proliferation of human intestinal crypt cells via a predominant RhoA/ROCK-dependent mechanism.

BACKGROUND: Integrins are transmembrane alphabeta heterodimer receptors that function as structural and functional bridges between the cytoskeleton and ECM (extracellular matrix) molecules. The RGD (arginine-glycine-aspartate tripeptide motif)-dependent integrin alpha8beta1 has been shown to be involved in various cell functions in neuronal and mesenchymal-derived cell types. Its role in epithelial cells remains unknown. RESULTS: Integrin alpha8beta1 was found to be expressed in the crypt cell population of the human intestine but was absent from differentiating and mature epithelial cells of the villus. The function of alpha8beta1 in epithelial crypt cells was investigated at the cellular level using normal HIECs (human intestinal epithelial cells). Specific knockdown of alpha8 subunit expression using an shRNA (small-hairpin RNA) approach showed that alpha8beta1 plays important roles in RGD-dependent cell adhesion, migration and proliferation via a RhoA/ROCK (Rho-associated kinase)-dependent mechanism as demonstrated by active RhoA quantification and pharmacological inhibition of ROCK. Moreover, loss of alpha8beta1, through RhoA/ROCK, impairs FA (focal adhesion) complex integrity as demonstrated by faulty vinculin recruitment. CONCLUSIONS: Integrin alpha8beta1 is expressed in epithelial cells. In intestinal crypt cells, alpha8beta1 is closely involved in the regulation of adhesion, migration and cell proliferation via a predominant RhoA/ROCK-dependent mechanism. These results suggest an important role for this integrin in intestinal crypt cell homoeostasis.

Hepatocyte nuclear factor 4alpha contributes to an intestinal epithelial phenotype in vitro and plays a partial role in mouse intestinal epithelium differentiation.

Hepatocyte nuclear factor 4alpha (HNF4alpha) is a regulator of hepatocyte and pancreatic transcription. Hnf4alpha deletion in the mouse is embryonically lethal with severe defects in visceral endoderm formation. It has been concluded in the past that the role of Hnf4alpha in the developing colon was much less important than in the liver. However, the precise role of Hnf4alpha in the homeostasis of the small intestinal epithelium remains unclear. Our aim was to evaluate the potential of Hnf4alpha to support an intestinal epithelial phenotype. First, Hnf4alpha potential to dictate this phenotype was assessed in nonintestinal cell lines in vitro. Forced expression of Hnf4alpha in fibroblasts showed an induction of features normally restricted to epithelial cells. Combinatory expression of Hnf4alpha with specific transcriptional regulators of the intestine resulted in the induction of intestinal epithelial genes in this context. Second, the importance of Hnf4alpha in maintaining the homeostasis of the intestinal epithelium was investigated in mice. Mice conditionally deficient for intestinal Hnf4alpha developed normally throughout adulthood with an epithelium displaying normal morphological and functional structures with minor alterations. Subtle but statistical differences were observed at the proliferation and the cytodifferentiation levels. Hnf4alpha mutant mice displayed an increase in the number of goblet and enteroendocrine cells compared with controls. Given the fundamental role of this transcription factor in other tissues, these findings dispute the crucial role for this regulator in the maintenance of intestinal epithelial cell function at a period of time that follows cytodifferentiation but may suggest a functional role in instructing cells to become specific to the intestinal epithelium.

Hepatocyte nuclear factor-4alpha promotes differentiation of intestinal epithelial cells in a coculture system.

Normal cellular models able to efficiently recapitulate intestinal epithelial cell differentiation in culture are not yet available. The aim of this work was to establish and genetically characterize a mesenchymal-epithelial coculture system to identify transcriptional regulators involved in this process. The deposition of rat intestinal epithelial cells on human intestinal mesenchymal cells led to the formation of clustered structures that expanded shortly after seeding. These structures were composed of polarized epithelial cells with brush borders and cell junction complexes. A rat GeneChip statistical analysis performed at different time points during this process identified hepatocyte nuclear factor-4alpha (HNF-4alpha) and hepatocyte nuclear factor-1alpha (HNF-1alpha) as being induced coincidently with the apparition of polarized epithelial structures. Stable introduction of HNF-4alpha in undifferentiated epithelial cells alone led to the rapid induction of HNF-1alpha and several intestinal-specific markers and metabolism-related genes for which mRNA was identified to be upregulated during epithelial differentiation. HNF-4alpha was capable to transactivate the calbindin 3 gene promoter, a process that was synergistically increased in the presence of HNF-1alpha. When HNF-4alpha-expressing cells were plated on mesenchymal cells, an epithelial monolayer formed rapidly with the apparition of dome structures that are characteristics of vectorial ion transport. Forced expression of HNF-1alpha alone did not result in dome structures formation. In sum, this novel coculture system functionally identified for the first time HNF-4alpha as an important modulator of intestinal epithelial differentiation and offers an innovative opportunity to investigate molecular mechanisms involved in this process.

Loss of cathepsin L activity promotes claudin-1 overexpression and intestinal neoplasia.

Intestinal epithelial integrity and polarity are maintained by cohesive interactions between cells via the formation of tight junctions. Irregularities in tight junctions have only recently been found to be associated with the initiation and progression of intestinal neoplasia. The claudin family of proteins is integral to the structure and function of the tight junction but little is known of the molecular events that regulate the expression of these components. The present report identifies cathepsin L, classically a lysosomal cysteine protease, as being induced during intestinal epithelial cell polarization and differentiation. Inhibition of intracellular cathepsin L activity results in the accumulation of disorganized cell layers and a decline in the expression of differentiation markers in cultured intestinal epithelial cells. This coincides with a rapid up-regulation of claudin-1 protein accumulation. Mutant mice defective in cathepsin L activity (furless) display an elevated level of intestinal claudin-1 and claudin-2 expression. Loss of cathepsin L activity leads to a marked increase in tumor multiplicity in the intestine of Apc(Min) mice. Given the traditionally viewed biological role of cathepsin L in the processing of lysosomal content as well as in pathological extracellular matrix remodeling, the results here demonstrate an as yet unsuspected intracellular role for this protease in normal intestinal epithelial polarization and initiation of neoplasia.

Nuclear expression of E2F4 induces cell death via multiple pathways in normal human intestinal epithelial crypt cells but not in colon cancer cells.

E2F transcription factors control cell cycle progression. The localization of E2F4 in intestinal epithelial cells is cell cycle dependent, being cytoplasmic in quiescent differentiated cells but nuclear in proliferative cells. However, whether nuclear translocation of E2F4 alone is sufficient to trigger intestinal epithelial cell proliferation remains to be established. Adenoviruses expressing fusion proteins between green fluorescent protein (GFP) and wild-type (wt)E2F4 or GFP and nuclear localization signal (NLS)-tagged E2F4 were used to infect normal human intestinal epithelial crypt cells (HIEC). In contrast to expression of wtE2F4, persistent expression of E2F4 into the nucleus of HIEC triggered phosphatidylserine exposure, cytoplasmic shrinkage, zeiosis, formation of apoptotic bodies, and activation of caspase 9 and caspase 3. Inhibition of caspase activities by zVAD-fmk partially inhibited cell death induced by E2F4-NLS. An induction of p53, phosphorylated Ser15-p53, PUMA, FAS, BAX, RIP, and phosphorylated JNK1 was also observed in HIEC expressing E2F4-NLS compared with wtE2F4-expressing cells. E2F1 and p14ARF expression remained unaltered. Downregulation of p53 expression by RNA interference attenuated cell death induced by E2F4-NLS. By contrast, the level of cell death was negligible in colon cancer cells despite the strong expression of E2F4 into the nucleus. In conclusion, deregulated nuclear E2F4 expression induces apoptosis via multiple pathways in normal intestinal epithelial cells but not in colon cancer cells. Hence, mutations that deregulate E2F4 localization may provide an initial proliferative advantage but at the same time accelerate cell death. However, intestinal cells acquiring mutations (e.g., p53, Bax loci, etc.) may escape apoptosis, thereby revealing the full mitogenic potential of the E2F4 transcription factor.