Alessandro Porrello

Hedgehog signaling regulates epithelial-mesenchymal transition during biliary fibrosis in rodents and humans

Epithelial-mesenchymal transitions (EMTs) play an important role in tissue construction during embryogenesis, and evidence suggests that this process may also help to remodel some adult tissues after injury. Activation of the hedgehog (Hh) signaling pathway regulates EMT during development. This pathway is also induced by chronic biliary injury, a condition in which EMT has been suggested to have a role. We evaluated the hypothesis that Hh signaling promotes EMT in adult bile ductular cells (cholangiocytes). In liver sections from patients with chronic biliary injury and in primary cholangiocytes isolated from rats that had undergone bile duct ligation (BDL), an experimental model of biliary fibrosis, EMT was localized to cholangiocytes with Hh pathway activity. Relief of ductal obstruction in BDL rats reduced Hh pathway activity, EMT, and biliary fibrosis. In mouse cholangiocytes, coculture with myofibroblastic hepatic stellate cells, a source of soluble Hh ligands, promoted EMT and cell migration. Addition of Hh-neutralizing antibodies to cocultures blocked these effects. Finally, we found that EMT responses to BDL were enhanced in patched-deficient mice, which display excessive activation of the Hh pathway. Together, these data suggest that activation of Hh signaling promotes EMT and contributes to the evolution of biliary fibrosis during chronic cholestasis.

B-Raf(V600E) and thrombospondin-1 promote thyroid cancer progression.

Although B-RafV600E is the most common somatic mutation in papillary thyroid carcinoma (PTC), how it induces tumor aggressiveness is not fully understood. Using gene set enrichment analysis and in vitro and in vivo functional studies, we identified and validated a B-RafV600E gene set signature associated with tumor progression in PTCs. An independent cohort of B-RafV600E-positive PTCs showed significantly higher expression levels of many extracellular matrix genes compared with controls. We performed extensive in vitro and in vivo validations on thrombospondin-1 (TSP-1), because it has been previously shown to be important in the regulation of tumor angiogenesis and metastasis and is present in abundance in tumor stroma. Knockdown of B-RafV600E resulted in TSP-1 down-regulation and a reduction of adhesion and migration/invasion of human thyroid cancer cells. Knockdown of TSP-1 resulted in a similar phenotype. B-RafV600E cells in which either B-RafV600E or TSP-1 were knocked down were implanted orthotopically into the thyroids of immunocompromised mice, resulting in significant reduction in tumor size and fewer pulmonary metastases from the primary carcinoma as compared with the control cells. Treatment of orthotopic thyroid tumors, initiated 1 week after tumor cell implantation with PLX4720, an orally available selective inhibitor of B-RafV600E, caused a significant tumor growth delay and decreased distant metastases, without evidence of toxicity. In conclusion, B-RafV600E plays an important role in PTC progression through genes (i.e., TSP-1) important in tumor invasion and metastasis. Testing of a patients thyroid cancer for B-RafV600E will yield important information about potential tumor aggressiveness and also allow for future use of targeted therapies with selective B-RafV600E inhibitors, such as PLX4720.

The Isopeptidase USP2a Protects Human Prostate Cancer from Apoptosis

Deubiquitinating enzymes can prevent the destruction of protein substrates prior to proteasomal degradation. The ubiquitin-specific protease 2a (USP2a) deubiquitinates the antiapoptotic proteins Fatty Acid Synthase and Mdm2. Here, we show that when USP2a is overexpressed in nontransformed cells, it exhibits oncogenic behavior both in vitro and in vivo and prevents apoptosis induced by chemotherapeutic agents. Notably, USP2a silencing in several human cancer cell lines results in apoptosis. Gene set enrichment analysis, which focuses on groups of genes sharing biological function or regulatory pathways, was done on microarray expression data from human prostate cancers. The cell death-related gene set, as well as a selected cluster of validated p53 target genes, were significantly enriched in the low USP2a expression group of tumors. Conversely, genes implicated in fatty acid metabolism were significantly associated with tumors expressing high USP2a (44%). The expression profile analysis is consistent with the effects of USP2a on its known targets, i.e., Fatty Acid Synthase and Mdm2, defining a subset of prostate tumors resistant to apoptosis. USP2a thus represents a therapeutic target in prostate cancer.

Repair-related activation of hedgehog signaling promotes cholangiocyte chemokine production.

The mechanisms mediating hepatic accumulation of inflammatory cells in cholestatic liver disease remain enigmatic. Our thesis is that Hedgehog (Hh) pathway activation promotes hepatic accumulation of immune cells that interact with cholangiocytes. We believe that myofibroblastic hepatic stellate cells (MF‐HSCs) release soluble Hh ligands that stimulate cholangiocytes to express chemokines that recruit mononuclear cell types with cognate receptors for these chemokines, thereby orchestrating a repair‐related mechanism for liver inflammation. To address this thesis, we used three experimental systems that allow the definition of Hh‐dependent mechanisms that induce phenotypic changes in cholangiocytes. First, cholangiocytes were cultured alone or in the presence of Hh‐producing MF‐HSCs in a transwell coculture system and/or treated with MF‐HSC–conditioned medium with or without Hh‐neutralizing antibodies. Changes in the cholangiocyte phenotype were then evaluated by microarray analysis, quantitative reverse‐transcriptase polymerase chain reaction (QRT‐PCR), and/or enzyme‐linked immunosorbent assay for chemokine (C‐X‐C) motif ligand 16 (Cxcl16). Bile duct ligation was chosen to model biliary fibrosis in mice with an overly active Hh pathway, control littermates, and healthy rats, and the gene profile was evaluated by QRT‐PCR in whole liver tissue. Second, a transwell chemotaxis assay was used to examine natural killer T (NKT) cell migration in response to cholangiocytes and particularly cholangiocyte‐derived Cxcl16. Finally, we studied liver samples from primary biliary cirrhosis patients and controls by QRT‐PCR to compare differences in the Hh pathway and Cxcl16. Co‐immunostaining of cytokeratin‐7 and Cxcl16 was then performed to localize the phenotypic source of Cxcl16. We found that MF‐HSCs release soluble Hh ligands that stimulate cholangiocytes to produce Cxcl16 and recruit NKT cells. Hh pathway activation during cholestatic liver injury also induces cholangiocyte expression of Cxcl16. Conclusion: During biliary injury, Hh pathway activation induces cholangiocyte production of chemokines that recruit NKT cells to portal tracts. (HEPATOLOGY 2009.)

Analysis of sample set enrichment scores

MOTIVATION Gene expression profiling experiments in cell lines and animal models characterized by specific genetic or molecular perturbations have yielded sets of genes annotated by the perturbation. These gene sets can serve as a reference base for interrogating other expression datasets. For example, a new dataset in which a specific pathway gene set appears to be enriched, in terms of multiple genes in that set evidencing expression changes, can then be annotated by that reference pathway. We introduce in this paper a formal statistical method to measure the enrichment of each sample in an expression dataset. This allows us to assay the natural variation of pathway activity in observed gene expression data sets from clinical cancer and other studies. RESULTS Validation of the method and illustrations of biological insights gleaned are demonstrated on cell line data, mouse models, and cancer-related datasets. Using oncogenic pathway signatures, we show that gene sets built from a model system are indeed enriched in the model system. We employ ASSESS for the use of molecular classification by pathways. This provides an accurate classifier that can be interpreted at the level of pathways instead of individual genes. Finally, ASSESS can be used for cross-platform expression models where data on the same type of cancer are integrated over different platforms into a space of enrichment scores. AVAILABILITY Versions are available in Octave and Java (with a graphical user interface). Software can be downloaded at http://people.genome.duke.edu/assess.

A genomic approach to identify molecular pathways associated with chemotherapy resistance

Resistance to chemotherapy in cancer is common. As gene expression profiling has been shown to anticipate chemotherapeutic resistance, we sought to identify cellular pathways associated with resistance to facilitate effective combination therapy. Gene set enrichment analysis was used to associate pathways with resistance in two data sets: the NCI-60 cancer cell lines deemed sensitive and resistant to specific chemotherapeutic agents (Adriamycin, cyclophosphamide, docetaxel, etoposide, 5-fluorouracil, paclitaxel, and topotecan) and a series of 40 lung cancer cell lines for which sensitivity to cisplatin and docetaxel was determined. Candidate pathways were further screened in silico using the Connectivity Map. The lead candidate pathway was functionally validated in vitro. Gene set enrichment analysis associated the matrix metalloproteinase, p53, methionine metabolism, and free pathways with cytotoxic resistance in the NCI-60 cell lines across multiple agents, but no gene set was common to all drugs. Analysis of the lung cancer cell lines identified the bcl-2 pathway to be associated with cisplatin resistance and the AKT pathway enriched in cisplatin- and docetaxel-resistant cell lines. Results from Connectivity Map supported an association between phosphatidylinositol 3-kinase/AKT and docetaxel resistance but did not support the association with cisplatin. Targeted inhibition of the phosphatidylinositol 3-kinase/AKT pathway with LY294002, in combination with docetaxel, resulted in a synergistic effect in previously docetaxel-resistant cell lines but not with cisplatin. These results support the use of a genomic approach to identify drug-specific targets associated with the development of chemotherapy resistance and underscore the importance of disease context in identifying these pathways. [Mol Cancer Ther 2008;7(10):3141–9]

MYC Activity Mitigates Response to Rapamycin in Prostate Cancer through Eukaryotic Initiation Factor 4E–Binding Protein 1–Mediated Inhibition of Autophagy

Loss of PTEN and activation of phosphoinositide 3-kinase are commonly observed in advanced prostate cancer. Inhibition of mammalian target of rapamycin (mTOR), a downstream target of phosphoinositide 3-kinase signaling, results in cell cycle arrest and apoptosis in multiple in vitro and in vivo models of prostate cancer. However, single-agent use of mTOR inhibition has limited clinical success, and the identification of molecular events mitigating tumor response to mTOR inhibition remains a critical question. Here, using genetically engineered human prostate epithelial cells (PrEC), we show that MYC, a frequent target of genetic gain in prostate cancers, abrogates sensitivity to rapamycin by decreasing rapamycin-induced cytostasis and autophagy. Analysis of MYC and the mTOR pathway in human prostate tumors and PrEC showed selective increased expression of eukaryotic initiation factor 4E-binding protein 1 (4EBP1) with gain in MYC copy number or forced MYC expression, respectively. We have also found that MYC binds to regulatory regions of the 4EBP1 gene. Suppression of 4EBP1 expression resulted in resensitization of MYC-expressing PrEC to rapamycin and increased autophagy. Taken together, our findings suggest that MYC expression abrogates sensitivity to rapamycin through increased expression of 4EBP1 and reduced autophagy.

Wild-type p53 gene transfer is not detrimental to normal cells in vivo : implications for tumor gene therapy

The p53 oncosuppressor is strictly maintained in an inactive form under normal conditions, while it is post-translationally activated by a variety of stresses, enacting different protective biological functions. Since one critical issue in cancer gene therapy is tumor specificity, we asked whether the tight p53 regulation applies also to exogenously transferred p53. In principle, this type of regulation could allow p53 gene transfer in both normal and tumor cells to produce detrimental effects only in the latter ones. Here, we report that primary bone marrow cells infected with a p53 recombinant retrovirus and transplanted into irradiated mice reconstitute the hematopoietic system, with no detectable alterations in any of its compartments. Furthermore, simultaneous infection of leukemia and bone marrow cells depleted the neoplastic contamination, allowing lifelong, disease-free survival of 65% of the transplanted animals. These results show that exogenous p53 is controlled as tightly as the endogenous one, and opens the way to p53 gene therapy, without requiring tumor targeting.

Ser58 of mouse p53 is the homologue of human Ser46 and is phosphorylated by HIPK2 in apoptosis.

Ser58 of mouse p53 is the homologue of human Ser46 and is phosphorylated by HIPK2 in apoptosis

Discrimination of single amino acid mutations of the p53 protein by means of deterministic singularities of recurrence quantification analysis

p53 is mutated in roughly 50% of all human tumors, predominantly in the DNA‐binding domain codons. Structural, biochemical, and functional studies have reported that the different p53 mutants possess a broad range of behaviors that include the elimination of the tumor‐suppression function of wild‐type protein, the acquisition of dominant‐negative function over the wild‐type form, and the establishment of gain‐of‐function activities. The contribution of each of these types of mutations to tumor progression, grade of malignancy, and response to anticancer treatments has been so far analyzed only for a few “hot‐spots.” In an attempt to identify new approaches to systematically characterize the complete spectrum of p53 mutations, we applied recurrence quantification analysis (RQA), a non‐linear signal analysis technique, to p53 primary structure. Moving from the study of the p53 hydrophobicity pattern, which revealed important similarities with the singular deterministic structuring of prions, we could statistically discriminate, on a pure amino acid sequence basis, between experimentally characterized DNA‐contact defective and conformational p53 mutants with a very high percentage of success. This result indicates that RQA is a mathematical tool particularly advantageous for the development of a database of p53 mutations that integrates epidemiological data with structural and functional categorizations. Proteins 2004;55:000–000.