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1.  Multifactorial ERβ and NOTCH1 control of squamous differentiation and cancer 
The Journal of Clinical Investigation  2014;124(5):2260-2276.
Downmodulation or loss-of-function mutations of the gene encoding NOTCH1 are associated with dysfunctional squamous cell differentiation and development of squamous cell carcinoma (SCC) in skin and internal organs. While NOTCH1 receptor activation has been well characterized, little is known about how NOTCH1 gene transcription is regulated. Using bioinformatics and functional screening approaches, we identified several regulators of the NOTCH1 gene in keratinocytes, with the transcription factors DLX5 and EGR3 and estrogen receptor β (ERβ) directly controlling its expression in differentiation. DLX5 and ERG3 are required for RNA polymerase II (PolII) recruitment to the NOTCH1 locus, while ERβ controls NOTCH1 transcription through RNA PolII pause release. Expression of several identified NOTCH1 regulators, including ERβ, is frequently compromised in skin, head and neck, and lung SCCs and SCC-derived cell lines. Furthermore, a keratinocyte ERβ–dependent program of gene expression is subverted in SCCs from various body sites, and there are consistent differences in mutation and gene-expression signatures of head and neck and lung SCCs in female versus male patients. Experimentally increased ERβ expression or treatment with ERβ agonists inhibited proliferation of SCC cells and promoted NOTCH1 expression and squamous differentiation both in vitro and in mouse xenotransplants. Our data identify a link between transcriptional control of NOTCH1 expression and the estrogen response in keratinocytes, with implications for differentiation therapy of squamous cancer.
PMCID: PMC4002597  PMID: 24743148
2.  Loss of mesenchymal CSL signaling leads to field cancerization and multifocal epithelial tumors 
Cell  2012;149(6):1207-1220.
It is currently unclear whether tissue changes surrounding multifocal epithelial tumors are a cause or consequence of cancer. Here, we provide evidence that loss of mesenchymal Notch/CSL signaling causes tissue alterations, including stromal atrophy and inflammation, which precede and are potent triggers for epithelial tumors. Mice carrying a mesenchymal-specific deletion of CSL/RBP-Jκ, a key Notch effector, exhibit spontaneous multifocal keratinocyte tumors that develop after dermal atrophy and inflammation. CSL-deficient dermal fibroblasts promote increased tumor cell proliferation through up-regulation of c-Jun and c-Fos expression and consequently higher levels of diffusible growth factors, inflammatory cytokines, and matrix remodeling enzymes. In human skin samples, stromal fields adjacent to cutaneous squamous cell carcinomas and multifocal premalignant actinic keratosis lesions exhibit decreased Notch/CSL signaling and associated molecular changes. Importantly, these changes in gene expression are also induced by UVA, a known environmental cause of cutaneous field cancerization and skin cancer.
PMCID: PMC3578441  PMID: 22682244
epithelial-mesenchymal interactions; epithelial cancer; Cancer Associated Fibroblasts; in situ carcinoma; actinic keratosis; Notch; AP-1
3.  Cross-Analysis of Gene and miRNA Genome-Wide Expression Profiles in Human Fibroblasts at Different Stages of Transformation 
We have developed a cellular system constituted of human telomerase immortalized fibroblasts that gradually underwent neoplastic transformation during propagation in culture. We exploited this cellular system to investigate gene and miRNA transcriptional programs in cells at different stages of propagation, representing five different phases along the road to transformation, from non-transformed cells up to tumorigenic and metastatic ones. Here we show that gene and miRNA expression profiles were both able to divide cells according to their transformation phase. We identified more than 1,700 genes whose expression was highly modulated in cells at at least one propagation stage and we found that the number of modulated genes progressively increased at successive stages of transformation. These genes identified processes significantly deregulated in tumorigenic cells, such as cell differentiation, cell movement and extracellular matrix remodeling, cell cycle and apoptosis, together with upregulation of several cancer testis antigens. Alterations in cell cycle, apoptosis, and cancer testis antigen expression were particular hallmarks of metastatic cells. A parallel deregulation of a panel of 43 miRNAs strictly connected to the p53 and c-Myc pathways and with oncogenic/oncosuppressive functions was also found. Our results indicate that cen3tel cells can be a useful model for human fibroblast neoplastic transformation, which appears characterized by complex and peculiar alterations involving both genetic and epigenetic reprogramming, whose elucidation could provide useful insights into regulatory networks underlying cancerogenesis.
PMCID: PMC3275799  PMID: 22321013
5.  Overexpression of CD157 Contributes to Epithelial Ovarian Cancer Progression by Promoting Mesenchymal Differentiation 
PLoS ONE  2012;7(8):e43649.
Epithelial ovarian carcinoma (EOC) is an aggressive tumor often diagnosed at an advanced stage, when there is little or no prospect of cure. Despite advances in surgical and chemotherapeutic strategies, only marginal improvements in patient outcome have been obtained. Hence, unraveling the biological mechanisms underpinning EOC progression is critical for improving patients’ survival. Recently, we reported that CD157 (an ectoenzyme regulating leukocyte diapedesis) is expressed in EOC and that high expression of the molecule is negatively correlated with the disease outcome in patients. Here, we demonstrate that forced overexpression of CD157 in OVCAR-3, TOV-21G, A2780 and OV-90 ovarian cancer cell lines promotes morphological and phenotypic changes characterized by disruption of intercellular junctions, downregulation of epithelial markers and upregulation of mesenchymal ones. These changes in cell shape and phenotype bring to reduced sensitivity to anoikis, increased anchorage-independent growth, cell motility and mesothelial invasion. Conversely, knockdown of CD157 in OV-90 and OC314 cells reverts the mesenchymal phenotype and reduces the cells’ migratory potential. Transcriptome profiling analysis highlighted 378 significantly differentially expressed genes, representing the signature of CD157-overexpressing OVCAR-3 and OV-90 cells. The modulation of selected genes translates into alteration of protein expression that give cells a highly malignant phenotype. The overall picture deduced from the analysis of the modulated transcripts is that high expression of CD157 strengthens a number of biological processes favoring tumor progression (including development and cell motility), and weakens several biological processes hindering tumor progression (such as apoptosis, cell death and response to stress). Together, these findings implicate CD157 in the progression of EOC to metastatic disease and suggest that CD157 may represent a valuable therapeutic target.
PMCID: PMC3423388  PMID: 22916288
6.  A Systems Biology Approach to Characterize the Regulatory Networks Leading to Trabectedin Resistance in an In Vitro Model of Myxoid Liposarcoma 
PLoS ONE  2012;7(4):e35423.
Trabectedin, a new antitumor compound originally derived from a marine tunicate, is clinically effective in soft tissue sarcoma. The drug has shown a high selectivity for myxoid liposarcoma, characterized by the translocation t(12;16)(q13; p11) leading to the expression of FUS-CHOP fusion gene. Trabectedin appears to act interfering with mechanisms of transcription regulation. In particular, the transactivating activity of FUS-CHOP was found to be impaired by trabectedin treatment. Even after prolonged response resistance occurs and thus it is important to elucidate the mechanisms of resistance to trabectedin. To this end we developed and characterized a myxoid liposarcoma cell line resistant to trabectedin (402-91/ET), obtained by exposing the parental 402-91 cell line to stepwise increases in drug concentration. The aim of this study was to compare mRNAs, miRNAs and proteins profiles of 402-91 and 402-91/ET cells through a systems biology approach. We identified 3,083 genes, 47 miRNAs and 336 proteins differentially expressed between 402-91 and 402-91/ET cell lines. Interestingly three miRNAs among those differentially expressed, miR-130a, miR-21 and miR-7, harbored CHOP binding sites in their promoter region. We used computational approaches to integrate the three regulatory layers and to generate a molecular map describing the altered circuits in sensitive and resistant cell lines. By combining transcriptomic and proteomic data, we reconstructed two different networks, i.e. apoptosis and cell cycle regulation, that could play a key role in modulating trabectedin resistance. This approach highlights the central role of genes such as CCDN1, RB1, E2F4, TNF, CDKN1C and ABL1 in both pre- and post-transcriptional regulatory network. The validation of these results in in vivo models might be clinically relevant to stratify myxoid liposarcoma patients with different sensitivity to trabectedin treatment.
PMCID: PMC3327679  PMID: 22523595
8.  A positive FGFR3/FOXN1 feedback loop underlies benign skin keratosis versus squamous cell carcinoma formation in humans 
The Journal of Clinical Investigation  2009;119(10):3127-3137.
Seborrheic keratoses (SKs) are common, benign epithelial tumors of the skin that do not, or very rarely, progress into malignancy, for reasons that are not understood. We investigated this by gene expression profiling of human SKs and cutaneous squamous cell carcinomas (SCCs) and found that several genes previously connected with keratinocyte tumor development were similarly modulated in SKs and SCCs, whereas the expression of others differed by only a few fold. In contrast, the tyrosine kinase receptor FGF receptor–3 (FGFR3) and the transcription factor forkhead box N1 (FOXN1) were highly expressed in SKs, and close to undetectable in SCCs. We also showed that increased FGFR3 activity was sufficient to induce FOXN1 expression, counteract the inhibitory effect of EGFR signaling on FOXN1 expression and differentiation, and induce differentiation in a FOXN1-dependent manner. Knockdown of FOXN1 expression in primary human keratinocytes cooperated with oncogenic RAS in the induction of SCC-like tumors, whereas increased FOXN1 expression triggered the SCC cells to shift to a benign SK-like tumor phenotype, which included increased FGFR3 expression. Thus, we have uncovered a positive regulatory loop between FGFR3 and FOXN1 that underlies a benign versus malignant skin tumor phenotype.
PMCID: PMC2752067  PMID: 19729838

Results 1-8 (8)