Paola Ostano

Multifocal Epithelial Tumors and Field Cancerization from Loss of Mesenchymal CSL Signaling

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 upregulation 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 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.

iASPP/p63 autoregulatory feedback loop is required for the homeostasis of stratified epithelia

iASPP, an inhibitory member of the ASPP (apoptosis stimulating protein of p53) family, is an evolutionarily conserved inhibitor of p53 which is frequently upregulated in human cancers. However, little is known about the role of iASPP under physiological conditions. Here, we report that iASPP is a critical regulator of epithelial development. We demonstrate a novel autoregulatory feedback loop which controls crucial physiological activities by linking iASPP to p63, via two previously unreported microRNAs, miR‐574‐3p and miR‐720. By investigating its function in stratified epithelia, we show that iASPP participates in the p63‐mediated epithelial integrity program by regulating the expression of genes essential for cell adhesion. Silencing of iASPP in keratinocytes by RNA interference promotes and accelerates a differentiation pathway, which also affects and slowdown cellular proliferation. Taken together, these data reveal iASPP as a key regulator of epithelial homeostasis.

The FoxO3a gene is a key negative target of canonical Notch signalling in the keratinocyte UVB response.

Notch signalling has an important role in skin homeostasis, promoting keratinocyte differentiation and suppressing tumorigenesis. Here we show that this pathway also has an essential anti‐apoptotic function in the keratinocyte UVB response. Notch1 expression and activity are significantly induced, in a p53‐dependent manner, by UVB exposure of primary keratinocytes as well as intact epidermis of both mouse and human origin. The apoptotic response to UVB is increased by deletion of the Notch1 gene or down‐modulation of Notch signalling by pharmacological inhibition or genetic suppression of ‘canonical’ Notch/CSL/MAML1‐dependent transcription. Conversely, Notch activation protects keratinocytes against apoptosis through a mechanism that is not linked to Notch‐induced cell cycle withdrawal or NF‐κB activation. Rather, transcription of FoxO3a, a key pro‐apoptotic gene, is under direct negative control of Notch/HERP transcription in keratinocytes, and upregulation of this gene accounts for the increased susceptibility to UVB of cells with suppressed Notch signalling. Thus, the canonical Notch/HERP pathway functions as a protective anti‐apoptotic mechanism in keratinocytes through negative control of FoxO3a expression.

A miR-34a-SIRT6 axis in the squamous cell differentiation network

Squamous cell carcinomas (SCCs) are highly heterogeneous tumours, resulting from deranged expression of genes involved in squamous cell differentiation. Here we report that microRNA‐34a (miR‐34a) functions as a novel node in the squamous cell differentiation network, with SIRT6 as a critical target. miR‐34a expression increases with keratinocyte differentiation, while it is suppressed in skin and oral SCCs, SCC cell lines, and aberrantly differentiating primary human keratinocytes (HKCs). Expression of this miRNA is restored in SCC cells, in parallel with differentiation, by reversion of genomic DNA methylation or wild‐type p53 expression. In normal HKCs, the pro‐differentiation effects of increased p53 activity or UVB exposure are miR‐34a‐dependent, and increased miR‐34a levels are sufficient to induce differentiation of these cells both in vitro and in vivo. SIRT6, a sirtuin family member not previously connected with miR‐34a function, is a direct target of this miRNA in HKCs, and SIRT6 down‐modulation is sufficient to reproduce the miR‐34a pro‐differentiation effects. The findings are of likely biological significance, as SIRT6 is oppositely expressed to miR‐34a in normal keratinocytes and keratinocyte‐derived tumours.

Combined CSL and p53 downregulation promotes cancer-associated fibroblast activation

Stromal fibroblast senescence has been linked to ageing-associated cancer risk. However, density and proliferation of cancer-associated fibroblasts (CAFs) are frequently increased. Loss or downmodulation of the Notch effector CSL (also known as RBP-Jκ) in dermal fibroblasts is sufficient for CAF activation and ensuing keratinocyte-derived tumours. We report that CSL silencing induces senescence of primary fibroblasts from dermis, oral mucosa, breast and lung. CSL functions in these cells as a direct repressor of multiple senescence- and CAF-effector genes. It also physically interacts with p53, repressing its activity. CSL is downmodulated in stromal fibroblasts of premalignant skin actinic keratosis lesions and squamous cell carcinomas, whereas p53 expression and function are downmodulated only in the latter, with paracrine FGF signalling as the probable culprit. Concomitant loss of CSL and p53 overcomes fibroblast senescence, enhances expression of CAF effectors and promotes stromal and cancer cell expansion. The findings support a CAF activation–stromal co-evolution model under convergent CSL–p53 control.

Patient-derived ovarian tumor xenografts recapitulate human clinicopathology and genetic alterations

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. On the basis of its histopathology and molecular-genomic changes, ovarian cancer has been divided into subtypes, each with distinct biology and outcome. The aim of this study was to develop a panel of patient-derived EOC xenografts that recapitulate the molecular and biologic heterogeneity of human ovarian cancer. Thirty-four EOC xenografts were successfully established, either subcutaneously or intraperitoneally, in nude mice. The xenografts were histologically similar to the corresponding patient tumor and comprised all the major ovarian cancer subtypes. After orthotopic transplantation in the bursa of the mouse ovary, they disseminate into the organs of the peritoneal cavity and produce ascites, typical of ovarian cancer. Gene expression analysis and mutation status indicated a high degree of similarity with the original patient and discriminate different subsets of xenografts. They were very responsive, responsive, and resistant to cisplatin, resembling the clinical situation in ovarian cancer. This panel of patient-derived EOC xenografts that recapitulate the recently type I and type II classification serves to study the biology of ovarian cancer, identify tumor-specific molecular markers, and develop novel treatment modalities.

A positive FGFR3/FOXN1 feedback loop underlies benign skin keratosis versus squamous cell carcinoma formation in humans

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.

ETS transcription factor ESE1/ELF3 orchestrates a positive feedback loop that constitutively activates NF-κB and drives prostate cancer progression

Chromosomal translocations leading to deregulated expression of ETS transcription factors are frequent in prostate tumors. Here, we report a novel mechanism leading to oncogenic activation of the ETS factor ESE1/ELF3 in prostate tumors. ESE1/ELF3 was overexpressed in human primary and metastatic tumors. It mediated transforming phenotypes in vitro and in vivo and induced an inflammatory transcriptome with changes in relevant oncogenic pathways. ESE1/ELF3 was induced by interleukin (IL)-1β through NF-κB and was a crucial mediator of the phenotypic and transcriptional changes induced by IL-1β in prostate cancer cells. This linkage was mediated by interaction of ESE1/ELF3 with the NF-κB subunits p65 and p50, acting by enhancing their nuclear translocation and transcriptional activity and by inducing p50 transcription. Supporting these findings, gene expression profiling revealed an enrichment of NF-κB effector functions in prostate cancer cells or tumors expressing high levels of ESE1/ELF3. We observed concordant upregulation of ESE1/ELF3 and NF-κB in human prostate tumors that was associated with adverse prognosis. Collectively, our results define an important new mechanistic link between inflammatory signaling and the progression of prostate cancer.

Multifactorial ERβ and NOTCH1 control of squamous differentiation and cancer

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.

Nicotinamide phosphoribosyltransferase (NAMPT) is over‐expressed in melanoma lesions

To take out a personal subscription, please click here More information about Pigment Cell & Melanoma Research at www.pigment.org Nicotinamide phosphoribosyltransferase (NAMPT) is over-expressed in melanoma lesions Elena Maldi, Cristina Travelli, Antonio Caldarelli, Nicolò Agazzone, Sara Cintura, Ubaldina Galli, Maria Scatolini, Paola Ostano, Benedetta Miglino, Giovanna Chiorino, Renzo Boldorini and Armando A. Genazzani