Giovanna Chiorino

Telomere damage induced by the G-quadruplex ligand RHPS4 has an antitumor effect

Functional telomeres are required for the replicability of cancer cells. The G-rich strand of telomeric DNA can fold into a 4-stranded structure known as the G-quadruplex (G4), whose stabilization alters telomere function limiting cancer cell growth. Therefore, the G4 ligand RHPS4 may possess antitumor activity. Here, we show that RHPS4 triggers a rapid and potent DNA damage response at telomeres in human transformed fibroblasts and melanoma cells, characterized by the formation of several telomeric foci containing phosphorylated DNA damage response factors gamma-H2AX, RAD17, and 53BP1. This was dependent on DNA repair enzyme ATR, correlated with delocalization of the protective telomeric DNA-binding protein POT1, and was antagonized by overexpression of POT1 or TRF2. In mice, RHPS4 exerted its antitumor effect on xenografts of human tumor cells of different histotype by telomere injury and tumor cell apoptosis. Tumor inhibition was accompanied by a strong DNA damage response, and tumors overexpressing POT1 or TRF2 were resistant to RHPS4 treatment. These data provide evidence that RHPS4 is a telomere damage inducer and that telomere disruption selectively triggered in malignant cells results in a high therapeutic index in mice. They also define a functional link between telomere damage and antitumor activity and reveal the key role of telomere-protective factors TRF2 and POT1 in response to this anti-telomere strategy.

High Commitment of Embryonic Keratinocytes to Terminal Differentiation through a Notch1-caspase 3 Regulatory Mechanism

Embryonic cells are expected to possess high growth/differentiation potential, required for organ morphogenesis and expansion during development. However, little is known about the intrinsic properties of embryonic epithelial cells due to difficulties in their isolation and cultivation. We report here that pure keratinocyte populations from E15.5 mouse embryos commit irreversibly to differentiation much earlier than newborn cells. Notch signaling, which promotes keratinocyte differentiation, is upregulated in embryonic keratinocyte and epidermis, and elevated caspase 3 expression, which we identify as a transcriptional Notch1 target, accounts in part for the high commitment of embryonic keratinocytes to terminal differentiation. In vivo, lack of caspase 3 results in increased proliferation and decreased differentiation of interfollicular embryonic keratinocytes, together with decreased activation of PKC-delta, a caspase 3 substrate which functions as a positive regulator of keratinocyte differentiation. Thus, a Notch1-caspase 3 regulatory mechanism underlies the intrinsically high commitment of embryonic keratinocytes to terminal differentiation.

Association between miR-200c and the survival of patients with stage I epithelial ovarian cancer: a retrospective study of two independent tumour tissue collections.

BACKGROUND International Federation of Gynecology and Obstetrics stage I epithelial ovarian cancer (EOC) has a significantly better prognosis than stage III/IV EOC, with about 80% of patients surviving at 5 years (compared with about 20% of those with stage III/IV EOC). However, 20% of patients with stage I EOC relapse within 5 years. It is therefore crucial that the biological properties of stage I EOCs are further elucidated. MicroRNAs (miRNAs) have shown diagnostic and prognostic potential in stage III and IV EOCs, but the small number of patients diagnosed with stage I EOC has so far prevented an investigation of its molecular features. We profiled miRNA expression in stage I EOC tumours to assess whether there is a miRNA signature associated with overall and progression-free survival (PFS) in stage I EOC. METHODS We analysed tumour samples from 144 patients (29 of whom relapsed) with stage I EOC gathered from two independent tumour tissue collections (A and B), both with a median follow-up of 9 years. 89 samples from tumour tissue collection A were stratified into a training set (51 samples, 15 of which were from patients who relapsed) for miRNA signature generation, and into a validation set (38 samples, seven of which were from patients who relapsed) for signature validation. Tumour tissue collection B (55 samples, seven of which were from patients who relapsed) was used as an independent test set. The Cox proportional hazards model and the log-rank test were used to assess the correlation of quantitative reverse transcription PCR (qRT-PCR)-validated miRNAs with overall survival and PFS. FINDINGS A signature of 34 miRNAs associated with survival was generated by microarray analysis in the training set. In both the training set and validation set, qRT-PCR analysis confirmed that 11 miRNAs (miR-214, miR-199a-3p, miR-199a-5p, miR-145, miR-200b, miR-30a, miR-30a*, miR-30d, miR-200c, miR-20a, and miR-143) were expressed differently in relapsers compared with non-relapsers. Three of these miRNAs (miR-200c, miR-199a-3p, miR-199a-5p) were associated with PFS, overall survival, or both in multivariate analysis. qRT-PCR analysis in the test set confirmed the downregulation of miR-200c in relapsers compared with non-relapsers, but not the upregulation of miR-199a-3p and miR-199a-5p. Multivariate analysis confirmed that downregulation of miR-200c in the test set was associated with overall survival (HR 0·094, 95% CI 0·012-0·766, p=0·0272) and PFS (0·035, 0·004-0·311; p=0·0026), independent of clinical covariates. INTERPRETATION miR-200c has potential as a predictor of survival, and is a biomarker of relapse, in stage I EOC. FUNDING Nerina and Mario Mattioli Foundation, Cariplo Foundation (Grant Number 2010-0744), and the Italian Association for Cancer Research.

ETS Transcription Factors Control Transcription of EZH2 and Epigenetic Silencing of the Tumor Suppressor Gene Nkx3.1 in Prostate Cancer

Background ETS transcription factors regulate important signaling pathways involved in cell differentiation and development in many tissues and have emerged as important players in prostate cancer. However, the biological impact of ETS factors in prostate tumorigenesis is still debated. Methodology/Principal Findings We performed an analysis of the ETS gene family using microarray data and real-time PCR in normal and tumor tissues along with functional studies in normal and cancer cell lines to understand the impact in prostate tumorigenesis and identify key targets of these transcription factors. We found frequent dysregulation of ETS genes with oncogenic (i.e., ERG and ESE1) and tumor suppressor (i.e., ESE3) properties in prostate tumors compared to normal prostate. Tumor subgroups (i.e., ERGhigh, ESE1high, ESE3low and NoETS tumors) were identified on the basis of their ETS expression status and showed distinct transcriptional and biological features. ERGhigh and ESE3low tumors had the most robust gene signatures with both distinct and overlapping features. Integrating genomic data with functional studies in multiple cell lines, we demonstrated that ERG and ESE3 controlled in opposite direction transcription of the Polycomb Group protein EZH2, a key gene in development, differentiation, stem cell biology and tumorigenesis. We further demonstrated that the prostate-specific tumor suppressor gene Nkx3.1 was controlled by ERG and ESE3 both directly and through induction of EZH2. Conclusions/Significance These findings provide new insights into the role of the ETS transcriptional network in prostate tumorigenesis and uncover previously unrecognized links between aberrant expression of ETS factors, deregulation of epigenetic effectors and silencing of tumor suppressor genes. The link between aberrant ETS activity and epigenetic gene silencing may be relevant for the clinical management of prostate cancer and design of new therapeutic strategies.

Resistance to platinum-based chemotherapy is associated with epithelial to mesenchymal transition in epithelial ovarian cancer

BACKGROUND The present study is aimed to identify genetic pathways correlated with chemoresistance in epithelial ovarian cancer (EOC). METHODS We compared the molecular profiles of 23 tumour biopsies of stage III-IV (training set) at primary surgery, before chemotherapy, to the profile from the same patients at second surgery, after several lines of platinum (Pt)-based chemotherapy when the tumours were resistant. In the hypothesis that identified markers were related to Pt-resistance and to prognosis, we validated this signature in 52 EOC taken at primary surgery (validation set) selected to be either very sensitive to the first line therapy, i.e. not relapsing before one year from the end of therapy, or resistant, i.e. relapsing within 6 months from the end of therapy. RESULTS In the training set, we identified a resistance signature indicative of the activation of epithelial to mesenchymal transition (EMT) by transforming growth factor (TGF)-beta pathway. We then validated this signature in 52 EOC taken at primary surgery (validation set). Some genes involved in EMT, such as BMP and activin membrane-bound inhibitor (BAMBI), and mir-141 resulted in association with overall or progression free survival. CONCLUSION Some genes involved in EMT were associated to overall or progression free survival, suggesting EMT as vital to the resistance mechanisms.

Current understanding of the thrombospondin-1 interactome

The multifaceted action of thrombospondin-1 (TSP-1) depends on its ability to physically interact with different ligands, including structural components of the extracellular matrix, other matricellular proteins, cell receptors, growth factors, cytokines and proteases. Through this network, TSP-1 regulates the ligand activity, availability and structure, ultimately tuning the cell response to environmental stimuli in a context-dependent manner, contributing to physiological and pathological processes. Complete mapping of the TSP-1 interactome is needed to understand its diverse functions and to lay the basis for the rational design of TSP-1-based therapeutic approaches. So far, large-scale approaches to identify TSP-1 ligands have been rarely used, but many interactions have been identified in small-scale studies in defined biological systems. This review, based on information from protein interaction databases and the literature, illustrates current knowledge of the TSP-1 interactome map.

ESE3/EHF Controls Epithelial Cell Differentiation and Its Loss Leads to Prostate Tumors with Mesenchymal and Stem-like Features

Cancer stem cells (CSC) play a significant role in tumor progression, disease recurrence, and treatment failure. Here, we show that the endogenously expressed ETS transcription factor ESE3/EHF controls prostate epithelial cell differentiation and stem-like potential. We found that loss of ESE3/EHF induced epithelial-to-mesenchymal transition (EMT), stem-like features, and tumor-initiating and metastatic properties in prostate epithelial cells, and reexpression of ESE3/EHF inhibited the stem-like properties and tumorigenic potential of prostate cancer cells. Mechanistically, ESE3/EHF repressed the expression of key EMT and CSC genes, including TWIST1, ZEB2, BMI1, and POU5F1. Analysis of human tissue microarrays showed that reduced ESE3/EHF expression is an early event in tumorigenesis, frequently occurring independently of other ETS gene alterations. Additional analyses linked loss of ESE3/EHF expression to a distinct group of prostate tumors with distinctive molecular and biologic characteristics, including increased expression of EMT and CSC genes. Low ESE3/EHF expression was also associated with increased biochemical recurrence of prostate cancer and reduced overall survival after prostatectomy. Collectively, our findings define a key role for ESE3/EHF in the development of a subset of prostate tumors and highlight the clinical importance of identifying molecularly defined tumor subgroups.

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.

Altered molecular pathways in melanocytic lesions.

To identify gene expression changes in melanocytic lesions, biopsies from 18 common nevi (CMN), 11 dysplastic nevi (DN), 8 radial and 15 vertical growth phase melanomas (RGPM, VGPM), and 5 melanoma metastases (MTS) were analyzed using whole genome microarrays. The comparison between CMN and RGPM showed an enrichment of Gene Ontology terms related to inter and intracellular junctions, whereas the transition from RGPM to VGPM underlined the alteration of apoptosis. Upregulation of genes involved in dsDNA break repair and downregulation of cellular adhesion genes were observed in MTS with respect to VGPM. DN exhibited rather heterogeneous molecular profiles, with some proliferation genes expressed at higher levels than in CMN, altered regulation of transcription compared to RGPM and a subset of processes, such as mismatch repair, equally expressed as in VGPM. Furthermore, the expression profile of genes involved into cellular detoxification and antigen presentation split them into two classes, with different proliferation potential. Finally, molecular profiling of individual lesions identified altered biological processes, such as regulation of apoptosis, regulation of transcription and T‐cell activation, not associated with specific histological classes but rather with subgroups of samples without apparent relationship. This holds true for dysplastic nevi in particular. Our data indicate that generally the intersection between stage specific and sample specific molecular alterations may lead to a more precise determination of the individual progression risk of melanocytic lesions.