Cell death and neuronal differentiation of glioblastoma stem-like cells induced by neurogenic transcription factors.

Glioblastoma multiform (GBM) are devastating brain tumors containing a fraction of multipotent stem-like cells which are highly tumorigenic. These cells are resistant to treatments and are likely to be responsible for tumor recurrence. One approach to eliminate GBM stem-like cells would be to force their terminal differentiation. During development, neurons formation is controlled by neurogenic transcription factors such as Ngn1/2 and NeuroD1. We found that in comparison with oligodendrogenic genes, the expression of these neurogenic genes is low or absent in GBM tumors and derived cultures. We thus explored the effect of overexpressing these neurogenic genes in three CD133(+) Sox2(+) GBM stem-like cell cultures and the U87 glioma line. Introduction of Ngn2 in CD133(+) cultures induced massive cell death, proliferation arrest and a drastic reduction of neurosphere formation. Similar effects were observed with NeuroD1. Importantly, Ngn2 effects were accompanied by the downregulation of Olig2, Myc, Shh and upregulation of Dcx and NeuroD1 expression. The few surviving cells adopted a typical neuronal morphology and some of them generated action potentials. These cells appeared to be produced at the expense of GFAP(+) cells which were radically reduced after differentiation with Ngn2. In vivo, Ngn2-expressing cells were unable to form orthotopic tumors. In the U87 glioma line, Ngn2 could not induce neuronal differentiation although proliferation in vitro and tumoral growth in vivo were strongly reduced. By inducing cell death, cell cycle arrest or differentiation, this work supports further exploration of neurogenic proteins to oppose GBM stem-like and non-stem-like cell growth.

Variations in the mRNA expression of poly(ADP-ribose) polymerases, poly(ADP-ribose) glycohydrolase and ADP-ribosylhydrolase 3 in breast tumors and impact on clinical outcome.

In order to assess the variation in expression of poly(ADP-ribose) polymerase (PARP) family members and the hydrolases that degrade the poly(ADP-ribose) polymers they generate and possible associations with classical pathological parameters, including long-term outcome, the mRNA levels of PARP1, PARP2, PARP3, poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3) were examined using quantitative reverse transcription polymerase chain reaction in 443 unilateral invasive breast cancers and linked to hormonal status, tumor proliferation and clinical outcome. PARP1 mRNA levels were the highest among these five genes in both normal and tumor tissues, with a 2.45-fold higher median level in tumors compared to normal tissues. Tumors (34.1%) showed PARP1 overexpression (>3 fold relative to normal breast tissues) compared to underexpression (<0.33 fold) in only 0.5%. This overexpression was seen in all breast tumor subgroups, with the highest fraction (51%) seen in the HR-positive/ERBB2-positive subgroup and was not highly associated with any other classical predictive factors. No correlation was seen between PARP1 mRNA and PARP-1 protein levels in a subset of 31 tumors. PARP3 was underexpressed in 10.4% of tumors, more frequently in the HR-negative tumors (25.4%) than the HR-positive tumors (5.9%). This PARP3 underexpression was mutually exclusive with a PARP1 overexpression. PARP2 levels were unchanged between normal and tumor tissues and few tumors showed overexpression of PARG (3.8%) or ARH3 (3.4%). Within the subgroup of triple negative tumors, PARG mRNA levels below the median were associated with a higher risk of developing metastases (p = 0.039) raising the possibility this might be marker of clinical outcome.

microRNA expression profile in a large series of bladder tumors: identification of a 3-miRNA signature associated with aggressiveness of muscle-invasive bladder cancer.

The aim of this study was to evaluate the expression levels of microRNAs (miRNAs) in bladder tumors in order to identify miRNAs involved in bladder carcinogenesis with potential prognostic implications. Expression levels of miRNAs were assessed by quantitative real-time RT-PCR in 11 human normal bladder and 166 bladder tumor samples (86 non-muscle-invasive bladder cancer (NMIBC) and 80 muscle-invasive bladder cancer (MIBC)). The expression level of 804 miRNAs was initially measured in a well-defined series of seven NMIBC, MIBC and normal bladder samples (screening set). The most strongly deregulated miRNAs in tumor samples compared to normal bladder tissue were then selected for RT-PCR validation in a well-characterized independent series of 152 bladder tumors (validation set), and in six bladder cancer cell lines. Expression levels of these miRNAs were tested for their association with clinical outcome. A robust group of 15 miRNAs was found to be significantly deregulated in bladder cancer. Except for two miRNAs, miR-146b and miR-9, which were specifically upregulated in MIBC, the majority of miRNAs (n = 13) were deregulated in the same way in the two types of bladder tumors, irrespective of pathological stage : three miRNAs were upregulated (miR-200b, miR-182 and miR-138) and the other 10 miRNAs were downregulated (miR-1, miR-133a, miR-133b, miR-145, miR-143, miR-204, miR-921, miR-1281, miR-199a and miR-199b). A 3-miRNA signature (miR-9, miR-182 and miR-200b) was found to be related to MIBC tumor aggressiveness and was associated with both recurrence-free and overall survival in univariate analysis with a trend to significance in the multivariate analysis (p = 0.05). Our results suggested a promising individual prognostic value of these new markers.

miRNA expression profiling of inflammatory breast cancer identifies a 5-miRNA signature predictive of breast tumor aggressiveness.

IBC (inflammatory breast cancer) is a rare but very aggressive form of breast cancer with a particular phenotype. The molecular mechanisms responsible for IBC remain largely unknown. In particular, genetic and epigenetic alterations specific to IBC remain to be identified. MicroRNAs, a class of small noncoding RNAs able to regulate gene expression, are deregulated in breast cancer and may therefore serve as tools for diagnosis and prediction. This study was designed to determine miRNA expression profiling (microRNAome) in IBC. Quantitative RT-PCR was used to determine expression levels of 804 miRNAs in a screening series of 12 IBC compared to 31 non-stage-matched non-IBC and 8 normal breast samples. The differentially expressed miRNAs were then validated in a series of 65 IBC and 95 non-IBC. From a set of 18 miRNAs of interest selected from the screening series, 13 were differentially expressed with statistical significance in the validation series of IBC compared to non-IBC. Among these, a 5-miRNA signature comprising miR-421, miR-486, miR-503, miR-720 and miR-1303 was shown to be predictive for IBC phenotype with an overall accuracy of 89%. Moreover, multivariate analysis showed that this signature was an independent predictor of poor Metastasis-Free Survival in non-IBC patients.

PIK3R1 underexpression is an independent prognostic marker in breast cancer.

BACKGROUND: The present study focused on the prognostic roles of PIK3CA and PIK3R1 genes and additional PI3K pathway-associated genes in breast cancer. METHODS: The mutational and mRNA expression status of PIK3CA, PIK3R1 and AKT1, and expression status of other genes involved in the PI3K pathway (EGFR, PDK1, PTEN, AKT2, AKT3, GOLPH3, WEE1, P70S6K) were assessed in a series of 458 breast cancer samples. RESULTS: PIK3CA mutations were identified in 151 samples (33.0%) in exons 1, 2, 9 and 20. PIK3R1 mutations were found in 10 samples (2.2%) and underexpression in 283 samples (61.8%). AKT1 mutations were found in 15 samples (3.3%) and overexpression in 116 samples (25.3%). PIK3R1 underexpression tended to mutual exclusivity with PIK3CA mutations (p = 0.00097). PIK3CA mutations were associated with better metastasis-free survival and PIK3R1 underexpression was associated with poorer metastasis-free survival (p = 0.014 and p = 0.00028, respectively). By combining PIK3CA mutation and PIK3R1 expression status, four prognostic groups were identified with significantly different metastasis-free survival (p = 0.00046). On Cox multivariate regression analysis, the prognostic significance of PIK3R1 underexpression was confirmed in the total population (p = 0.0013) and in breast cancer subgroups. CONCLUSIONS: PIK3CA mutations and PIK3R1 underexpression show opposite effects on patient outcome and could become useful prognostic and predictive factors in breast cancer.

PIK3CA mutation impact on survival in breast cancer patients and in ERα, PR and ERBB2-based subgroups.

INTRODUCTION: PIK3CA is the oncogene showing the highest frequency of gain-of-function mutations in breast cancer, but the prognostic value of PIK3CA mutation status is controversial. METHODS: We investigated the prognostic significance of PIK3CA mutation status in a series of 452 patients with unilateral invasive primary breast cancer and known long-term outcome (median follow-up 10 years). RESULTS: PIK3CA mutations were identified in 151 tumors (33.4%). The frequency of PIK3CA mutations differed markedly according to hormone receptor (estrogen receptor alpha [ERα] and progesterone receptor [PR]) and ERBB2 status, ranging from 12.5% in the triple-negative subgroup (ER-/PR-/ERBB2-) to 41.1% in the HR+/ERBB2- subgroup. PIK3CA mutation was associated with significantly longer metastasis-free survival in the overall population (P = 0.0056), and especially in the PR-positive and ERBB2-positive subgroups. In Cox multivariate regression analysis, the prognostic significance of PIK3CA mutation status persisted only in the ERBB2-positive subgroup. CONCLUSIONS: This study confirms the high prevalence of PIK3CA mutations in breast cancer. PIK3CA mutation is an emerging tumor marker which might become used in treatment-choosing process. The independent prognostic value of PIK3CA mutation status in ERBB2-positive breast cancer patients should be now confirmed in larger series of patients included in randomized prospective ERBB2-based clinical trials.

Quantification of PKC family genes in sporadic breast cancer by qRT-PCR: evidence that PKCι/λ overexpression is an independent prognostic factor.

Drugs targeting protein kinase C (PKC) show promising therapeutic activity. However, little is known about the expression patterns of the 11 PKC genes in human tumors, and the clinical significance of most PKC genes is unknown. We used qRT-PCR assays to quantify mRNA levels of the 11 PKC genes in 458 breast tumors from patients with known clinical/pathological status and long-term outcome. The proportion of tumors in which the expression of the different genes was altered varied widely, from 9.6% for PKN2 to 40.2% for PKCι/λ. In breast tumors, overexpression was the main alteration observed for PKCι/λ (33.4%), PKCδ (29.5%) and PKCζ (9.6%), whereas underexpression was the main alteration observed for PKCα (27.3%), PKCε (11.6%), PKCη (8.7%) and PKN2 (8.1%). Both overexpression and underexpression were observed for PKCß (underexpression 15.5%, overexpression 13.8%), PKCθ (underexpression 14.8%, overexpression 10.0%) and PKN1 (underexpression 6.6%, overexpression 7.4%). Several links were found between different PKC genes; and also between the expression patterns of PKC genes and several classical pathological and clinical parameters. PKCι/λ alone was found to have prognostic significance (p = 0.043), whereas PKCα showed a trend towards an influence on relapse-free survival (p = 0.052). PKCι/λ retained its prognostic significance in Cox multivariate regression analysis (p = 0.031). These results reveal very complex expression patterns of PKC genes in breast tumors, and suggest that their expression should be considered together when evaluating anti-tumoral drugs. PKCι/λ seems to be the most promising therapeutic target in breast cancer.

Identification of new candidate therapeutic target genes in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a subgroup of breast cancer that is negative for estrogen and progesterone receptor and ERBB2 protein expression. It is characterized by its aggressive behavior and by the lack of targeted therapies. To identify new therapeutic targets in TNBC, we used real-time quantitative RT-PCR to analyze 63 TNBC samples in terms of their mRNA expression of 26 genes coding for the major proteins currently targeted by drugs used to treat other cancers or undergoing clinical trials in breast cancer. Six of the 26 genes tested (VEGFA, SRC, PARP1, PTK2, RAF1, and FGFR3) were significantly upregulated in 13% to 46% of the TNBCs. None of the 6 genes was specifically upregulated in the TNBCs compared with 3 other classical breast tumor subtypes. No association was observed between overexpression of these 6 genes (except for FGFR3) and PIK3CA mutation status. These results confirm the interest of targeting VEGFA and PARP1 in ongoing clinical trials in TNBC patients and also identify new target genes (SRC, PTK2, RAF1, and FGFR3). Clinical trials could be initiated easily with existing drugs. Our results also suggest that these target genes might serve as predictive biomarkers of the TNBC treatment response.

Clinical relevance of tumor cells with stem-like properties in pediatric brain tumors.

BACKGROUND: Primitive brain tumors are the leading cause of cancer-related death in children. Tumor cells with stem-like properties (TSCs), thought to account for tumorigenesis and therapeutic resistance, have been isolated from high-grade gliomas in adults. Whether TSCs are a common component of pediatric brain tumors and are of clinical relevance remains to be determined. METHODOLOGY/PRINCIPAL FINDINGS: Tumor cells with self-renewal properties were isolated with cell biology techniques from a majority of 55 pediatric brain tumors samples, regardless of their histopathologies and grades of malignancy (57% of embryonal tumors, 57% of low-grade gliomas and neuro-glial tumors, 70% of ependymomas, 91% of high-grade gliomas). Most high-grade glioma-derived oncospheres (10/12) sustained long-term self-renewal akin to neural stem cells (>7 self-renewals), whereas cells with limited renewing abilities akin to neural progenitors dominated in all other tumors. Regardless of tumor entities, the young age group was associated with self-renewal properties akin to neural stem cells (P = 0.05, chi-square test). Survival analysis of the cohort showed an association between isolation of cells with long-term self-renewal abilities and a higher patient mortality rate (P = 0.013, log-rank test). Sampling of low- and high-grade glioma cultures showed that self-renewing cells forming oncospheres shared a molecular profile comprising embryonic and neural stem cell markers. Further characterization performed on subsets of high-grade gliomas and one low-grade glioma culture showed combination of this profile with mesenchymal markers, the radio-chemoresistance of the cells and the formation of aggressive tumors after intracerebral grafting. CONCLUSIONS/SIGNIFICANCE: In brain tumors affecting adult patients, TSCs have been isolated only from high-grade gliomas. In contrast, our data show that tumor cells with stem cell-like or progenitor-like properties can be isolated from a wide range of histological sub-types and grades of pediatric brain tumors. They suggest that cellular mechanisms fueling tumor development differ between adult and pediatric brain tumors.

Gene expression profiling reveals new aspects of PIK3CA mutation in ERalpha-positive breast cancer: major implication of the Wnt signaling pathway.

BACKGROUND: The PI3K/AKT pathway plays a pivotal role in breast cancer development and maintenance. PIK3CA, encoding the PI3K catalytic subunit, is the oncogene exhibiting a high frequency of gain-of-function mutations leading to PI3K/AKT pathway activation in breast cancer. PIK3CA mutations have been observed in 30% to 40% of ERα-positive breast tumors. However the physiopathological role of PIK3CA mutations in breast tumorigenesis remains largely unclear. METHODOLOGY/PRINCIPAL FINDINGS: To identify relevant downstream target genes and signaling activated by aberrant PI3K/AKT pathway in breast tumors, we first analyzed gene expression with a pangenomic oligonucleotide microarray in a series of 43 ERα-positive tumors with and without PIK3CA mutations. Genes of interest were then investigated in 249 ERα-positive breast tumors by real-time quantitative RT-PCR. A robust collection of 19 genes was found to be differently expressed in PIK3CA-mutated tumors. PIK3CA mutations were associated with over-expression of several genes involved in the Wnt signaling pathway (WNT5A, TCF7L2, MSX2, TNFRSF11B), regulation of gene transcription (SEC14L2, MSX2, TFAP2B, NRIP3) and metal ion binding (CYP4Z1, CYP4Z2P, SLC40A1, LTF, LIMCH1). CONCLUSION/SIGNIFICANCE: This new gene set should help to understand the behavior of PIK3CA-mutated cancers and detailed knowledge of Wnt signaling activation could lead to novel therapeutic strategies.