GJB5 association with BRAF mutation and survival in cutaneous malignant melanoma.

BACKGROUND: Gap-junctional intercellular communication is crucial for epidermal cellular homeostasis. Inability to establish melanocyte-keratinocyte contact and loss of the intercellular junction's integrity may contribute to melanoma development. Connexins, laminins and desmocollins have been implicated in the control of melanoma growth, where their reduced expression has been reported in metastatic lesions. OBJECTIVES: The aim of this study was to investigate connexin 31·1 (GJB5) expression and identify any association with BRAF mutational status, prognosis of patients with melanoma and mitogen-activated protein kinase (MAPK) inhibitor (MAPKi) treatment. METHODS: GJB5 expression was measured at RNA and protein level in melanoma clinical samples and established cell lines treated (or not) with BRAF and MEK inhibitors (MEKi), as well as in cell lines which developed MAPKi resistance. Findings were further validated and confirmed by analysis of independent datasets. RESULTS: Our analysis reveals significant downregulation of GJB5 expression in metastatic melanoma lesions compared with primary ones and in BRAF-mutated vs. BRAF-wildtype (BRAFWT ) melanomas. Likewise, GJB5 expression is significantly lower in BRAFV600E compared with BRAFWT cell lines and increases on MAPKi treatment. MAPKi-resistant melanoma cells display a similar expression pattern compared with BRAFWT cells, with increased GJB5 expression associated with morphological changes. Enhancement of BRAFV600E expression in BRAFWT melanoma cells significantly upregulates miR-335-5p expression with consequent downregulation of GJB5, one of its targets. Furthermore, overexpression of miR-335-5p in two BRAFWT cell lines confirms specific GJB5 protein downregulation. Reverse transcriptase quantitative polymerase chain reaction analysis also revealed upregulation of miR-335 in BRAFV600E melanoma cells, which is significantly downregulated in cells resistant to MEKi. Our data were further validated using the TCGA_SKCM dataset, where BRAF mutations associate with increased miR-335 expression and inversely correlate with GJB5 expression. In clinical samples, GJB5 underexpression is also associated with patient overall worse survival, especially at early stages. CONCLUSIONS: We identified a significant association between metastases/BRAF mutation and low GJB5 expression in melanoma. Our results identify a novel mechanism of gap-junctional protein regulation, suggesting a prognostic role for GJB5 in cutaneous melanoma.

Transcriptomic analysis and mutational status of IDH1 in paired primary-recurrent intrahepatic cholangiocarcinoma.

BACKGROUND: Effective target therapies for intrahepatic cholangiocarcinoma (ICC) have not been identified so far. One of the reasons may be the genetic evolution from primary (PR) to recurrent (REC) tumors. We aim to identify peculiar characteristics and to select potential targets specific for recurrent tumors. Eighteen ICC paired PR and REC tumors were collected from 5 Italian Centers. Eleven pairs were analyzed for gene expression profiling and 16 for mutational status of IDH1. For one pair, deep mutational analysis by Next Generation Sequencing was also carried out. An independent cohort of patients was used for validation. RESULTS: Two class-paired comparison yielded 315 differentially expressed genes between REC and PR tumors. Up-regulated genes in RECs are involved in RNA/DNA processing, cell cycle, epithelial to mesenchymal transition (EMT), resistance to apoptosis, and cytoskeleton remodeling. Down-regulated genes participate to epithelial cell differentiation, proteolysis, apoptotic, immune response, and inflammatory processes. A 24 gene signature is able to discriminate RECs from PRs in an independent cohort; FANCG is statistically associated with survival in the chol-TCGA dataset. IDH1 was mutated in the RECs of five patients; 4 of them displayed the mutation only in RECs. Deep sequencing performed in one patient confirmed the IDH1 mutation in REC. CONCLUSIONS: RECs are enriched for genes involved in EMT, resistance to apoptosis, and cytoskeleton remodeling. Key players of these pathways might be considered druggable targets in RECs. IDH1 is mutated in 30% of RECs, becoming both a marker of progression and a target for therapy.

Characterization of a new trabectedin-resistant myxoid liposarcoma cell line that shows collateral sensitivity to methylating agents.

Myxoid Liposarcomas (MLS), characterized by the expression of FUS-CHOP fusion gene are clinically very sensitive to the DNA binding antitumor agent, trabectedin. However, resistance eventually occurs, preventing disease eradication. To investigate the mechanisms of resistance, a trabectedin resistant cell line, 402-91/ET, was developed. The resistance to trabectedin was not related to the expression of MDR related proteins, uptake/efflux of trabectedin or GSH levels that were similar in parental and resistant cells. The 402-91/ET cells were hypersensitive to UV light because of a nucleotide excision repair defect: XPG complementation decreased sensitivity to UV rays, but only partially to trabectedin. 402-91/ET cells showed collateral sensitivity to temozolomide due to the lack of O(6) -methylguanine-DNA-methyltransferase (MGMT) activity, related to the hypermethylation of MGMT promoter. In 402-91 cells chromatin immunoprecipitation (ChIP) assays showed that FUS-CHOP was bound to the PTX3 and FN1 gene promoters, as previously described, and trabectedin caused FUS-CHOP detachment from DNA. Here we report that, in contrast, in 402-91/ET cells, FUS-CHOP was not bound to these promoters. Differences in the modulation of transcription of genes involved in different pathways including signal transduction, apoptosis and stress response between the two cell lines were found. Trabectedin activates the transcription of genes involved in the adipogenic-program such as c/EBPα and ß, in 402-91 but not in 402-91/ET cell lines. The collateral sensitivity of 402-91/ET to temozolomide provides the rationale to investigate the potential use of methylating agents in MLS patients resistant to trabectedin.

From single gene to integrative molecular concept MAPS: pitfalls and potentials of microarray technology.

Microarray experiments have a large variety of applications and several important achievements have been obtained by means of this technology, especially within the field of whole genome expression profiling, which undoubtedly is the most diffused world-wide. Nevertheless, care must be taken in unconditionally applying such high-throughput techniques and in extracting/interpreting their results. Both the validity and the reproducibility of microarray-based clinical research have recently been challenged. Pitfalls and potentials of the microarray technology for gene expression profiling are critically reviewed in this paper.

Transcriptional Remodeling in Primary Hippocampal Astrocytes from an Alzheimer's Disease Mouse Model.

BACKGROUND: It is well known that alterations in astrocytes occur in Alzheimer's disease and reactive astrogliosis is one of the hallmarks of the disease. Recently, data has emerged that suggests that alterations in astrocytes may also occur early in the pathogenesis of the disease. OBJECTIVE: The aim of present work was to characterize the transcriptional alterations occurring in cultured astrocytes from 3xTg-AD mouse pups compared to control non-transgenic mice. Furthermore, we also compared these changes to those reported by others in astrocytes from symptomatic AD mice. METHOD: We conducted a whole-genome microarray study on primary cultured astrocytes from the hippocampus of 3xTg-AD and non-transgenic mouse newborn pups. We used cross-platform normalization and an unsupervised hierarchical clustering algorithm to compare our results with other datasets of cultured or freshly isolated astrocytes, including those isolated from plaque-stage APPswe/PS1dE9 AD mice. RESULTS: We found a set of 993 genes differentially expressed in 3xTg-AD as compared with non-Tg astrocytes. Over-represented gene ontology terms were related to calcium, cell-cell communication, mitochondria, transcription, nucleotide binding and phosphorylation. Of note, no genes related to inflammation were found in cultured 3xTg-AD astrocytes. Comparison with astrocytes isolated from plaque stage APPswe/PS1dE9 showed that 882 out of 993 genes were selectively changed in primary 3xTg-AD astrocytes while 50 genes were co-regulated and 61 were anti-regulated (regulated in the opposite direction in the datasets). CONCLUSION: Our data show that in cultured astrocytes from an AD mouse model, transcriptional changes occur and are different from those reported in models mimicking later stages of the disease.

GPR55 signalling promotes proliferation of pancreatic cancer cells and tumour growth in mice, and its inhibition increases effects of gemcitabine.

The life expectancy for pancreatic cancer patients has seen no substantial changes in the last 40 years as very few and mostly just palliative treatments are available. As the five years survival rate remains around 5%, the identification of novel pharmacological targets and development of new therapeutic strategies are urgently needed. Here we demonstrate that inhibition of the G protein-coupled receptor GPR55, using genetic and pharmacological approaches, reduces pancreatic cancer cell growth in vitro and in vivo and we propose that this may represent a novel strategy to inhibit pancreatic ductal adenocarcinoma (PDAC) progression. Specifically, we show that genetic ablation of Gpr55 in the KRASWT/G12D/TP53WT/R172H/Pdx1-Cre+/+ (KPC) mouse model of PDAC significantly prolonged survival. Importantly, KPC mice treated with a combination of the GPR55 antagonist Cannabidiol (CBD) and gemcitabine (GEM, one of the most used drugs to treat PDAC), survived nearly three times longer compared to mice treated with vehicle or GEM alone. Mechanistically, knockdown or pharmacologic inhibition of GPR55 reduced anchorage-dependent and independent growth, cell cycle progression, activation of mitogen-activated protein kinase (MAPK) signalling and protein levels of ribonucleotide reductases in PDAC cells. Consistent with this, genetic ablation of Gpr55 reduced proliferation of tumour cells, MAPK signalling and ribonucleotide reductase M1 levels in KPC mice. Combination of CBD and GEM inhibited tumour cell proliferation in KPC mice and it opposed mechanisms involved in development of resistance to GEM in vitro and in vivo. Finally, we demonstrate that the tumour suppressor p53 regulates GPR55 protein expression through modulation of the microRNA miR34b-3p. Our results demonstrate the important role played by GPR55 downstream of p53 in PDAC progression. Moreover our data indicate that combination of CBD and GEM, both currently approved for medical use, might be tested in clinical trials as a novel promising treatment to improve PDAC patients' outcome.

Molecular characterisation of two human cancer cell lines selected in vitro for their chemotherapeutic drug resistance to ET-743.

ET-743 (Yondelis(TM), Trabectedin) isolated from the tunicate Ecteinascidia turbinata, is being tested in phase II clinical trials in Europe and the United States of America (USA). Studies with different solid tumours have shown antitumour activity in advanced, pre-treated sarcomas as well as in drug-resistant breast and ovarian cancer. The primary mechanism of action for ET-743 has not been fully elucidated and different models have been suggested to explain its molecular mechanism of action. ET-743 binds tightly to the minor groove of DNA and previous data have suggested that ET-743 acts by interfering with RNA transcription. To further investigate the mechanism of in vitro drug resistance, we evaluated the gene expression profile in ovarian and chondrosarcoma cell lines selected for resistance to ET-743. We found 70 genes whose expression was modulated in both drug-resistant cell lines when compared with their respective parental drug-sensitive cell lines. This pattern of gene expression seems to be selective for ET-743-resistant cells, since ovarian cancer cells resistant to paclitaxel did not share the same gene expression changes. Data presented in this study reveal different molecular pathways that could be involved in the cellular mechanism of ET-743 resistance.

Interpretation of expression-profiling results obtained from different platforms and tissue sources: examples using prostate cancer data.

The analysis of expression signatures is a powerful tool for the classification of cancer and other tissue samples. Several protocols and platforms are available on the market, and these lead to both confirmatory and complementary results. We review the main processing techniques for cross-platform comparisons and the different tissue sources for cancer profiling. Some examples and the cross-interpretation of bibliographic data related to prostate cancer are also presented.

Changes in gene expression profile induced by the anticancer agent Aplidine in Molt-4 leukemic cell lines.

Microarray technique was employed to study differences in gene expression profile induced by Aplidine treatment in the Molt-4 human leukemic T cell line. Aplidine is a novel marine compound purified from caribbean tunicate (sea squirt) Aplidium Albicans. Despite promising anti-tumor activity, few data are available on its mechanism of action. Exponentially growing cells were treated with Aplidine concentrations close to its 5IC50 for 1 hour and RNA samples collected after 0.5, 1, 6 and 24 hours of recovery in drug free medium. 32P labelled cDNAs were hybridized against Atlas Human Cancer arrays onto which 588 cDNAs were spotted. Genes involved in different cellular pathways, (such as growth factors, signal transduction or transcription factors) were found modulated by the drug. Even if the data obtained in the present study cannot be conclusive, several hypothesis on Aplidine's mechanism of action are indicated that will be the subject of future studies.

Variability in the timing of G(1)/S transition.

Cell cycle duration and phase transition times are not fixed, even within homogeneous cell populations growing under optimal environmental conditions. We investigate G(1) phase variability from the molecular point of view and propose a mathematical approach to model the protein interactions regulating the transition from the G(1) phase to the phase of DNA synthesis. The mathematical model has some connections with flow cytometry experimental data.

Behavioral choices based on patch selection: a model using aggregation methods.

The aim of this work is to study the influence of patch selection on the dynamics of a system describing the interactions between two populations, generically called 'population N' and 'population P'. Our model may be applied to prey-predator systems as well as to certain host-parasite or parasitoid systems. A situation in which population P affects the spatial distribution of population N is considered. We deal with a heterogeneous environment composed of two spatial patches: population P lives only in patch 1, while individuals belonging to population N migrate between patch 1 and patch 2, which may be a refuge. Therefore they are divided into two patch sub-populations and can migrate according to different migration laws. We make the assumption that the patch change is fast, whereas the growth and interaction processes are slower. We take advantage of the two time scales to perform aggregation methods in order to obtain a global model describing the time evolution of the total populations, at a slow time scale. At first, a migration law which is independent on population P density is considered. In this case the global model is equivalent to the local one, and under certain conditions, population P always gets extinct. Then, the same model, but in which individuals belonging to population N leave patch 1 proportionally to population P density, is studied. This particular behavioral choice leads to a dynamically richer global system, which favors stability and population coexistence. Finally, we study a third example corresponding to the addition of an aggregative behavior of population N on patch 1. This leads to a more complicated situation in which, according to initial conditions, the global system is described by two different aggregated models. Under certain conditions on parameters a stable limit cycle occurs, leading to periodic variations of the total population densities, as well as of the local densities on the spatial patches.

Aberrant expression of the neuronal-specific protein DCDC2 promotes malignant phenotypes and is associated with prostate cancer progression.

By integrating gene profiling and immunohistochemical data with functional experiments in cell lines in this study we show for the first time that doublecortin (DCX) domain containing 2 (DCDC2), a protein belonging to the DCX family and involved in neuronal cell migration, is aberrantly expressed in prostate tumors whereas absent in normal prostate. Furthermore, in patients treated with radical prostatectomy, high levels of DCDC2 RNA were significantly associated with increased biochemical relapse (LogRank Mantel-Cox=0.012). Mechanistically, we found that the ETS transcription factor ESE3/EHF, which is expressed in normal prostate and frequently lost in prostate tumors, maintained DCDC2 repressed by binding to a novel identified ETS binding site in the gene promoter. Consistently, in prostate tumors and in cellular models of gain and loss of ESE3/EHF, the expression of DCDC2 and ESE3/EHF were inversely correlated. In prostate cancer cells, DCDC2 colocalized with microtubules and promoted cell migration and resistance to the microtubule-targeting drug taxol. Collectively, this study establishes DCDC2 as a novel ESE3/EHF oncogenic target in prostate cancer. These findings may be relevant for the clinical management of prostate cancer as DCDC2 may signal tumors more prone to relapse and resistant to taxol treatment.

Targeting of the adaptor protein Tab2 as a novel approach to revert tamoxifen resistance in breast cancer cells.

Pharmacological resistance is a serious threat to the clinical success of hormone therapy for breast cancer. The antiproliferative response to antagonistic drugs such as tamoxifen (Tam) critically depends on the recruitment of NCoR/SMRT corepressors to estrogen receptor alpha (ERα) bound to estrogen target genes. Under certain circumstances, as demonstrated in the case of interleukin-1ß (IL-1ß) treatment, the protein Tab2 interacts with ERα/NCoR and causes dismissal of NCoR from these genes, leading to loss of the antiproliferative response. In Tam-resistant (TamR) ER-positive breast cancer cells, we observed that Tab2 presents a shift in mobility on sodium dodecyl sulfate--PAGE (SDS-PAGE) similar to that seen in MCF7 wt upon stimulation with IL-1ß, suggesting constitutive activation. Accordingly, TamR treatment with Tab2-specific short interfering RNA, restored the antiproliferative response to Tam in these cells. As Tab2 is known to directly interact with the N-terminal domain of ERα, we synthesized a peptide composed of a 14-aa motif of this domain, which effectively competes with ERα/Tab2 interaction in pull-down and co-immunoprecipitation experiments, fused to the carrier TAT peptide to allow internalization. Treatment of TamR cells with this peptide resulted in partial recovery of the antiproliferative response to Tam, suggesting a strategy to revert pharmacological resistance in breast cancer. Silencing of Tab2 in TamR cells by siRNA caused modulation of a gene set related to the control of cell cycle and extensively connected to BRCA1 in a functional network. These genes were able to discern two groups of patients, from a published data set of Tam-treated breast cancer profiles, with significantly different disease-free survival. Altogether, our data implicate Tab2 as a mediator of resistance to endocrine therapy and as a potential new target to reverse pharmacological resistance and potentiate antiestrogen action.

The SRA protein UHRF1 promotes epigenetic crosstalks and is involved in prostate cancer progression.

Epigenetic silencing of tumour suppressor genes is an important mechanism involved in cell transformation and tumour progression. The Set and RING-finger-associated domain-containing protein UHRF1 might be an important link between different epigenetic pathways. Here, we report that UHRF1 is frequently overexpressed in human prostate tumours and has an important role in prostate cancer pathogenesis and progression. Analysis of human prostate cancer samples by microarrays and immunohistochemistry showed increased expression of UHRF1 in about half of the cases. Moreover, UHRF1 expression was associated with reduced overall survival after prostatectomy in patients with organ-confined prostate tumours (P < 0.0001). UHRF1 expression was negatively correlated with several tumour suppressor genes and positively with the histone methyltransferase (HMT) EZH2 both in prostate tumours and cell lines. UHRF1 knockdown reduced proliferation, clonogenic capability and anchorage-independent growth of prostate cancer cells. Depletion of UHRF1 resulted in reactivation of several tumour suppressor genes. Gene reactivation upon UHRF1 depletion was associated with changes in histone H3K9 methylation, acetylation and DNA methylation, and impaired binding of the H3K9 HMT Suv39H1 to the promoter of silenced genes. Co-immunoprecipitation experiments showed direct interaction between UHRF1 and Suv39H1. Our data support the notion that UHRF1, along with Suv39H1 and DNA methyltransferases, contributes to epigenetic gene silencing in prostate tumours. This could represent a parallel and convergent pathway to the H3K27 methylation catalyzed by EZH2 to synergistically promote inactivation of tumour suppressor genes. Deregulated expression of UHRF1 is involved in the prostate cancer pathogenesis and might represent a useful marker to distinguish indolent cancer from those at high risk of lethal progression.

Reduced expression and tumor suppressor function of the ETS transcription factor ESE-3 in prostate cancer.

Deregulated expression of ETS transcription factors has emerged as an important event in prostate cancer pathogenesis. Here we show that the expression of epithelial-specific ETS (ESE)-3 factor is frequently reduced at the RNA and protein level in prostate cancer clinical samples compared to normal prostate. In PC3 and DU145 cells, ESE-3 was silenced by methylation of an evolutionarily conserved CpG site in its promoter and treatment with 5-aza-2'-deoxycytidine restored its expression. In a prostate epithelial cell transformation model, methylation of this site was inversely correlated with ESE-3 expression and occurred only in Ras-transformed and tumorigenic cells and not in normal and immortalized cells suggesting that ESE-3 silencing was functionally linked to oncogenic transformation. Consistent with a tumor suppressor function, re-expression of ESE-3 in prostate cancer cells inhibited clonogenic survival and induced apoptotic cell death. ESE-3 increased the level of procaspase-3, a key element in the apoptotic cascade. This effect was mediated at the transcriptional level by direct binding of ESE-3 to the caspase-3 promoter. Collectively, our findings implicate ESE-3 as a candidate tumor suppressor in prostate cancer. Decreased expression of ESE-3 may result in loss of important regulatory mechanisms in prostate epithelial cells and contribute to the pathogenesis of prostate cancer.

Desynchronization rate in cell populations: mathematical modeling and experimental data.

We characterize the kinetics of two cancer cell lines: IGROV1 (ovarian carcinoma) and MOLT4 (leukemia). By means of flow cytometry, we selected two populations from exponentially growing in vitro cell lines, depending on the cells' DNA synthesis activity during a preceding labeling period. For these populations we determined the time course of the percentages of cells in different phases of the cycles, sampling every 3 hr for 60 hr. Initially, semi-synchronous populations quickly converged to a stable age distribution, which is typical of the cell line (at equilibrium); this desynchronization reflects the intercell variability in cell cycle duration. By matching these experimental observations to mathematical modelling, we related the convergence rate toward the asymptotic distribution (R) and the period of the phase-percentage oscillations (T), to the mean cell cycle duration and its coefficient of variation. We give two formulas involving the above-mentioned parameters. Since T and R can be drawn by fitting our data to an asymptotic formula obtained from the model, we can estimate the other two kinetic parameters. IGROV1 cells have a shorter mean cell cycle time, but higher intercell variability than the leukemia line, which takes longer to lose synchrony.

Cell cycle phase perturbations and apoptosis in tumour cells induced by aplidine.

Aplidine, dehydrodidemnin B, is a marine depsipeptide isolated from the Mediterranean tunicate Aplidium albicans currently in phase II clinical trial. In human Molt-4 leukaemia cells Aplidine was found to be cytotoxic at nanomolar concentrations and to induce both a G(1) arrest and a G(2) blockade. The drug-induced cell cycle perturbations and subsequent cell death do not appear to be related to macromolecular synthesis (protein, RNA, DNA) since the effects occur at concentrations (e.g. 10 nM) in which macromolecule synthesis was not markedly affected. Ten nM Aplidine for 1 h inhibited ornithine decarboxylase activity, with a subsequently strong decrease in putrescine levels. This finding has questionable relevance since addition of putrescine did not significantly reduce the cell cycle perturbations or the cytotoxicity of Aplidine. The cell cycle perturbations caused by Aplidine were also not due to an effect on the cyclin-dependent kinases. Although the mechanism of action of Aplidine is still unclear, the cell cycle phase perturbations and the rapid induction of apoptosis in Molt-4 cells appear to be due to a mechanism different from that of known anticancer drugs.