A Proteomics Approach Identifies RREB1 as a Crucial Molecular Target of Imidazo-Pyrazole Treatment in SKMEL-28 Melanoma Cells.

Cutaneous melanoma is the most dangerous and deadly form of human skin malignancy. Despite its rarity, it accounts for a staggering 80% of deaths attributed to cutaneous cancers overall. Moreover, its final stages often exhibit resistance to drug treatments, resulting in unfavorable outcomes. Hence, ensuring access to novel and improved chemotherapeutic agents is imperative for patients grappling with this severe ailment. Pyrazole and its fused systems derived thereof are heteroaromatic moieties widely employed in medicinal chemistry to develop effective drugs for various therapeutic areas, including inflammation, pain, oxidation, pathogens, depression, and fever. In a previous study, we described the biochemical properties of a newly synthesized group of imidazo-pyrazole compounds. In this paper, to improve our knowledge of the pharmacological properties of these molecules, we conduct a differential proteomic analysis on a human melanoma cell line treated with one of these imidazo-pyrazole derivatives. Our results detail the changes to the SKMEL-28 cell line proteome induced by 24, 48, and 72 h of 3e imidazo-pyrazole treatment. Notably, we highlight the down-regulation of the Ras-responsive element binding protein 1 (RREB1), a member of the zinc finger transcription factors family involved in the tumorigenesis of melanoma. RREB1 is a downstream element of the MAPK pathway, and its activation is mediated by ERK1/2 through phosphorylation.

Identification of Molecular Markers Associated with Prostate Cancer Subtypes: An Integrative Bioinformatics Approach.

Prostate cancer (PCa) is characterised by androgen dependency. Unfortunately, under anti-androgen treatment pressure, castration-resistant prostate cancer (CRPC) emerges, characterised by heterogeneous cell populations that, over time, lead to the development of different androgen-dependent or -independent phenotypes. Despite important advances in therapeutic strategies, CRPC remains incurable. Context-specific essential genes represent valuable candidates for targeted anti-cancer therapies. Through the investigation of gene and protein annotations and the integration of published transcriptomic data, we identified two consensus lists to stratify PCa patients' risk and discriminate CRPC phenotypes based on androgen receptor activity. ROC and Kaplan-Meier survival analyses were used for gene set validation in independent datasets. We further evaluated these genes for their association with cancer dependency. The deregulated expression of the PCa-related genes was associated with overall and disease-specific survival, metastasis and/or high recurrence risk, while the CRPC-related genes clearly discriminated between adeno and neuroendocrine phenotypes. Some of the genes showed context-specific essentiality. We further identified candidate drugs through a computational repositioning approach for targeting these genes and treating lethal variants of PCa. This work provides a proof-of-concept for the use of an integrative approach to identify candidate biomarkers involved in PCa progression and CRPC pathogenesis within the goal of precision medicine.

Untargeted lipidomics reveal association of elevated plasma C18 ceramide levels with reduced survival in metastatic castration-resistant prostate cancer patients.

Emerging evidence highlights the potential prognostic relevance of circulating lipids in metastatic castration-resistant prostate cancer (mCRPC), with a proposed 3-lipid signature. This study aims to analyze the lipidomic profiles of individuals with mCRPC to identify lipid species that could serve as predictive indicators of prognosis and therapeutic response. Plasma samples were collected from mCRPC patients initiating first-line treatment (1 L) (n = 29) and those previously treated with at least two lines of therapy (> 2 L) (n = 19), including an androgen-receptor signaling inhibitor and a taxane. Employing an untargeted lipidomic approach, lipids were extracted from the plasma samples and subjected to analysis. A comprehensive identification and quantification of 789 plasma lipids was achieved. Notably, 75 species displayed significant dysregulation in > 2 L patients in comparison to the 1 L group. Among these, 63 species exhibited elevated levels, while 12 were reduced. Patients included in > 2 L cohort showed elevated levels of acylcarnitines (CAR), diacylglycerols (DG), phosphatidylethanolamines (PE), triacylglycerols (TG), and ceramides (Cer). Notably, some upregulated lipids, including CAR 14:0, CAR 24:1, Cer d18:1/16:0, Cer d18:1/18:0 (C18 Cer), Cer d18:2/18:0, Cer d18:1/24:1, and Cer d20:1/24:1, showed significant associations with overall survival (OS) in univariate models. Specifically, increased levels of C18 Cer remained significantly associated with poorer OS in the multivariate model, even after adjusting for treatment line and PSA levels (Hazard Ratio: 3.59 [95% Confidence Interval 1.51-8.52], p = 0.004). Employing quantitative mass spectrometry, our findings underscore the independent prognostic significance of C18 Cer in individuals with mCRPC. This discovery opens avenues for further studies within this field.

Gene's expression underpinning the divergent predictive value of [18F]F-fluorodeoxyglucose and prostate-specific membrane antigen positron emission tomography in primary prostate cancer: a bioinformatic and experimental study.

BACKGROUND: Positron Emission Tomography (PET) imaging with Prostate-Specific Membrane Antigen (PSMA) and Fluorodeoxyglucose (FDG) represent promising biomarkers for risk-stratification of Prostate Cancer (PCa). We verified whether the expression of genes encoding for PSMA and enzymes regulating FDG cellular uptake are independent and additive prognosticators in PCa. METHODS: mRNA expression of genes involved in glucose metabolism and PSMA regulation obtained from primary PCa specimens were retrieved from open-source databases and analyzed using an integrative bioinformatics approach. Machine Learning (ML) techniques were used to create predictive Progression-Free Survival (PFS) models. Cellular models of primary PCa with different aggressiveness were used to compare [18F]F-PSMA-1007 and [18F]F-FDG uptake kinetics in vitro. Confocal microscopy, immunofluorescence staining, and quantification analyses were performed to assess the intracellular and cellular membrane PSMA expression. RESULTS: ML analyses identified a predictive functional network involving four glucose metabolism-related genes: ALDOB, CTH, PARP2, and SLC2A4. By contrast, FOLH1 expression (encoding for PSMA) did not provide any additive predictive value to the model. At a cellular level, the increase in proliferation rate and migratory potential by primary PCa cells was associated with enhanced FDG uptake and decreased PSMA retention (paralleled by the preferential intracellular localization). CONCLUSIONS: The overexpression of a functional network involving four glucose metabolism-related genes identifies a higher risk of disease progression since the earliest phases of PCa, in agreement with the acknowledged prognostic value of FDG PET imaging. By contrast, the prognostic value of PSMA PET imaging is independent of the expression of its encoding gene FOLH1. Instead, it is influenced by the protein docking to the cell membrane, regulating its accessibility to tracer binding.

Fraisinib: a calixpyrrole derivative reducing A549 cell-derived NSCLC tumor in vivo acts as a ligand of the glycine-tRNA synthase, a new molecular target in oncology.

Background and purpose: Lung cancer is the leading cause of death in both men and women, constituting a major public health problem worldwide. Non-small-cell lung cancer accounts for 85%-90% of all lung cancers. We propose a compound that successfully fights tumor growth in vivo by targeting the enzyme GARS1. Experimental approach: We present an in-depth investigation of the mechanism through which Fraisinib [meso-(p-acetamidophenyl)-calix(4)pyrrole] affects the human lung adenocarcinoma A549 cell line. In a xenografted model of non-small-cell lung cancer, Fraisinib was found to reduce tumor mass volume without affecting the vital parameters or body weight of mice. Through a computational approach, we uncovered that glycyl-tRNA synthetase is its molecular target. Differential proteomics analysis further confirmed that pathways regulated by Fraisinib are consistent with glycyl-tRNA synthetase inhibition. Key results: Fraisinib displays a strong anti-tumoral potential coupled with limited toxicity in mice. Glycyl-tRNA synthetase has been identified and validated as a protein target of this compound. By inhibiting GARS1, Fraisinib modulates different key biological processes involved in tumoral growth, aggressiveness, and invasiveness. Conclusion and implications: The overall results indicate that Fraisinib is a powerful inhibitor of non-small-cell lung cancer growth by exerting its action on the enzyme GARS1 while displaying marginal toxicity in animal models. Together with the proven ability of this compound to cross the blood-brain barrier, we can assess that Fraisinib can kill two birds with one stone: targeting the primary tumor and its metastases "in one shot." Taken together, we suggest that inhibiting GARS1 expression and/or GARS1 enzymatic activity may be innovative molecular targets for cancer treatment.

Two calix[4]pyrroles as potential therapeutics for castration-resistant prostate cancer.

Macrocyclic compounds meso-(p-acetamidophenyl)-calix[4]pyrrole and meso-(m-acetamidophenyl)-calix[4]pyrrole have previously been reported to exhibit cytotoxic properties towards lung cancer cells. Here, we report pre-clinical in vitro and in vivo studies showing that these calixpyrrole derivatives can inhibit cell growth in both PC3 and DU145 prostatic cancer cell lines. We explored the impact of these compounds on programmed cell death, as well as their ability to inhibit cellular invasion. In this study we have demonstrated the safety of these macrocyclic compounds by cytotoxicity tests on ex-vivo human peripheral blood mononuclear cells (PBMCs), and by in vivo subcutaneous administration. Preliminary in vivo tests demonstrated no hepato-, no nephro- and no genotoxicity in Balb/c mice compared to controls treated with cisplatin. These findings suggest these calixpyrroles might be novel therapeutic tools for the treatment of prostate cancer and of particular interest for the treatment of androgen-independent castration-resistant prostate cancer.

The prognostic value of aspartate beta-hydroxylase in early breast cancer.

PURPOSE: Aspartate beta-hydroxylase (ASPH) is a transmembrane protein involved in cancer progression, which has been shown to imply a worse prognosis in several solid tumors. The aim of the present study was to further investigate the prognostic value of ASPH in early breast cancer. METHODS: ASPH expression was investigated through immunohistochemistry in a cohort of 153 breast cancer patients with long-term follow-up, and correlated with clinical-pathological features plus all-cause and breast-cancer-specific mortality. Appropriate statistics were utilized. RESULTS: ASPH negatively correlated with all-cause and breast-cancer-specific mortality. CONCLUSIONS: The results of this cohort study support the prognostic value of ASPH in early breast cancer.

Etoposide and topoisomerase II inhibition for aggressive prostate cancer: Data from a translational study.

BACKGROUND: Etoposide phosphate (VP-16) is a topoisomerase 2 (TOP2) inhibitor that demonstrated activity in patients with metastatic castration-resistant prostate cancer (mCRPC). We investigated the sensitivity of prostate cancer (PCa) cells (LNCaP, 22Rv1, PC3, DU145, PDB and MDB) to VP-16 and the possible relationship between VP-16 activity and TOP2 expression. The activity of VP-16 was compared with that of docetaxel, enzalutamide and olaparib. The prevalence and clinical significance of TOP2 genetic and transcriptomic alterations was also explored in mCRPC. METHODS: Cell cultures and crystal violet cell proliferation assays were performed. Specific antibodies were used in western blots analyses of cell protein extracts. Datasets were analyzed in cBioportal. RESULTS: VP-16 was active in all PCa cell lines analyzed and demonstrated increased activity in PC3 and DU145 cells. VP-16 was more cytotoxic compared to the other treatments, except for LNCaP and 22Rv1, which were more sensitive to docetaxel. Maintenance of antiandrogen treatment in MDB and PDB increased sensitivity to VP-16, docetaxel and enzalutamide. TOP2A was found overexpressed in 22Rv1, DU145 and PC3, whereas TOP2B was overexpressed in 22Rv1 and PDB. In the mCRPC datasets analysis, TOP2A mRNA overexpression was associated with worse patients' prognosis, with the molecular features of neuroendocrine prostate cancer (NEPC) and with lower androgen receptor (AR) score. Patients overexpressing TOP2A mRNA were more likely to harbor RB1 loss. CONCLUSIONS: Specific subpopulations of patients with aggressive variant prostate cancer (AVPC) could benefit from VP-16 treatment. TOP2A overexpression, rather than TOP2B, might be a good biomarker to predict response to VP-16.

Epidemiological Characteristics and Survival in Patients with De Novo Metastatic Prostate Cancer.

The real-world outcomes of patients with metastatic prostate cancer (mPCa) are largely unexplored. We investigated the trends in overall survival (OS) and cancer-specific survival (CSS) in patients with de novo mPCa according to distinct time periods. The U.S. Surveillance, Epidemiology, and End Results (SEER) Research Data (2000-2017) were analyzed using the SEER*Stat software. The Kaplan-Meier method and Cox regression were used. Patients with de novo mPCa were allocated to three cohorts based on the year of diagnosis: A (2000-2003), B (2004-2010), and C (2011-2014). The maximum follow-up was fixed to 5 years. Overall, 26,434 patients were included. Age, race, and metastatic stage (M1) significantly affected OS and CSS. After adjustment for age and race, patients in Cohort C showed a 9% reduced risk of death (hazard ratio (HR): 0.91 (95% confidence interval [CI] 0.87-0.95), p < 0.001) and an 8% reduced risk of cancer-specific death (HR: 0.92 (95% CI 0.88-0.96), p < 0.001) compared with those in Cohort A. After adjustment for age, race, and metastatic stage, patients in Cohort C showed an improvement in OS and CSS compared with Cohort B (HR: 0.94 (95% CI 0.91-0.97), p = 0.001; HR: 0.89 (95% CI 0.85-0.92), p < 0.001). Patients with M1c disease had a more pronounced improvement in OS and CSS compared with the other stages. No differences were found between Cohorts B and C. In conclusion, the real-world survival of de novo mPCa remains poor, with a median OS and CSS improvement of only 4 months in the latest years.

Aspartate ß-hydroxylase targeting in castration-resistant prostate cancer modulates the NOTCH/HIF1α/GSK3ß crosstalk.

Castration-resistant prostate cancer (CRPC) is an incurable stage of the disease. A multivariate principal component analysis on CRPC in vitro models identified aspartyl (asparaginyl) ß hydrolase (ASPH) as the most relevant molecule associated with the CRPC phenotype. ASPH is overexpressed in various malignant neoplasms and catalyzes the hydroxylation of aspartyl and asparaginyl residues in the epidermal growth factor (EGF)-like domains of proteins like NOTCH receptors and ligands, enhancing cell motility, invasion and metastatic spread. Bioinformatics analyses of ASPH in prostate cancer (PCa) and CRPC datasets indicate that ASPH gene alterations have prognostic value both in PCa and CRPC patients. In CRPC cells, inhibition of ASPH expression obtained through specific small interfering RNA or culturing cells in hypoxic conditions, reduced cell proliferation, invasion and cyclin D1 expression through modulation of the NOTCH signaling. ASPH and HIF1α crosstalk, within a hydroxylation-regulated signaling pathway, might be transiently driven by the oxidative stress evidenced inside CRPC cells. In addition, increased phosphorylation of GSK3ß by ASPH silencing demonstrates that ASPH regulates GSK3ß activity inhibiting its interactions with upstream kinases. These findings demonstrate the critical involvement of ASPH in CRPC development and may represent an attractive molecular target for therapy.

Erratum: Benelli, R., et al. Aspartate-ß-Hydroxylase: A Promising Target to Limit the Local Invasiveness of Colorectal Cancer. Cancers 2020, 12, 971.

The authors wish to make the following corrections to this paper [...].

Integrative Analysis of Periostin in Primary and Advanced Prostate Cancer.

Periostin (POSTN) is an extracellular matrix protein associated with tumor progression and shorter survival in prostate cancer (PCa). Here, we performed an integrative analysis of POSTN's role in patients with PCa. Clinical and POSTN data from large-scale datasets were analyzed. POSTN cutoffs were identified with X-Tile, and STRING was used for protein-protein interaction analysis. In a cohort of 48 patients with metastatic castration-resistant prostate cancer (mCRPC), we used the AdnaTest platform to isolate circulating tumor cells and extract POSTN mRNA. Plasma samples were also tested for POSTN protein expression by dot blot assay. Data from large-scale datasets did not reveal any association between POSTN genetic alterations and outcome. In primary tumors, we found a significant correlation between POSTN mRNA overexpression, worse baseline prognostic features, and shorter disease-free survival. POSTN was overexpressed in mCRPC and correlated with aggressive features. In our cohort of mCRPC patients, we found a positive correlation between POSTN plasma levels and androgen-receptor variant 7 positivity and an association with shorter overall survival. Our integrative analysis shows that POSTN is associated with poor clinical features and worse outcome in patients with PCa. Further studies are warranted to uncover the function of POSTN in PCa progression and to validate the prognostic significance of POSTN in mCRPC.

Aspartate-ß-Hydroxylase: A Promising Target to Limit the Local Invasiveness of Colorectal Cancer.

Colorectal cancer's (CRC) ability to invade local tissues and lymph nodes and generate distant metastases is the key for TNM classification. Aspartate-ß-hydroxylase (ASPH), a transmembrane protein that catalyzes Notch receptors and ligand activation, is involved in tumor invasion. Because Notch is involved in gut homeostasis, it could be a target for CRC therapy. ASPH mRNA and protein expression, promoter methylation and gene copy numbers were evaluated using the TCGA and CPTAC human CRC datasets. Using digital pathology, ASPH was scored in the luminal area (LM), center tumor (CT) and invasive margin (IM) of 100 human CRCs. The effect of ASPH targeting on invasiveness and viability was tested by siRNA knockdown and small molecule inhibitors (SMI). Bioinformatics analysis showed increased expression of ASPH mRNA and protein in CRC, paired with a decreased methylation profile. ASPH genetic gain or amplification was frequent (56%), while deletion was rare (0.03%). Digital pathology analysis showed that ASPH exerted its pathological activity in the invasive margin of the tumor, affecting invasive front morphology, tumor budding and patients' overall survival. In vitro, ASPH targeting by siRNA or SMI reduced cell invasion and growth and caused Notch-1 downregulation. This study demonstrates that ASPH targeting by specific inhibitors could improve CRC treatment strategies.

Multifocal Signal Modulation Therapy by Celecoxib: A Strategy for Managing Castration-Resistant Prostate Cancer.

BACKGROUND: Prostate cancer (PCa) is a significant health concern throughout the world. Standard therapy for advanced disease consists of anti-androgens, however, almost all prostate tumors become castration resistant (CRPC). Progression from androgen-sensitive PCa to CRPC is promoted by inflammatory signaling through cyclooxygenase-2 (COX-2) expression and ErbB family receptors/AKT activation, compensating androgen receptor inactivity. METHODS: Making use of CRPC cell lines, we investigated the effects of the anti-inflammatory drug celecoxib. Biochemical data obtained using immunoblotting, enzyme-linked immunosorbent assay (ELISA), invasion, and xenografts were further integrated by bioinformatic analyses. RESULTS: Celecoxib reduced cell growth and induced apoptosis through AKT blockade, cleavage of poly (ADP-ribose) polymerase-1 (PARP-1), and proteasomal degradation of the anti-apoptotic protein Mcl-1. Epidermal growth factor receptor (EGFR), ErbB2, and ErbB3 degradation, and heterogeneous nuclear ribonucleoprotein K (hnRNP K) downregulation, further amplified the inhibition of androgen signaling. Celecoxib reduced the invasive phenotype of CRPC cells by modulating NF-κB activity and reduced tumor growth in mice xenografts when administered in association with the anti-EGFR receptor antibody cetuximab. Bioinformatic analyses on human prostate cancer datasets support the relevance of these pathways in PCa progression. CONCLUSIONS: Signaling nodes at the intersection of pathways implicated in PCa progression are simultaneously modulated by celecoxib treatment. In combination therapies with cetuximab, celecoxib could represent a novel therapeutic strategy to curb signal transduction during CRPC progression.

Role of Circulating Tumor Cells (CTC), Androgen Receptor Full Length (AR-FL) and Androgen Receptor Splice Variant 7 (AR-V7) in a Prospective Cohort of Castration-Resistant Metastatic Prostate Cancer Patients.

BACKGROUND: Circulating tumor cells (CTC), androgen receptor full-length (AR-FL), and androgen receptor splice variant 7 (AR-V7) are prognostic in patients (pts) with metastatic castration-resistant prostate cancer (mCRPC). AR-V7 seems to predict resistance to androgen-receptor signaling inhibitors (ARSi). METHODS: We assessed the association of CTC, AR-FL, and AR-V7 with prostate-specific antigen (PSA) response and overall survival (OS). We used a modified AdnaTest CTC-based AR-FL and AR-V7 mRNA assay. Chi-square test, Fisher Exact test, Kaplan-Meier method, log-rank test, Cox proportional hazards models were used as appropriate. RESULTS: We enrolled 39 mCRPC pts, of those 24 started a first-line treatment for mCRPC (1L subgroup) and 15 had received at least two lines for mCRPC (>2L subgroup). CTC, AR-FL, and AR-V7 were enriched in >2L compared to 1L subgroup. Detection of these biomarkers was associated with a lower percentage of biochemical responses. Only 1/7 AR-V7+ pts had a PSA response and received cabazitaxel. Median OS was 4.7 months (95% CI 0.6-8.9) in AR-V7+ pts and not reached in AR-V7- pts. AR-V7 was the only variable with prognostic significance in the Cox model. CONCLUSION: AR-V7, CTC, and AR-FL are associated with advanced mCRPC and AR-V7+ predicts for shorter OS.

A hnRNP K⁻AR-Related Signature Reflects Progression toward Castration-Resistant Prostate Cancer.

The major challenge in castration-resistant prostate cancer (CRPC) remains the ability to predict the clinical responses to improve patient selection for appropriate treatments. The finding that androgen deprivation therapy (ADT) induces alterations in the androgen receptor (AR) transcriptional program by AR coregulators activity in a context-dependent manner, offers the opportunity for identifying signatures discriminating different clinical states of prostate cancer (PCa) progression. Gel electrophoretic analyses combined with western blot showed that, in androgen-dependent PCa and CRPC in vitro models, the subcellular distribution of spliced and serine-phosphorylated heterogeneous nuclear ribonucleoprotein K (hnRNP K) isoforms can be associated with different AR activities. Using mass spectrometry and bioinformatic analyses, we showed that the protein sets of androgen-dependent (LNCaP) and ADT-resistant cell lines (PDB and MDB) co-immunoprecipitated with hnRNP K varied depending on the cell type, unravelling a dynamic relationship between hnRNP K and AR during PCa progression to CRPC. By comparing the interactome of LNCaP, PDB, and MDB cell lines, we identified 51 proteins differentially interacting with hnRNP K, among which KLK3, SORD, SPON2, IMPDH2, ACTN4, ATP1B1, HSPB1, and KHDRBS1 were associated with AR and differentially expressed in normal and tumor human prostate tissues. This hnRNP K⁻AR-related signature, associated with androgen sensitivity and PCa progression, may help clinicians to better manage patients with CRPC.

Proteomics study of silver nanoparticles on Caco-2 cells.

Silver nanoparticles (AgNPs) have been incorporated into several consumer products. While these advances in technology are promising and exciting, the effects of these nanoparticles have not equally been studied. Due to the size, AgNPs can penetrate the body through oral exposure and reach the gastrointestinal tract. The present study was designed as a comparative proteomic analysis of Caco-2 cells, used as an in vitro model of the small intestine, exposed to 30 nm citrate stabilized-silver nanoparticles (AgNPs) for 24 or 72 h. Using two complementary proteomic approaches, 2D gel-based and label-free mass spectrometry, we present insight into the effects of AgNPs at proteins level. Exposure of 1 or 10 µg/mL AgNPs to Caco-2 cells resulted in 56 and 88 altered proteins at 24 h and 72 h respectively, by 2D gel-based technique. Ten of these proteins were found to be common between the two time-points. Using label-free mass spectrometry technique, 291 and 179 altered proteins were found at 24 h and 72 h, of which 24 were in common. Analysis of the proteomes showed several major biological processes altered, from which, cell cycle, cell morphology, cellular function and maintenance were the most affected.

Adaptive phenotype drives resistance to androgen deprivation therapy in prostate cancer.

BACKGROUND: Prostate cancer (PCa), the second most common cancer affecting men worldwide, shows a broad spectrum of biological and clinical behaviour representing the epiphenomenon of an extreme heterogeneity. Androgen deprivation therapy is the mainstay of treatment for advanced forms but after few years the majority of patients progress to castration-resistant prostate cancer (CRPC), a lethal form that poses considerable therapeutic challenges. METHODS: Western blotting, immunocytochemistry, invasion and reporter assays, and in vivo studies were performed to characterize androgen resistant sublines phenotype in comparison to the parental cell line LNCaP. RNA microarray, mass spectrometry, integrative transcriptomic and proteomic differential analysis coupled with GeneOntology and multivariate analyses were applied to identify deregulated genes and proteins involved in CRPC evolution. RESULTS: Treating the androgen-responsive LNCaP cell line for over a year with 10 µM bicalutamide both in the presence and absence of 0.1 nM 5-α-dihydrotestosterone (DHT) we obtained two cell sublines, designated PDB and MDB respectively, presenting several analogies with CRPC. Molecular and functional analyses of PDB and MDB, compared to the parental cell line, showed that both resistant cell lines were PSA low/negative with comparable levels of nuclear androgen receptor devoid of activity due to altered phosphorylation; cell growth and survival were dependent on AKT and p38MAPK activation and PARP-1 overexpression; their malignant phenotype increased both in vitro and in vivo. Performing bioinformatic analyses we highlighted biological processes related to environmental and stress adaptation supporting cell survival and growth. We identified 15 proteins that could direct androgen-resistance acquisition. Eleven out of these 15 proteins were closely related to biological processes involved in PCa progression. CONCLUSIONS: Our models suggest that environmental factors and epigenetic modulation can activate processes of phenotypic adaptation driving drug-resistance. The identified key proteins of these adaptive phenotypes could be eligible targets for innovative therapies as well as molecules of prognostic and predictive value.

The ErbB family and androgen receptor signaling are targets of Celecoxib in prostate cancer.

Inflammation plays a central role in prostate cancer (PCa) development through significant crosstalk between the COX-2-ErbB family receptor network and androgen receptor (AR)-EGFR signaling pathways. The purpose of this work was to determine the ability of the COX-2 inhibitor Celecoxib to modulate the EGFR-AR signaling pathway in androgen-dependent PCa cells and to provide a rationale for its beneficial use in chemopreventive strategies. Functional studies of Celecoxib activity were performed on LNCaP prostate cancer cells. Western blotting, gene expression analysis, dual-luciferase reporter assay and ELISA were applied to assess the Celecoxib mechanisms of action. We found that Celecoxib, through EGF and amphiregulin (AREG) induction, caused EGFR and ErbB2 activation and consequent degradation associated with the inhibition of androgenic signaling. By upregulating the E3 ubiquitin ligase Nrdp1, Celecoxib also efficiently downregulated ErbB3, which is strongly implicated in castration-resistant prostate cancer. Lastly, Celecoxib directly regulated AR transcription and translation independent of ErbB activation by downregulating the RNA binding protein heterogeneous nuclear ribonucleoprotein K (hnRNP K). The simultaneous suppression of ErbB kinases and androgen signaling by Celecoxib represents a novel strategy to interrupt the vicious cycle of AR/ErbB cross-talk with the primary purpose of undermining their resilient signaling in prostate cancer progression. Our data provide important premises for the chemopreventive use of Celecoxib in the clinical management of prostate cancer.

A combined proteomics and metabolomics approach to assess the effects of gold nanoparticles in vitro.

Omics technologies, such as proteomics or metabolomics, have to date been applied in the field of nanomaterial safety assessment to a limited extent. To address this dearth, we developed an integrated approach combining the two techniques to study the effects of two sizes, 5 and 30 nm, of gold nanoparticles (AuNPs) in Caco-2 cells. We observed differences in cells exposed for 72 h to each size of AuNPs: 61 responsive (up/down-regulated) proteins were identified and 35 metabolites in the cell extract were tentatively annotated. Several altered biological pathways were highlighted by integrating the obtained multi-omics data with bioinformatic tools. This provided a unique set of molecular information on the effects of nanomaterials at cellular level. This information was supported by complementary data obtained by immunochemistry, microscopic analysis, and multiplexed assays. A part from increasing our knowledge on how the cellular processes and pathways are affected by nanomaterials (NMs), these findings could be used to identify specific biomarkers of toxicity or to support the safe-by-design concept in the development of new nanomedicines.