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Acquired resistance to platinum (Pt)‐based therapies is an urgent unmet need in the management of epithelial ovarian cancer (EOC) patients. Here, we characterized by an unbiased proteomics method three isogenic EOC models of acquired Pt resistance (TOV‐112D, OVSAHO, and MDAH‐2774). Using this approach, we identified several differentially expressed proteins in Pt‐resistant (Pt‐res) compared to parental cells and the chaperone HSP90 as a central hub of these protein networks. Accordingly, up‐regulation of HSP90 was observed in all Pt‐res cells and heat‐shock protein 90 alpha isoform knockout resensitizes Pt‐res cells to cisplatin (CDDP) treatment. Moreover, pharmacological HSP90 inhibition using two different inhibitors [17‐(allylamino)‐17‐demethoxygeldanamycin (17AAG) and ganetespib] synergizes with CDDP in killing Pt‐res cells in all tested models. Mechanistically, genetic or pharmacological HSP90 inhibition plus CDDP ‐induced apoptosis and increased DNA damage, particularly in Pt‐res cells. Importantly, the antitumor activities of HSP90 inhibitors (HSP90i) were confirmed both ex vivo in primary cultures derived from Pt‐res EOC patients ascites and in vivo in a xenograft model. Collectively, our data suggest an innovative antitumor strategy, based on Pt compounds plus HSP90i, to rechallenge Pt‐res EOC patients that might warrant further clinical evaluation., By unbiased proteomics, we identified HSP90 as a central hub in platinum (Pt)‐resistant ovarian cancer models. Indeed, genetic and pharmacologial HSP90 inhibition sensitized resistant cells to CDDP treatment, potentiating DNA‐damage and apoptosis. Combined CDDP plus anti‐HSP90 therapy is effective also ex vivo on primary cultures from Pt‐resistant (Pt‐res) ovarian cancer patients and in vivo Pt‐res ovarian cancer xenograft mouse model.

Although fluoropyrimidines were introduced as anticancer agents over 60 years ago, they are still the backbone of many combination chemotherapy regimens for the treatment of solid cancers. Like other chemotherapeutic agents, the therapeutic efficacy of fluoropyrimidines can be affected by drug resistance and severe toxicities; thus, novel therapeutic approaches are required to potentiate their efficacy and overcome drug resistance. In the last 20 years, the deregulation of epigenetic mechanisms has been shown to contribute to cancer hallmarks. Histone modifications play an important role in directing the transcriptional machinery and therefore represent interesting druggable targets. In this review, we focused on histone deacetylase inhibitors (HDACis) that can increase antitumor efficacy and overcome resistance to fluoropyrimidines by targeting specific genes or proteins. Our preclinical data showed a strong synergistic interaction between HDACi and fluoropyrimidines in different cancer models, but the clinical studies did not seem to confirm these observations. Most likely, the introduction of increasingly complex preclinical models, both in vitro and in vivo, cannot recapitulate human complexity; however, our analysis of clinical studies revealed that most of them were designed without a mechanistic approach and, importantly, without careful patient selection.

INTRODUCTION: Epithelial ovarian cancer (EOC) represents the most lethal gynecological disease, with a 5-year relative survival rate of 46% after the diagnosis. The standard treatment of advanced EOC is based on surgery, followed by platinum (Pt)-based chemotherapy. However, the development of platinum resistant disease could occur and strongly impact on the survival of EOC patients for whom we still do not have valid therapeutic options. By using a proteomic approach followed by a bioinformatic analysis, we previously validated the role of HSP90 in the mechanism of platinum-resistance. Here, we propose a novel therapeutic strategy based on the combined pharmacologic inhibition of HSP90 and mTOR to further potentiate Pt-based chemotherapy and to revert Pt-resistance in EOC and non-small-cell lung cancer (NSCLC) models. METHODS: TOV-112D parental and Pt-resistant cells were characterized by phosphoproteomics followed by functional analysis and proteins validation by western blot. Synergistic anti-tumor effect was evaluated in Pt-sensitive and Pt-resistant EOC and NSCLC cell lines by calculating combination indexes (CI), colony formation assay, apoptosis and DNA damage, as well as on 3D in vitro microtissues obtained by co-culturing cancer cells with normal fibroblasts and in vivo EOC and NSCLC xenograft models. RESULTS: 542 differentially phosphorylated expressed proteins were identified in Pt-resistant TOV-112D compared with parental cells, and mTOR and the transcription factor HSF1 emerged as the most enriched pathways. The up-regulation of the phosphorylated form of PDK1, AKT, mTOR and rpS6 was observed in Pt-resistant compared to parental cells. Moreover, we also demonstrated the up-regulation of the activity of HSF1 along with the elevation of its targets such as heat shock proteins HSP90, HSP70 and HSP40, crucial components of chaperone complex machinery. Interestingly, among the differentially expressed proteins, we identified the kinase DYRK2 able to phosphorylate HSF1, supporting its transactivation. Accordingly, the combination of HSP90 inhibitor ganetespib and the mTOR inhibitor temsirolimus plus cisplatin, synergistically reduced colony formation, cancer cells and microtissues cell growth in vitro by increasing DNA-damage and apoptosis and in vivo by enhancing mouse survival. Mechanistically, the triple combination treatment, impaired the proteins involved in mTOR signalling and HSF1 transactivation. Notably, all these data were confirmed in Pt-resistant NSCLC models, supporting the possibility that the same mechanism is present in different tumor types. CONCLUSIONS: Our findings identify a promising new antitumor strategy based on the combination of HSP90 and mTOR inhibitors to revert Pt-resistance that that warrant further clinical evaluation. Citation Format: Rita Lombardi, Laura Addi, Biagio Pucci, Maura Sonego, Maria Serena Roca, Francesca Capone, Federica Iannelli, Francesca Bruzzese, Gustavo Baldassarre, Alfredo Budillon. Combined inhibition of mTOR and HSP90 potentiates cisplatin antitumor effect and reverts cisplatin resistance in vitro and in vivo models of epithelial ovarian cancer by modulating HSF1-dependent transactivation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3232.

Background Despite the introduction of several novel therapeutic approaches that improved survival, metastatic castration-resistant prostate cancer (mCRPC) remains an incurable disease. Herein we report the synergistic antitumor interaction between two well-known drugs used for years in clinical practice, the antiepileptic agent with histone deacetylase inhibitory activity valproic acid and the cholesterol lowering agent simvastatin, in mCRPC models. Methods Synergistic anti-tumor effect was assessed on PC3, 22Rv1, DU145, DU145R80, LNCaP prostate cancer cell lines and EPN normal prostate epithelial cells, by calculating combination index (CI), caspase 3/7 activation and colony formation assays as well as on tumor spheroids and microtissues scored with luminescence 3D-cell viability assay. Cancer stem cells (CSC) compartment was studied evaluating specific markers by RT-PCR, western blotting and flow cytometry as well as by limiting dilution assay. Cholesterol content was evaluated by 1H-NMR. Overexpression of wild-type YAP and constitutively active YAP5SA were obtained by lipofectamine-based transfection and evaluated by immunofluorescence, western blotting and RT-PCR. 22Rv1 R_39 docetaxel resistant cells were selected by stepwise exposure to increasing drug concentrations. In vivo experiments were performed on xenograft models of DU145R80, 22Rv1 parental and docetaxel resistant cells, in athymic mice. Results We demonstrated the capacity of the combined approach to target CSC compartment by a novel molecular mechanism based on the inhibition of YAP oncogene via concurrent modulation of mevalonate pathway and AMPK. Because both CSCs and YAP activation have been associated with chemo-resistance, we tested if the combined approach can potentiate docetaxel, a standard of care in mCRCP treatment. Indeed, we demonstrated, both in vitro and in vivo models, the ability of valproic acid/simvastatin combination to sensitize mCRPC cells to docetaxel and to revert docetaxel-resistance, by mevalonate pathway/YAP axis modulation. Conclusion Overall, mCRPC progression and therapeutic resistance driven by CSCs via YAP, can be tackled by the combined repurposing of two generic and safe drugs, an approach that warrants further clinical development in this disease.

With the aim to develop alternative therapeutic tools for the treatment of resistant cancers, here we propose targeted Pluronic(®) P123/F127 mixed micelles (PMM) delivering niclosamide (NCL) as a repositioning strategy to treat multidrug resistant non-small lung cancer cell lines. To build multifunctional PMM for targeting and imaging, Pluronic(®) F127 was conjugated with biotin, while Pluronic(®) P123 was fluorescently tagged with rhodamine B, in both cases at one of the two hydroxyl end groups. This design intended to avoid any interference of rhodamine B on biotin exposition on PMM surface, which is a key fundamental for cell trafficking studies. Biotin-decorated PMM were internalized more efficiently than non-targeted PMM in A549 lung cancer cells, while very low internalization was found in NHI3T3 normal fibroblasts. Biotin-decorated PMM entrapped NCL with good efficiency, displayed sustained drug release in protein-rich media and improved cytotoxicity in A549 cells as compared to free NCL (P

INTRODUCTION: Epithelial ovarian cancer (EOC) represents the gynecologic cancer with the highest mortality rate. Despite a high initial response to platinum (PT)-based chemotherapy, the majority of patients with advanced EOC relapses and develops chemo-resistance that results in treatment failure and poor prognosis. Therefore, understanding the mechanisms underlying PT-resistance and finding strategies to overcome them are urgently needed. METHODS: Three isogenic models of PT-resistance generated by our group from EOC cell lines (TOV-112D, OVSAHO and MDAH) (Sonego M. et al, 2017), were characterized by a proteomic approach taking advantage of two-dimensional differential in gel electrophoresis (2-D DIGE) followed by Mass Spectrometry. Proteomics data were also analyzed by the ingenuity pathway analysis (IPA) software. Synergistic anti-proliferative activity was evaluated by calculating combination indexes (CI) by the Chou-Talalay method and colony formation assay. Apoptosis was measured by flow-cytometry and by western blotting evaluation of caspase-3 and PARP cleavage. In vivo experiments were performed on xenograft model in athymic mice. RESULTS: We identified 23, 24 and 20 differentially expressed proteins in PT-resistant TOV-112D, OVSAHO and MDAH respectively, compared with parental cells. A relevant relationship between all the identified proteins in the three models was highlighted by IPA. Interestingly, eight of the identified proteins, HSP7C, HNRPL, ATPA, EFTU, PHB, HNRPK, LMNA and PHGDH, shared at least within two cell lines, are all included in one main network related with cancer. Notably, the protein chaperone HSP90 emerged as a central hub of this network and its up-regulation was observed in all the PT-resistant cell lines compared with parental counterparts. Further molecular characterization also showed overexpression of HSF1 transcription factor, a relevant HSP90 activator and client protein, in PT-resistant cells. On this bases, we evaluated the effect of cisplatin in combination with two HSP90 inhibitors (17AAG or ganetespib), both in phase II/III clinical development in cancer patients, observing synergistic anti-proliferative activity and reduced colony formation, particularly in PT-resistant cells. These data were confirmed in primary cultures from PT-Resistant ovarian cancer patients. Furthermore we confirmed for the first time in vivo the synergistic antitumor effect of cisplatin and ganetespib combination in TOV112D PT-resistant xenograft model. Increased pro-apoptotic effect and DNA-damage induction by the combination treatment compared to untreated or single agents treated tumors was confirmed both in vitro and in vivo. CONCLUSIONS: Our data suggest an innovative antitumor strategy based on the combination of platinum compounds with HSP90 inhibitors, to re-challenge PT-resistant EOC, that warrants further clinical evaluation. Citation Format: Rita Lombardi, Maura Sonego, Laura Addi, Federica Iannelli, Francesca Capone, Maria Serena Roca, Maria Rita Milone, Alice Costa, Francesca Bruzzese, Biagio Pucci, Gustavo Baldassarre, Alfredo Budillon. HSP90 identified by a proteomic approach as druggable target to reverse platinum-resistance in ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5270.

Background Modifications of lipid metabolism have been progressively accepted as a hallmark of tumor cells and in particular, an elevated lipogenesis has been described in various types of cancers. Objective Important or deregulated activity of the mevalonate pathway has been demonstrated in different tumors and a wide range of studies have suggested that tumor cells are more dependent on the unceasing availability of mevalonate pathway metabolites than their non-malignant complements. Methods This study provides an overview of the state of the art of statins treatment on human cancer. Results In recent times, various actions have been proposed for statins in different physiological and pathological conditions beyond anti-inflammation and neuroprotection activity. Statins have been shown to act through mevalonate-dependent and -independent mechanisms able to affect several tissue functions and modulating specific signal transduction pathways that could account for statin pleiotropic effect. Based on their characteristics, statins represent ideal candidates for repositioning in cancer therapy. Conclusion In this review article, we provide an overview of the current preclinical and clinical status of statins as antitumor agents. In addition, we evaluated various patents that describe the role of mevalonate pathway inhibitors and methods to determine if cancer cells are sensitive to statins treatment.

Vorinostat demonstrated preclinical and clinical efficacy in human cancers and is the first histone deacetylase inhibitor (HDACi) approved for cancer treatment. Tissue transglutaminase (TG2) is a multifunctional enzyme that catalyzes a Ca2+ dependent transamidating reaction resulting in covalent cross-links between proteins. TG2 acts also as G-protein in trans-membrane signaling and as a cell surface adhesion mediator. TG2 up-regulation has been demonstrated in several cancers and its expression levels correlate with resistance to chemotherapy and metastatic potential. We demonstrated that the anti-proliferative effect of the HDACi vorinostat is paralleled by the induction of TG2 mRNA and protein expression in cancer cells but not in ex vivo treated peripheral blood lymphocytes. This effect was also shared by other pan-HDACi and resulted in increased TG2 transamidating activity. Notably, high TG2 basal levels in a panel of cancer cell lines correlated with lower vorinostat antiproliferative activity. Notably, in TG2-knockdown cancer cells vorinostat anti-proliferative and pro-apoptotic effects were enhanced, whereas in TG2-full-length transfected cells were impaired, suggesting that TG2 could represent a mechanism of intrinsic or acquired resistance to vorinostat. In fact, co-treatment of tumor cells with inhibitors of TG2 transamidating activity potentiated the antitumor effect of vorinostat. Moreover, vorinostat-resistant MCF7 cells selected by stepwise increasing concentrations of the drug, significantly overexpressed TG2 protein compared to parental cells, and co-treatment of these cells with TG2 inhibitors reversed vorinostat-resistance. Taken together, our data demonstrated that TG2 is involved in the resistance of cancer cells to vorinostat, as well as to other HDACi.

The need to explore new alternative therapeutic strategies and chemoprevention methods for hepatocellular carcinoma is growing significantly. Selenium is a trace element that plays a critical role in physiological processes, and is used in cancer chemoprevention. The aim of this work was to test in vitro the effect of sodium selenite on the human hepatoma cell lines, HepG2 and Huh7, to assess its effect on the expression of GPX1, SELK and SELENBP1 and also to evaluate its action on inflammation determinants such as cytokines. Our results show that: (i) the increase observed for the GPX1 and SELK expression is correlated with an increase in the sodium selenite concentration, also evidencing an inverse association between the levels of these two proteins and SELENBP1; (ii) the selenium concentrations evaluated in protein extracts increase in proportional way with the selenite concentrations used in the treatment, suggesting that other selenoproteins can also be modulated and should be evaluated in further studies, and (iii) some cytokines, VEGF and three pro-inflammatory cytokines, i.e., IL-6, IL-8, and IL-17, decreased with an increasing selenite concentration. Finally, interactomic studies show that GPX1 and SELK, and the four pro-inflammatory cytokines are functionally correlated evidencing a putative anti-inflammatory role for the selenite.

Several solid tumors are characterized by poor prognosis and few effective treatment options, other than palliative chemotherapy in the recurrent/metastatic setting. Epidermal growth factor receptor (EGFR) has been considered an important anticancer target because it is involved in the development and progression of several solid tumors; however, only a subset of patients show a clinically meaningful response to EGFR inhibition, particularly to EGFR tyrosine kinase inhibitors such as gefitinib. We have recently demonstrated synergistic antitumor effect of the histone deacetylase inhibitor vorinostat combined with gefitinib. To further characterize the interaction between these two agents, cellular extracts from Hep-2 cancer cells that were untreated or treated for 24 h with either vorinostat or gefitinib alone or with a vorinostat/gefitinib combination were analyzed using 2-D DIGE. Software analysis using DeCyder was performed, and numerous differentially expressed protein spots were visualized between the four examined settings. Using MALDI-TOF MS and ESI-Ion trap MS/MS, several differentially expressed proteins were identified; some of these were validated by Western blotting. Finally, a pathway analysis of experimental data performed using MetaCore highlighted a relevant relationship between the identified proteins and additional potential effectors. In conclusion, we performed a comprehensive analysis of proteins regulated by vorinostat and gefitinib, alone and in combination, providing a useful insight into their mechanisms of action as well as their synergistic interaction.

Introduction: docetaxel (DTX) represents the standard of care first line treatment of castration-resistant prostate cancer (PCa). However, the onset of systemic side effects hampers patient's compliance and DTX resistance invariably emerges, leading to disease relapse, suggesting the need of novel combination strategies. Cancer stem cells (CSC) drive PCa survival and metastasis and are thought to be responsible for the development of resistance to DTX. The mevalonate pathway (MVP) plays a critical role in PCa progression and is implicated in cell stemness, proliferation, and organ size regulation through the YAP-TAZ signaling axis. Here, we evaluate the combination treatment of valproic acid (VPA), an histone deacetylase (HDAC) inhibitor and the cholesterol-lowering drug simvastatin (SIM), an inhibitor of HMGCoA reductase (HMGCR), the rate limiting enzyme in MVP, alone and plus DTX in PCa models. Method: synergistic antiproliferative effects were assessed on LNCAP, 22Rv1, DU145, PC3 and DU14580 SIM-resistant cell lines and normal epithelial prostate EPN cells, by calculating combination index (CI) according to Chou and Talalay method. Apoptosis was measured by FACS analysis and caspase assay. Tumor spheroids were obtained by low attach systems and scored with luminescence 3D-cell viability assay. Proteins and genes expression was assessed by western blot and real time PCR analysis. In vivo experiments were performed on xenograft models in athymic mice. Cholesterol content was evaluated by nuclear magnetic resonance (1H-NMR). Results: synergistic antiproliferative and proapoptotic effect of VPA-SIM was observed in all PCa cell lines tested, except in EPN cells and was confirmed in PCa spheroids. SIM-dependent cholesterol content downmodulation was potentiated by VPA and mevalonic acid, the product of HMGCR activity, reverts all the antitumor combined effect, suggesting the involvement of MVP in the synergistic interaction. Mechanistically, the combination is able to induce a reduction of HMGCR mRNA expression and the inhibitory phosphorylation of HMGCR and YAP, followed by a reduction of CTGF, BRCA5 and Cyr61 YAP-target genes, as well as a reduction of a CSC-marker gene such as NANOg. Notably, the VPA-SIM combination, sensitizes PCa cells to DTX treatment in sensitive and DTX-resistant cells, as shown by CI calculation, apoptosis, and CSC enriched-spheroid experiments. The synergistic interaction of the triple combination was also confirmed in vivo in both DU145R80 and 22Rv1 xenograft models. Conclusions: Overall, this study suggests that the combination of two safe generic drugs such as VPA and SIM, may improve the therapeutic index of DTX and revert DTX-resistance, representing an innovative and feasible antitumor strategy for the treatment of prostate cancer that warrants further clinical evaluation. Citation Format: Federica Iannelli, Maria Serena Roca, Chiara Ciardiello, Simona De Rienzo, Rita Lombardi, Angela Sorice, Susan Costantini, Tania Moccia, Maria Rita Milone, Biagio Pucci, Alfredo Budillon, Francesca Bruzzese. Synergistic antitumor interaction of valproic acid and simvastatin sensitizes prostate cancer to docetaxel by targeting cancer stem cells compartment via YAP-pathway modulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2877.

The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases (SDRs) superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh gene was heterologously overexpressed in Escherichia coli, and the protein (SaADH) was purified to homogeneity and characterized. SaADH is a tetrameric enzyme consisting of identical 28,978-Da subunits, each composed of 264 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 75 degrees C and a 30-min half-inactivation temperature of ~90 degrees C, and shows good tolerance to common organic solvents. SaADH has a strict requirement for NAD(H) as the coenzyme, and displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and alpha-keto esters, but is poorly active on aliphatic, cyclic and aromatic alcohols, and shows no activity on aldehydes. The enzyme catalyses the reduction of alpha-methyl and alpha-ethyl benzoylformate, and methyl o-chlorobenzoylformate with 100% conversion to methyl (S)-mandelate [17% enantiomeric excess (ee)], ethyl (R)-mandelate (50% ee), and methyl (R)-o-chloromandelate (72% ee), respectively, with an efficient in situ NADH-recycling system which involves glucose and a thermophilic glucose dehydrogenase. This study provides further evidence supporting the critical role of the D37 residue in discriminating NAD(H) from NAD(P)H in members of the SDR superfamily.

The Mini-Chromosome Maintenance (MCM) proteins are candidates of replicative DNA helicase in eukarya and archaea. Here we report a 2.8 A crystal structure of the N-terminal domain (residues 1-268) of the Sulfolobus solfataricus MCM (Sso MCM) protein. The structure reveals single-hexameric ring-like architecture, at variance from the protein of Methanothermobacter thermoautotrophicus (Mth). Moreover, the central channel in Sso MCM seems significantly narrower than the Mth counterpart, which appears to more favorably accommodate single-stranded DNA than double-stranded DNA, as supported by DNA-binding assays. Structural analysis also highlights the essential role played by the zinc-binding domain in the interaction with nucleic acids and allows us to speculate that the Sso MCM N-ter domain may function as a molecular clamp to grasp the single-stranded DNA passing through the central channel. On this basis possible DNA unwinding mechanisms are discussed.

To protect their genetic material cells adopt different mechanisms linked to DNA replication, recombination and repair. Several proteins function at the interface of these DNA transactions. In the present study, we report on the identification of a novel archaeal DNA helicase. BlastP searches of the Sulfolobus solfataricus genome database allowed us to identify an open reading frame (SSO0112, 875 amino acid residues) having sequence similarity with the human RecQ5β. The corresponding protein, termed Hel112 by us, was produced in Escherichia coli in soluble form, purified to homogeneity and characterized. Gel-filtration chromatography and glycerol-gradient sedimentation analyses revealed that Hel112 forms monomers and dimers in solution. Biochemical characterization of the two oligomeric species revealed that only the monomeric form has an ATP-dependent 3′–5′ DNA-helicase activity, whereas, unexpectedly, both the monomeric and dimeric forms possess DNA strand-annealing capability. The Hel112 monomeric form is able to unwind forked and 3′-tailed DNA structures with high efficiency, whereas it is almost inactive on blunt-ended duplexes and bubble-containing molecules. This analysis reveals that S. solfataricus Hel112 shares some enzymatic features with the RecQ-like DNA helicases and suggests potential cellular functions of this protein.

// Valentina Bizzarro 1 , Raffaella Belvedere 1 , Maria Rita Milone 2 , Biagio Pucci 2 , Rita Lombardi 2 , Francesca Bruzzese 3 , Ada Popolo 1 , Luca Parente 1 , Alfredo Budillon 2, 3 , Antonello Petrella 1 1 Department of Pharmacy, University of Salerno, Fisciano (SA), Italy 2 Centro Ricerche Oncologiche Mercogliano, Istituto Nazionale Tumori Fondazione G. Pascale – IRCCS, Naples, Italy 3 Experimental Pharmacology Unit, Istituto Nazionale Tumori Fondazione G. Pascale – IRCCS, Naples, Italy Correspondence to: Antonello Petrella, e-mail: apetrella@unisa.it Alfredo Budillon, e-mail: a.budillon@istitutotumori.na.it Keywords: annexin A1, prostate cancer, cell invasion, EMT, cancer stem cells Received: May 18, 2015 Accepted: July 16, 2015 Published: July 28, 2015 ABSTRACT In this study, we have characterized the role of annexin A1 (ANXA1) in the acquisition and maintenance of stem-like/aggressive features in prostate cancer (PCa) cells comparing zoledronic acid (ZA)-resistant DU145R80 with their parental DU145 cells. ANXA1 is over-expressed in DU145R80 cells and its down-regulation abolishes their resistance to ZA. Moreover, ANXA1 induces DU145 and DU145R80 invasiveness acting through formyl peptide receptors (FPRs). Also, ANXA1 knockdown is able to inhibit epithelial to mesenchymal transition (EMT) and to reduce focal adhesion kinase (FAK) and metalloproteases (MMP)-2/9 expression in PCa cells. DU145R80 show a cancer stem cell (CSC)-like signature with a high expression of CSC markers including CD44, CD133, NANOG, Snail, Oct4 and ALDH7A1 and CSC-related genes as STAT3. Interestingly, ANXA1 knockdown induces these cells to revert from a putative prostate CSC to a more differentiated phenotype resembling DU145 PCa cell signature. Similar results are obtained concerning some drug resistance-related genes such as ATP Binding Cassette G2 (ABCG2) and Lung Resistant Protein (LRP). Our study provides new insights on the role of ANXA1 protein in PCa onset and progression.

Trematomus bernacchii immunoglobulin M concentration was determined in the serum by ELISA; the mean concentration value was 2.7 mg/ml corresponding to 9.6% of the total serum proteins. Purified IgM was analyzed by SDS-polyacrylamide gel electrophoresis, isoelectrofocusing and 2D electrophoresis. The relative molecular mass of the polymeric form was 830 kDa; that of separated H and L chains was, respectively, 78 and 25 kDa. The isoelectric points of the entire molecule ranged from 4.4 to 6.5, that of isolated H chains was between 4.0 and 6.0. Separated H chains were shown to reaggregate in tetrameric form. The cleavage site of trypsin was at the end of the CH1 domain, as confirmed by the N-terminal amino acid sequence of one of the resultant peptides. Immunoblotting was used to detect carbohydrates in the H and L chains labeled with digoxigenin. Glycosyl residues were detected only in the H chain. The carbohydrate content was evaluated to be 12.8% of the entire chain. Purified Igs were hydrolyzed by N-glycosidase F at different conditions and at least four different hydrolytic sites were revealed by limited deglycosylation. T. bernacchii IgM was also compared to those of five other polar fish species.

Patients with advanced prostate cancer (PCa) and multiple myeloma (MM) have limited long-term responses to available therapies. The histone deacetylase inhibitor panobinostat has shown significant preclinical and clinical anticancer activity in both hematological and solid malignancies and is currently in phase III trials for relapsed MM. Bisphosphonates (BPs), such as zoledronic acid (ZOL), inhibit osteoclast-mediated bone resorption and are indicated for the treatment of bone metastasis. BPs, including ZOL, have also shown anticancer activity in several preclinical and clinical studies. In the present report, we found a potent synergistic antiproliferative effect of panobinostat/ZOL treatment in three PCa and three MM cell lines as well as in a PCa ZOL-resistant subline, independently of p53/KRAS status, androgen dependency, or the schedule of administration. The synergistic effect was also observed in an anchorage-independent agar assay in both ZOL-sensitive and ZOL-resistant cells and was confirmed in vivo in a PCa xenograft model. The co-administration of the antioxidant N-acetyl-L-cysteine blocked the increased reactive oxygen species generation and apoptosis observed in the combination setting compared with control or single-agent treatments, suggesting that oxidative injury plays a functional role in the synergism. Proapoptotic synergy was also partially antagonized by the addition of geranyl-geraniol, which bypasses the inhibition of farnesylpyrophosphate synthase by ZOL in the mevalonate pathway, supporting the involvement of this pathway in the synergy. Finally, at the molecular level, the inhibition of basal and ZOL-induced activation of p38-MAPK by panobinostat in sensitive and ZOL-resistant cells and in tumor xenografts could explain, at least in part, the observed synergism.

Although docetaxel (DTX) remains a standard of care for advanced prostate cancer (PCa), limited long-term responses, side effects and resistant disease suggested the need of novel combination strategies. Increased expression of histone deacetylases (HDAC) and alteration of the mevalonate pathway (MVP) are common aberrations in PCa. In this study, we analyzed the antitumor effect of DTX in combination with valproic acid (VPA), an anticonvulsant with HDAC inhibitory activity, and simvastatin (SIM), a cholesterol-lowering drug inhibiting the rate-limiting enzyme of MVP HMG-CoA reductase, on androgen-dependent 22RV1 and LNCAP and androgen-independent PC3 and DU145 PCa cells, as well as on the highly aggressive SIM-resistant DU145R80 subline developed in our laboratory from DU145 cells (Milone MR et al. Cell Death Dis. 2013; Milone MR et al. Oncotarget 2014). We first showed a potent synergistic anti-proliferative effect of VPA/SIM combination, assessed by calculating combination index (CI) according to the method of Chou and Talalay, on all cell lines, including SIM-resistant cells, whatever schedule of administration (simultaneous vs sequential) we used, confirming our previous data on the combination between the HDAC inhibitor (HDACi) panobinostat and zoledronic acid, the latter also targeting the MVP pathway (Bruzzese F. et al, Cell Death Dis. 2013). Notably, exposure to triple combinations (VPA/SIM/DTX) resulted in a further strong synergistic anti-proliferative effect, with sequential exposure with 24h delay between VPA/SIM and DTX as the best schedule. The synergistic interaction of VPA/SIM and DTX combination involved apoptotic effect, measured by FACS analysis of sub-diploid DNA content and caspase 3/7 cleavage, and DNA damage induction, assessed by γH2AX expression. Synergistic effect of VPA/SIM and DTX combination was confirmed by soft agar clonogenic assay and by 3D culture on self-assembled PCa spheroids. Significantly, VPA/SIM combination was also able to revert DTX-resistance in DTX-resistant PC3 and DU145 sublines developed in our laboratory from the parental cells. All together these findings suggested that the combination of two safe generic drugs such as VPA and SIM can improve DTX efficacy, representing a novel therapeutic approach that warrant clinical investigation in advanced PCa patients. Our study also suggests a new strategy to overcome resistance to standard taxane-based therapy in PCa patients. Citation Format: Federica Iannelli, Rita Lombardi, Biagio Pucci, Maria Rita Milone, Chiara Ciardiello, Alessandra Leone, Elena Di Gennaro, Alfredo Budillon, Francesca Bruzzese. Repurposing of valproic acid and simvastatin combination as anticancer agents in prostate cancer: synergistic interaction with docetaxel and suppression of docetaxel resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4745.

Impairment of the immune response and aberrant expression of microRNAs are emerging hallmarks of tumour initiation/progression, in addition to driver gene mutations and epigenetic modifications. We performed a preliminary survey of independent adenoma and colorectal cancer (CRC) miRnoma data sets and, among the most dysregulated miRNAs, we selected miR-27a and disclosed that it is already upregulated in adenoma and further increases during the evolution to adenocarcinoma. To identify novel genes and pathways regulated by this miRNA, we employed a differential 2DE-DIGE proteome analysis. We showed that miR-27a modulates a group of proteins involved in MHC class I cell surface exposure and, mechanistically, demonstrated that calreticulin is a miR-27a direct target responsible for most downstream effects in epistasis experiments. In vitro miR-27a affected cell proliferation and angiogenesis; mouse xenografts of human CRC cell lines expressing different miR-27a levels confirmed the protein variations and recapitulated the cell growth and apoptosis effects. In vivo miR-27a inversely correlated with MHC class I molecules and calreticulin expression, CD8+ T cells infiltration and cytotoxic activity (LAMP-1 exposure and perforin release). Tumours with high miR-27a, low calreticulin and CD8+ T cells' infiltration were associated with distant metastasis and poor prognosis. Our data demonstrate that miR-27a acts as an oncomiRNA, represses MHC class I expression through calreticulin downregulation and affects tumour progression. These results may pave the way for better diagnosis, patient stratification and novel therapeutic approaches.

Immunogenic cell death (ICD) evoked by chemotherapeutic agents implies emission of selected damage-associated molecular patterns (DAMP) such as cell surface exposure of calreticulin, secretion of ATP and HMGB1. We sought to verify whether miR-27a is implicated in ICD, having demonstrated that it directly targets calreticulin. To this goal, we exposed colorectal cancer cell lines, genetically modified to express high or low miR-27a levels, to two bona fide ICD inducers (mitoxantrone and oxaliplatin). Low miR-27a-expressing cells displayed more ecto-calreticulin on the cell surface and increased ATP and HMGB1 secretion than high miR-27a-expressing ones in time-course experiments upon drug exposure. A calreticulin target protector counteracted the miR-27a effects while specific siRNAs mimicked them, confirming the results reported. In addition, miR-27a negatively influenced the PERK-mediated route and the late PI3K-dependent secretory step of the unfolded protein response to endoplasmic reticulum stress, suggesting that miR-27a modulates the entire ICD program. Interestingly, upon chemotherapeutic exposure, low miR-27a levels associated with an earlier and stronger induction of apoptosis and with morphological and molecular features of autophagy. Remarkably, in ex vivo setting, under the same chemotherapeutic induction, the conditioned media from high miR-27a-expressing cells impeded dendritic cell maturation while increased the secretion of specific cytokines (interleukin (IL)-4, IL-6, IL-8) and negatively influenced CD4(+) T-cell interferon γ production and proliferation, all markers of a tumor immunoevasion strategy. In conclusion, we provide the first evidence that miR-27a impairs the cell response to drug-induced ICD through the regulatory axis with calreticulin.

Medulloblastoma is the most common malignant brain tumor in children, and despite improvements in the overall survival rate, it still lacks an effective treatment. Src plays an important role in cancer, and recently high Src activity was documented in medulloblastoma. In this report, we examined the effects of novel pyrazolo-[3,4-d]-pyrimidine derivative Src inhibitors in medulloblastoma. By MTS assay, we showed that the pyrimidine derivatives indicated as S7, S29, and SI163 greatly reduce the growth rate of medulloblastoma cells by inhibiting Src phosphorylation, compared with HT22 non-neoplastic nerve cells. These compounds also halt cells in the G2/M phase, and this effect likely occurs through the regulation of cdc2 and CDC25C phosphorylation, as shown by Western blot. Moreover, the exposure to pyrimidine derivatives induces apoptosis, assayed by the supravital propidium iodide assay, through modulation of the apoptotic proteins Bax and Bcl2, and inhibits tumor growth in vivo in a mouse model. Notably, S7, S29, and SI163 show major inhibitory effects on medulloblastoma cell growth compared with the chemotherapeutic agents cisplatin and etoposide. In conclusion, our results suggest that S7, S29, and SI163 could be novel attractive candidates for the treatment of medulloblastoma or tumors characterized by high Src activity.—Rossi, A., Schenone, S., Angelucci, A., Cozzi, M., Caracciolo, V., Pentimalli, F., Puca, A., Pucci, B., La Montagna, R., Bologna, M., Botta, M., Giordano, A. New pyrazolo-[3,4-d]-pyrimidine derivative Src kinase inhibitors lead to cell cycle arrest and tumor growth reduction of human medulloblastoma cells.

The gene encoding a novel alcohol dehydrogenase (ADH) that belongs to the short-chain dehydrogenase/reductase (SDR) superfamily was identified in the extremely thermophilic, halotolerant gram-negative eubacterium Thermus thermophilus HB27. The T. thermophilus ADH gene ( adh Tt ) was heterologously overexpressed in Escherichia coli , and the protein (ADH Tt ) was purified to homogeneity and characterized. ADH Tt is a tetrameric enzyme consisting of identical 26,961-Da subunits composed of 256 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to ∼73°C and a 30-min half-inactivation temperature of ∼90°C, as well as good tolerance to common organic solvents. ADH Tt has a strict requirement for NAD(H) as the coenzyme, a preference for reduction of aromatic ketones and α-keto esters, and poor activity on aromatic alcohols and aldehydes. This thermophilic enzyme catalyzes the following reactions with Prelog specificity: the reduction of acetophenone, 2,2,2-trifluoroacetophenone, α-tetralone, and α-methyl and α-ethyl benzoylformates to ( S )-(−)-1-phenylethanol (>99% enantiomeric excess [ee]), ( R )-α-(trifluoromethyl)benzyl alcohol (93% ee), ( S )-α-tetralol (>99% ee), methyl ( R )-(−)-mandelate (92% ee), and ethyl ( R )-(−)-mandelate (95% ee), respectively, by way of an efficient in situ NADH-recycling system involving 2-propanol and a second thermophilic ADH. This study further supports the critical role of the D37 residue in discriminating NAD(H) from NADP(H) in members of the SDR superfamily.

Mini-chromosome maintenance (MCM) proteins form ring-like hexameric complexes that are commonly believed to act as the replicative DNA helicase at the eukaryotic/archaeal DNA replication fork. Because of their simplified composition with respect to the eukaryotic counterparts, the archaeal MCM complexes represent a good model system to use in analyzing the structural/functional relationships of these important replication factors. In this study the domain organization of the MCM-like protein from Sulfolobus solfataricus (Sso MCM) has been dissected by trypsin partial proteolysis. Three truncated derivatives of Sso MCM corresponding to protease-resistant domains were produced as soluble recombinant proteins and purified: the N-terminal domain (N-ter, residues 1-268); a fragment comprising the AAA+ and C-terminal domains (AAA+-C-ter, residues 269-686); and the C-terminal domain (C-ter, residues 504-686). All of the purified recombinant proteins behaved as monomers in solution as determined by analytical gel filtration chromatography, suggesting that the polypeptide chain integrity is required for stable oligomerization of Sso MCM. However, the AAA+-C-ter derivative, which includes the AAA+ motor domain and retains ATPase activity, was able to form dimers in solution when ATP was present, as analyzed by size exclusion chromatography and glycerol gradient sedimentation analyses. Interestingly, the AAA+-C-ter protein could displace oligonucleotides annealed to M13 single-stranded DNA although with a reduced efficiency in comparison with the full-sized Sso MCM. The implications of these findings for understanding the DNA helicase mechanism of the MCM complex are discussed.

The hyper-thermophilic archaeon Sulfolobus solfataricus possesses two functional DNA polymerases belonging to the B-family (Sso DNA pol B1) and to the Y-family (Sso DNA pol Y1). Sso DNA pol B1 recognizes the presence of uracil and hypoxanthine in the template strand and stalls synthesis 3-4 bases upstream of this lesion ("read-ahead" function). On the other hand, Sso DNA pol Y1 is able to synthesize across these and other lesions on the template strand. Herein we report evidence that Sso DNA pol B1 physically interacts with DNA pol Y1 by surface plasmon resonance measurements and immuno-precipitation experiments. The region of DNA pol B1 responsible for this interaction has been mapped in the central portion of the polypeptide chain (from the amino acid residue 482 to 617), which includes an extended protease hyper-sensitive linker between the N- and C-terminal modules (amino acid residues Asn482-Ala497) and the alpha-helices forming the "fingers" sub-domain (alpha-helices R, R' and S). These results have important implications for understanding the polymerase-switching mechanism on the damaged template strand during genome replication in S. solfataricus.

We have recently established a zoledronic acid-resistant prostate cancer cell line, DU145R80,which exhibits higher invasive capability compared to parental DU145 cells, and epithelial-to-mesenchymal transition (EMT)[Milone MR, Cell Death Dis. 2013]. To investigate the mechanism by which the cells become invasive, we compared DU145R80 and DU145 cells by a proteomic approach, unveiling a signaling network that links the interior of the nucleus to changes in cytoskeleton dynamics and to the extracellular matrix, dictating prostate cancer aggressiveness [Milone MR Oncotarget 2014].Cytoskeletal modifications allow cells to gain a more migratory/invasive behavior and to interact with the surrounding microenvironment triggering different kind of biological phenomena, including the formation of large oncosomes (LO), a bioactive class of extracellular vesicles (EVs) [Di Vizio D, Cancer Res. 2009]. In this study, we found a higher amount of spontaneously shed LO along with an increased gelatinase activity from DU145R80 cells compared to the parental counterpart. By applying low speed and discontinuous gradient ultracentrifugation to both DU145 and DU145R80 cell media, we obtained a pure preparation of LO, floating at a 1.15 g/mL density fraction and positive for LO markers such as as Cav-1, Ck18, GAPDH. Moreover, we treated DU145 parental cells with pure preparations of LO from either DU145 or DU145R80 cells and performed an invasion assay, showing that LO from the resistant, aggressive DU145R80 cell line increase the invading ability of DU145. Treatment of DU145 cells with LO originating from the parental cell line itself did not result in increased invasinevess, suggesting a specific role for EVs from aggressive cells. In addition, in order to investigate LO content, we focused our attention on a cell surface glycoprotein, CUB domain-containing protein 1 (CDCP-1), putatively linked to the network of proteins identified in DU145R80 cells and whose role in cancer is still under debate. Our results demonstrate that CDCP-1 expression is significantly reduced in DU145R80 compared to DU145 cells, suggesting a tumor-protective role for the protein in prostate cancer. We also observed, for the first time, that the CDCP-1 expression pattern displayed by both cell lines was reflected in their derived LO. These data intriguingly suggest that CDCP-1 may change its expression pattern upon modifications in tumor cells properties, playing a different role in different stages of cancer development. Overall, these findings highlight LO as a new biological component in the development of aggressive features by prostate cancer cells, connecting molecular alterations to changes in the ability to interact with the surrounding environment, and suggested new prognostic markers and/or therapeutical targets. Citation Format: Chiara Ciardiello, Valentina R. Minciacchi, Mariana Reis-Sobreiro, Maria R. Milone, Biagio Pucci, Rita Lombardi, Francesca Bruzzese, Dolores Di Vizio, Alfredo Budillon. Large oncosomes derived from the aggressive prostate cancer sub-line, DU145R80, can modify the biological behavior of the parental DU145 cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4154. doi:10.1158/1538-7445.AM2015-4154

Herein we report the identification of amino acids of the Sulfolobus solfataricus mini-chromosome maintenance (MCM)-like DNA helicase (SsoMCM), which are critical for DNA binding/remodeling. The crystallographic structure of the N-terminal portion (residues 2–286) of the Methanothermobacter thermoautotrophicum MCM protein revealed a dodecameric assembly with two hexameric rings in a head-to-head configuration and a positively charged central channel proposed to encircle DNA molecules. A structure-guided alignment of the M. thermoautotrophicum and S. solfataricus MCM sequences identified positively charged amino acids in SsoMCM that could point to the center of the channel. These residues (Lys-129, Lys-134, His-146, and Lys-194) were changed to alanine. The purified mutant proteins were all found to form homo-hexamers in solution and to retain full ATPase activity. K129A, H146A, and K194A SsoMCMs are unable to bind DNA either in single- or double-stranded form in band shift assays and do not display helicase activity. In contrast, the substitution of lysine 134 to alanine affects only binding to duplex DNA molecules, whereas it has no effect on binding to single-stranded DNA and on the DNA unwinding activity. These results have important implications for the understanding of the molecular mechanism of the MCM DNA helicase action.

The archaeal replication apparatus appears to be a simplified version of the eukaryotic one with fewer polypeptides and simpler protein complexes. Herein, we report evidence that a Cdc6-like factor from the hyperthermophilic crenarchaea Sulfolobus solfataricus stimulates binding of the homohexameric MCM-like complex to bubble- and fork-containing DNA oligonucleotides that mimic early replication intermediates. This function does not require the Cdc6 ATP and DNA binding activities. These findings may provide important clues to understanding how the DNA replication initiation process has evolved in the more complex eukaryotic organisms.

In the present paper, we report that a Cdc6 (cell-division control)-like factor from the hyperthermophilic crenarchaeon Sulfolobus solfataricus (referred to as SsoCdc6-2) has a modular organization of its biological functions. A reliable model of the SsoCdc6-2 three-dimensional structure was built up, based on the significant sequence identity with the Pyrobaculum aerophylum Cdc6 (PaeCdc6), whose crystallographic structure is known. This allowed us to design two truncated forms of SsoCdc6-2: the ΔC (residues 1–297, molecular mass 35 kDa) and the ΔN (residues 298–400, molecular mass 11 kDa) proteins. The ΔC protein contains the nucleotide-binding Rossmann fold and the Sensor-2 motif (Domains I and II in the PaeCdc6 structure), and retains the ability to bind and hydrolyse ATP. On the other hand, the ΔN protein contains the C-terminal WH (winged helix)-fold (Domain III), and is able to bind DNA molecules and to inhibit the DNA helicase activity of the SsoMCM (mini-chromosome maintenance) complex, although with lesser efficiency with respect to the full-sized SsoCdc6-2. These results provide direct biochemical evidence that the Cdc6 WH-domain is responsible for DNA-binding and inhibition of MCM DNA helicase activity.

Cdc6 proteins play an essential role in the initiation of chromosomal DNA replication in Eukarya. Genes coding for putative homologs of Cdc6 have been also identified in the genomic sequence of Archaea, but the properties of the corresponding proteins have been poorly investigated so far. Herein, we report the biochemical characterization of one of the three putative Cdc6-like factors from the hyperthermophilic crenarchaeon Sulfolobus solfataricus (SsoCdc6-1). SsoCdc6-1 was overproduced in Escherichia coli as a His-tagged protein and purified to homogeneity. Gel filtration and glycerol gradient ultracentrifugation experiments indicated that this protein behaves as a monomer in solution (molecular mass of about 45 kDa). We demonstrated that SsoCdc6-1 binds single- and double-stranded DNA molecules by electrophoretic mobility shift assays. SsoCdc6-1 undergoes autophosphorylation in vitro and possesses a weak ATPase activity, whereas the protein with a mutation in the Walker A motif (Lys-59 --> Ala) is completely unable to hydrolyze ATP and does not autophosphorylate. We found that SsoCdc6-1 strongly inhibits the ATPase and DNA helicase activity of the S. solfataricus MCM protein. These findings provide the first in vitro biochemical evidence of a functional interaction between a MCM complex and a Cdc6 factor and have important implications for the understanding of the Cdc6 biological function.

The aminobiphosphonate Zoledronic acid (ZOL) inhibit osteoclast-mediated bone resorption and it is used to prevent skeletal complications from bone metastases. Accumulating evidences in both preclinical and clinical studies indicated that ZOL might also have anticancer activity. We have recently selected for the first time the ZOL-resistant DU145R80 prostate cancer (PCa) cell line that demonstrated an antiapoptotic and proangiogenic phenotype with increased invasive capability and epithelial to mesenchymal transition (EMT), compared to parental DU145 cells. Interestingly, we also demonstrated that both the acquired resistance to ZOL and the aggressive phenotype are mediated by p38-MAPK (Milone et al. Cell Death Dis. 2013, 4:e641). To further investigate the mechanism of ZOL-resistance and of the parallel acquisition of an aggressive phenotype in this novel syngeneic model of PCa, we took advantage of a two-dimensional difference gel electrophoresis (2D-DIGE)/mass spectrometry (MS) proteomic approach, to investigate differential expressed proteins between DU145R80 and DU145 cell lines. We found 21 statistically differentially expressed protein spots between the two cell lines (average volume ratio threshold of ±1.4-fold and 95% level of significance, p Citation Format: Maria Rita Milone, Biagio Pucci, Federica Iannelli, Rita Lombardi, Katia Bifulco, Francesca Bruzzese, Elena Di Gennaro, Antonio Avallone, Maria Vincenza Carriero, Alfredo Budillon. Proteomic characterization of zoledronic acid-resistant prostate cancer cells identified key proteins in cytoskeleton organization and cancer stem cell markers associated with a very aggressive phenotype. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5311. doi:10.1158/1538-7445.AM2014-5311

The nitrogen-containing bisphosphonates (N-BP) zoledronic acid (ZOL) inhibits osteoclast-mediated bone resorption, and it is used to prevent skeletal complications from bone metastases. ZOL has also demonstrated anticancer activities in preclinical models and, recently, in cancer patients, highlighting the interest in determining eventual mechanisms of resistance against this agent. In our study, we selected and characterised a resistant subline of prostate cancer (PCa) cells to better understand the mechanisms, by which tumour cells can escape the antitumour effect of ZOL. DU145R80-resistant cells were selected in about 5 months using stepwise increasing concentrations of ZOL from DU145 parental cells. DU145R80 cells showed a resistance index value of 5.5 and cross-resistance to another N-BP, pamidronate, but not to the non-nitrogen containing BP clodronate. Notably, compared with DU145 parental cells, DU145R80 developed resistance to apoptosis and anoikis, as well as overexpressed the anti-apoptotic protein Bcl-2 and oncoprotein c-Myc. Moreover, DU145R80 cells underwent epithelial to mesenchymal transition (EMT) and showed increased expression of the metalloproteases MMP-2/9, as well as increased invading capability. Interestingly, compared with DU145, DU145R80 cells also increased the gene expression and protein secretion of VEGF and the cytokines Eotaxin-1 and IL-12. At the molecular level, DU145R80 cells showed strong activation of the p38-MAPK-dependent survival pathway compared with parental sensitive cells. Moreover, using the p38-inhibitor SB203580, we completely reversed the resistance to ZOL, as well as EMT marker expression and invasion. Furthermore, SB203580 treatment reduced the expression of VEGF, Eotaxin-1, IL-12, MMP-9, Bcl-2 and c-Myc. Thus, for the first time, we demonstrate that the p38-MAPK pathway can be activated under continuous extensive exposure to ZOL in PCa cells and that the p38-MAPK pathway has a critical role in the induction of resistance, as well as in the acquisition of a more aggressive and invasive phenotype.

Vorinostat has shown preclinical and clinical effects in human cancers and it is the first HDI approved by FDA for cutaneous T-cell-lymphoma treatment. TG2 is a multifunctional enzyme that catalyze a Ca2+-dependent transamidating reaction resulting in covalent cross-links between proteins. TG2 may also act as a G-protein in transmembrane signalling, as well as a cell surface adhesion mediator. TG2 upregulation has been demonstrated in several cancers and its expression levels correlate with resistance to chemotherapy and metastatic potential. In the present study, we demonstrated that the antiproliferative effects of vorinostat is paralleled by the induction of both TG2 mRNA and protein expression in breast, colorectal and oral cancer cell lines. This effect was also shared by other pan-HDIs and by class I HDIs but not by the specific inhibitor of HDAC-6. Vorinostat-induced TG2 upregulation correlated with an increased transamidating activity. Confocal microscopy analysis confirmed TG2 induction as well as TG2 cytosolic aggregation upon vorinostat treatment. Compartment analysis of TG2 protein expression revealed that vorinostat induces cytosolic, membrane and cytoskeleton but not nuclear upregulation of TG2. Apparently this effect seems confined to tumor cells, since same results were not observed in ex vivo vorinostat-treated peripheral blood lymphocytes from healthy donors. Notably high basal levels of TG2 protein correlated with lower antiproliferative activity of vorinostat. In order to confirm that TG2 play a role in vorinostat antitumor effect, we silenced by specific shRNA TG2 expression in high TG2 expressing HT29 colorectal cancer cells, and overexpressed full length TG2 in low TG2 expressing MCF7 breast cancer cells. We demonstrated that in TG2 silenced cells vorinostat antiproliferative and proapoptotic effects were enhanced whereas in TG2 overexpressed cells they were impaired, demonstrating that TG2 is directly involved in the mechanism of antitumor effect exerted by vorinostat. Moreover cotreatment of tumor cells with two specific inhibitors of TG2 transamidating activity (KCC009 and monodansylcadaverine) potentiated the antitumor effect of vorinostat, suggesting that TG2 cross-linking activity is important for vorinostat antitumor effect. Vorinostat also induced TG2 acetylation, however this posttranslational modification has no relevant effect on protein half-life as demonstrated by cotreatment with cycloheximide. Finally, vorinostat-resistant MCF-7 cell line selected by stepwise increasing concentrations of vorinostat up to 12 μM, significantly overexpressed TG2 protein compared with parental control cell line. Overall this study demonstrated that TG2 overexpression is a common mechanism of intrinsic or acquired resistance to vorinostat and that inhibition of TG2 transamidating activity may potentiate vorinostat antitumor effect. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2609. doi:10.1158/1538-7445.AM2011-2609

// Maria Rita Milone 1, * , Biagio Pucci 1, * , Katia Bifulco 3 , Federica Iannelli 1 , Rita Lombardi 1 , Chiara Ciardiello 2 , Francesca Bruzzese 2 , Maria Vincenza Carriero 3 , Alfredo Budillon 1, 2 1 Centro Ricerche Oncologiche Mercogliano, Istituto Nazionale Tumori Fondazione G. Pascale - IRCCS, Naples, Italy 2 Experimental Pharmacology Unit, Istituto Nazionale Tumori Fondazione G. Pascale - IRCCS, Naples, Italy 3 Neoplastic Progression Unit, Istituto Nazionale Tumori Fondazione G. Pascale - IRCCS, Naples, Italy * These authors contributed equally to this work Correspondence to: Alfredo Budillon, e-mail: a.budillon@istitutotumori.na.it Keywords: prostate cancer, zoledronic acid, cytoskeleton organization, αv integrin, urokinase receptor (uPAR) Received: July 25, 2014 Accepted: November 04, 2014 Published: November 24, 2014 ABSTRACT Proteomic analysis identified differentially expressed proteins between zoledronic acid-resistant and aggressive DU145R80 prostate cancer (PCa) cells and their parental DU145 cells. Ingenuity Pathway Analysis (IPA) showed a strong relationship between the identified proteins within a network associated with cancer and with homogeneous cellular functions prevalently related with regulation of cell organization, movement and consistent with the smaller and reduced cell-cell contact morphology of DU145R80 cells. The identified proteins correlated in publically available human PCa genomic data with increased tumor expression and aggressiveness. DU145R80 exhibit also a clear increase of alpha-v-(αv) integrin, and of urokinase receptor (uPAR), both included within the same network of the identified proteins. Interestingly, the actin-rich structures localized at the cell periphery of DU145R80 cells are rich of Filamin A, one of the identified proteins and uPAR which, in turn, co-localizes with αv-integrin, in podosomes and/or invadopodia. Notably, the invasive feature of DU145R80 may be prevented by blocking anti-αv antibody. Overall, we unveil a signaling network that physically links the interior of the nucleus via the cytoskeleton to the extracellular matrix and that could dictate PCa aggressiveness suggesting novel potential prognostic markers and therapeutic targets for PCa patients.