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Background Anthracycline-mediated adverse cardiovascular events are among the leading causes of morbidity and mortality in cancer patients. Cardioprotective strategies in primary and secondary prevention are still needed in clinical practice to improve cancer patient survival an to avoid drug therapy discontinuation. Sodium-glucose cotransporter 2 (SGLT2) inhibitors exerts multiple cardiometabolic benefits in patients with/without type 2 diabetes, chronic kidney disease, and heart failure with reduced ejection fraction. We hypothesized that Dapagliflozin, administered before and during doxorubicin therapy, could improve cardiac function and reduce pro-necrotic pathways in preclinical models Methods Female C57Bl/6 mice were untreated (Sham, n=6) or treated for 10 days with doxorubicin i.p at 2.17 mg/kg (DOXO, n=6), DAPA at 12 mg/kg (DAPA, n=6) or doxorubicin combined to DAPA (DOXO-DAPA, n=6). Ejection fraction, radial and longitudinal strain were analyzed through transthoracic echocardiography (Vevo 2100). Cardiac tissue expression of NLRP3 inflammasome, Myd88, DAMPs (galectine 3 and calgranulinS100) and systemic chemokines (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL17-α, IL-18, IFN-γ, TNF-α, G-CSF, and GM-CSF) were quantified through ELISA methods. Immunohistochemical stains (IHC) of NF-kB was performed in heart and kidney tissues. Results DAPA improved significantly the EF and prevented the reduction of radial and longitudinal strain after 10 days of treatment with doxorubicin. A reduced expression of NLRP3, MyD88, DAMPs and NF-kB in cardiac tissues was seen in DOXO-DAPA group compared to DOXO mice (p Conclusion DAPA is able to improve cardiac function and reduce biomarkers involved in heart failure and fibrosis. IHC analysis clearly indicates anti-inflammatory properties of DAPA in cardiac and renal tissues. The overall picture of the study pushes the use of DAPA in primary prevention of cardiomyopathies induced by anthracyclines in cancer patients.

Background. The C-terminal-binding protein 1/brefeldin A ADP-ribosylation substrate (CtBP1/BARS) acts both as an oncogenic transcriptional co-repressor and as a fission inducing protein required for membrane trafficking and Golgi complex partitioning during mitosis, hence for mitotic entry. CtBP1/BARS overexpression, in multiple cancers, has pro-tumorigenic functions regulating gene networks associated with “cancer hallmarks” and malignant behavior including: increased cell survival, proliferation, migration/invasion, epithelial-mesenchymal transition (EMT). Structurally, CtBP1/BARS belongs to the hydroxyacid-dehydrogenase family and possesses a NAD(H)-binding Rossmann fold, which, depending on ligands bound, controls the oligomerization of CtBP1/BARS and, in turn, its cellular functions. Here, we proposed to target the CtBP1/BARS Rossmann fold with small molecules as selective inhibitors of mitotic entry and pro-tumoral transcriptional activities. Methods. Structured-based screening of drug databases at different development stages was applied to discover novel ligands targeting the Rossmann fold. Among these identified ligands, N-(3,4-dichlorophenyl)-4-{[(4-nitrophenyl)carbamoyl]amino}benzenesulfonamide, called Comp.11, was selected for further analysis. Fluorescence spectroscopy, isothermal calorimetry, computational modelling and site-directed mutagenesis were employed to define the binding of Comp.11 to the Rossmann fold. Effects of Comp.11 on the oligomerization state, protein partners binding and pro-tumoral activities were evaluated by size-exclusion chromatography, pull-down, membrane transport and mitotic entry assays, Flow cytometry, quantitative real-time PCR, motility/invasion, and colony assays in A375MM and B16F10 melanoma cell lines. Effects of Comp.11 on tumor growth in vivo were analyzed in mouse tumor model. Results. We identify Comp.11 as a new, potent and selective inhibitor of CtBP1/BARS (but not CtBP2). Comp.11 directly binds to the CtBP1/BARS Rossmann fold affecting the oligomerization state of the protein (unlike other known CtBPs inhibitors), which, in turn, hinders interactions with relevant partners, resulting in the inhibition of both CtBP1/BARS cellular functions: i) membrane fission, with block of mitotic entry and cellular secretion; and ii) transcriptional pro-tumoral effects with significantly hampered proliferation, EMT, migration/invasion, and colony-forming capabilities. The combination of these effects impairs melanoma tumor growth in mouse models. Conclusions. This study identifies a potent and selective inhibitor of CtBP1/BARS active in cellular and melanoma animal models revealing new opportunities to study the role of CtBP1/BARS in tumor biology and to develop novel melanoma treatments.

Supplementary Figure 1 from Synergistic antitumor effect between vorinostat and topotecan in small cell lung cancer cells is mediated by generation of reactive oxygen species and DNA damage-induced apoptosis

Supplementary Figure Legends 1-2 from Synergistic antitumor effect between vorinostat and topotecan in small cell lung cancer cells is mediated by generation of reactive oxygen species and DNA damage-induced apoptosis

The topoisomerase-I (topo-I) inhibitor topotecan, derivative of camptothecin, is the only registered drug for relapsed small cell lung cancer (SCLC). The histone deacetylase inhibitor vorinostat has shown preclinical and clinical antitumor activities in hematologic malignancies and solid tumors, including SCLC, and has recently been approved for the treatment of cutaneous T-cell lymphomas. In this study, we analyzed the antitumor effect of vorinostat combined with topotecan or camptothecin in topo-I inhibitor-sensitive H209 and inhibitor-resistant H526 SCLC cells. Simultaneous or sequential exposure (24 h delay) to either agent resulted in strong synergistic cytotoxic effect in both cell lines, as shown by calculating combination index, and confirmed by growth in soft agar. Combination treatments increased S-phase cell cycle arrest paralleled by apoptosis as measured by hypodiploid peak formation, Annexin V binding, DNA fragmentation, and mitochondria destruction. The apoptotic process was triggered by a caspase-dependent mechanism and can be ascribed to the phosphorylation of H2AX, a reporter of DNA double-strand breaks. These effects were paralleled by an increase of topo-I/DNA covalent complexes induced by combination treatment and suggest a potentiation by vorinostat of topotecan-induced DNA damage. Finally, oxidative injury played a significant functional role in the observed enhanced lethality because coadministration of the antioxidant N-acetyl-l-cysteine blocked reactive oxygen species generation, apoptosis, and mitochondria destruction induced by the vorinostat/topotecan combination. To our knowledge, this is the first demonstration of a synergistic antitumor effect between topotecan and vorinostat in SCLC. Because no well-established treatment is available for recurrent SCLC patients, our results indicate that this drug combination should be explored clinically. [Mol Cancer Ther 2009;8(11):3075–87]

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PDF file - 207KB, GDF15/MIC-1 is expressed by LNCaP prostate cancer cells and NIH-GDF15 fibroblasts.

PDF file - 114KB, GDF15/MIC-1 expression in prostate tumor tissue.

PDF file - 66KB, Recombinant GDF15/MIC-1 stimulates prostate cancer cell growth and invasion.

PDF file - 166KB, Supplementary Table 1. Human and murine primer.

PDF file - 157KB, Fibroblast-derived GDF15/MIC-1 stimulates cytokine production by LNCaP cells.

PDF file - 117KB, Prostate cancer cells, NIH-GDF15 fibroblasts and CAFs secrete human GDF15/MIC-1.

PDF file - 275KB, GDF15/MIC-1 serum levels correlate with "responder" tumor growth.

Anthracyclines are essential adjuvant therapies for a variety of cancers, particularly breast, and gastric and esophageal cancers. Whilst prolonging cancer-related survival, these agents can in-duce drug-related cardiotoxicity. Spirulina, Reishi (Ganoderma Lucidum) and Moringa are three nutaceuticals with anti-inflammatory effects that are currently used in cancer patients as Com-plementary and Alternative Medicines to improve quality of life and fatigue. We hypothesize that the nutraceutical combination of Spirulina, Reishi and Moringa (Singo) could reduce in-flammation and cardiotoxicity induced by anthracyclines. Female C57Bl/6 mice were untreated (Sham, n=6) or treated for 10 days with short-term doxorubicin i.p at 2.17 mg/kg (DOXO, n=6), Singo at 12 mg/kg (Singo, n=6) or doxorubicin combined to Singo (DOXO-Singo, n=6). Ejection fraction, radial and longitudinal strain were analyzed through transthoracic echocardiography (Vevo 2100, Fujifilm). Myocardial expression of NLRP3, DAMPs (galectine 3 and calgranu-linS100) and 13 cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL17-α, IL-18, IFN-γ, TNF-α, G-CSF, and GM-CSF) were quantified through selective mouse ELISA methods. Myocadial fi-brosis, necrosis and hypertrophy were analyzed through Immunohistochemistry (IHC). Human cardiomyocytes (HFC cell line) were exposed to subclinical concentration of doxorubicin (200 nM) alone or in combination to Singo (at 10, 25 and 50 µg/ml) for 24 and 48h. Cell viability studies were performed through MTS assay. Quantification of malondialdehyde and 4-hydroxynonenal were performed through spectrophotometric methods. Anti-inflammatory studies (expression of NLRP3 and p65/NF-kB) were made through selective ELISA methods. In-tracellular concentration of IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL17-α, IL-18, IFN-γ, TNF-α, G-CSF, and GM-CSF were also performed. In preclinical models of short-term DOXO cardiotoxicity, Singo improved significantly EF and FS and prevented the reduction of radial and longitudinal strain after 10 days of treatment with DOXO. A reduced expression of myocardial NLRP3 and NF-kB levels in cardiac tissues were seen in DOXO-Singo group compared to DOXO mice (p

Anthracyclines are essential adjuvant therapies for a variety of cancers, particularly breast, gastric and esophageal cancers. Whilst prolonging cancer-related survival, these agents can induce drug-related cardiotoxicity. Spirulina, Reishi (

Introduction: Anthracyclines are essential adjuvant therapies for a variety of cancers, particularly breast, and gastric and esophageal cancers. Whilst prolonging cancer-related survival, these agents can induce drug-related cardiotoxicity. This causes an increased risk of heart failure. Cardioprotective agents used to mitigate cardiotoxicity, such as angiotensin antagonists, angiotensin receptor blockers and beta‐blockers, are often poorly tolerated in these patients due to intravascular volume fluctuations, which are further escalated by the hemodynamic side effects of these agents. Spirulina, Reishi (Ganoderma Lucidum) and Moringa are three nutaceuticals with anti-inflammatory effects that are currently used in cancer patients as Complementary and Alternative Medicines to improve quality of life and fatigue.Purpose: We hypothesize that the nutraceutical combination of Spirulina, Reishi and Moringa (Singo) could reduce inflammation and cardiotoxicity induced by anthracyclines. Methods: Female C57Bl/6 mice were untreated (Sham, n=6) or treated for 10 days with short-term doxorubicin i.p at 2.17 mg/kg (DOXO, n=6), Singo at 12 mg/kg (Singo, n=6) or doxorubicin combined to Singo (DOXO-Singo, n=6). Ejection fraction, radial and longitudinal strain were analyzed through transthoracic echocardiography (Vevo 2100, Fujifilm). Myocardial expression of NLRP3, DAMPs (galectine 3 and calgranulinS100) and 13 cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL17-α, IL-18, IFN-γ, TNF-α, G-CSF, and GM-CSF) were quantified through selective mouse ELISA methods. Myocadial fibrosis, necrosis and hypertrophy were analyzed through Immunohistochemistry (IHC). Human cardiomyocytes (HFC cell line) were exposed to subclinical concentration of doxorubicin (200 nM) alone or in combination to Singo ( at 10, 25 and 50 µg/ml) for 24 and 48h. Cell viability studies were performed through MTS assay. Quantification of malondialdehyde and 4-hydroxynonenal were performed through spectrophotometric methods. Anti-inflammatory studies (expression of NLRP3 and p65/NF-kB) were made through selective ELISA methods. Intracellular concentration of IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL17-α, IL-18, IFN-γ, TNF-α, G-CSF, and GM-CSF were also performed. Results: In preclinical models of short-term DOXO cardiotoxicity, Singo improved significantly EF and FS and prevented the reduction of radial and longitudinal strain after 10 days of treatment with DOXO. A reduced expression of myocardial NLRP3 and NF-kB levels in cardiac tissues were seen in DOXO-Singo group compared to DOXO mice (p

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.

Trimethylamine N-oxide (TMAO) is a microbial metabolite derived from nutrients, such as choline, L-carnitine, ergothioneine and betaine. Recently, it has come under the spotlight for its close interactions with gut microbiota and implications for gastrointestinal cancers, cardiovascular disease, and systemic inflammation. The culprits in the origin of these pathologies may be food sources, in particular, high fat meat, offal, egg yolk, whole dairy products, and fatty fish, but intercalated between these food sources and the production of pro-inflammatory TMAO, the composition of gut microbiota plays an important role in modulating this process. The aim of this review is to explain how the gut microbiota interacts with the conversion of specific compounds into TMA and its oxidation to TMAO. We will first cover the correlation between TMAO and various pathologies such as dysbiosis, then focus on cardiovascular disease, with a particular emphasis on pro-atherogenic factors, and then on systemic inflammation and gastrointestinal cancers. Finally, we will discuss primary prevention and therapies that are or may become possible. Possible treatments include modulation of the gut microbiota species with diets, physical activity and supplements, and administration of drugs, such as metformin and aspirin.

INTRODUCTION: Metastatic pancreatic ductal adenocarcinoma (PDAC) represents an unmet clinical need because the lack of effective treatment strategies and a very poor prognosis. Here we propose a novel therapeutic strategy, based on the repurposing of valproic acid (VPA), a safe and generic drug with epigenetic modulating activity, and simvastatin (SIM), a widely used generic cholesterol lowering drug, in combination with the standard gemcitabine/nab-paclitaxel (GEM/NP) treatment in metastatic PDAC setting. METHODS: Synergistic anti-tumor effect of the combined treatment was assessed in PANC1, ASPC1, PANC28 and BxPC3 PDAC cell lines in vitro by evaluating combination index, apoptosis, clonogenic capability, tumor spheroids and fibroblasts/tumor cells microtissues growth. Antitumor effect was confirmed in vivo on heterotopic and orthotropic xenograft PANC1 models in athymic mice. Expression and functional role of TGF-β and downstream epithelial-to-mesenchymal-transition (EMT) markers were studied by Ingenuity pathway analysis (IPA), mRNA and protein expression and by cell migration scratch assay. RESULTS: We showed, both in vitro and in vivo, the ability of VPA/SIM combination, used at low dosages, to synergistically improve the anti-proliferative and pro-apoptotic effect of chemotherapy (GEM/NP). Mechanistically, VPA/SIM treatment, alone or in combination with chemotherapy, induced e-Cadherin and impaired vimentin and ZEB-1 expression, functionally linked to the synergistic inhibition of cell migration. In line, IPA highlighted a protein network connecting HDACs and HMGCR, the targets of VPA and SIM respectively, with the two main EMT markers. Notably, the most significantly cancer enriched features associated with this network were “migration of tumor cell lines”, “fibrosis” and “invasion of tumor cell lines”, and TGFβ emerged as a hierarchical dominant network- node. Indeed, VPA/SIM inhibited TGFβ transcription and TGFβ-regulated EMT gene expression in PDAC cells. Consistently we also observed a significant reduction of circulating TGFβ1 levels in mice treated with VPA/SIM in combination with GEM/NP, paralleled by a reduced fibrosis on PDAC xenograft tumor sections, confirming the involvement of TGFβ functional down-modulation in the mechanism of VPA/SIM antitumor synergistic interaction and chemo-sensitization. CONCLUSIONS: Overall, we proposed a novel combination strategy, based on two safe and generic drugs, able to sensitize a widely employed regimen in metastatic PDAC patients, that warrant further clinical evaluation. Citation Format: Maria Serena Roca, federica iannelli, maria rita milone, veronica barile, cristina testa, tania moccia, carlo vitagliano, fabiana tatangelo, di gennaro elena, antonio avallone, francesca bruzzese, alessandra leone, alfredo budillon. Repurposing of valproic acid and simvastatin in pancreatic cancer: in vitro and in vivo synergistic antitumor interaction and sensitization to gemcitabine/nab-paclitaxel via inhibition of TGFβ-EMT signaling pathway [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 1840.

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.

Recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) is a devastating malignancy with a poor prognosis. The combination of cisplatin (CDDP) plus cetuximab (CX) is one of the standard first-line treatments in this disease. However, this therapeutic regimen is often associated with high toxicity and resistance, suggesting that new combinatorial strategies are needed to improve its therapeutic index. In our study, we evaluated the antitumor effects of valproic acid (VPA), a well-known antiepileptic agent with histone deacetylase inhibitory activity, in combination with CDDP/CX doublet in head and neck squamous cell carcinoma (HNSCC) models. We demonstrated, in HNSCC cell lines, but not in normal human fibroblasts, that simultaneous exposure to equitoxic doses of VPA plus CDDP/CX resulted in a clear synergistic antiproliferative and pro-apoptotic effects. The synergistic antitumor effect was confirmed in four different 3D-self-assembled spheroid models, suggesting the ability of the combined approach to affect also the cancer stem cells compartment. Mechanistically, VPA enhanced DNA damage in combination treatment by reducing the mRNA expression of ERCC Excision Repair 1, a critical player in DNA repair, and by increasing CDDP intracellular concentration via upregulation at transcriptional level of CDDP influx channel copper transporter 1 and downregulation of the ATPAse ATP7B involved in CDDP-export. Valproic acid also induced a dose-dependent downregulation of epidermal growth factor receptor (EGFR) expression and of MAPK and AKT downstream signaling pathways and prevent CDDP- and/or CX-induced EGFR nuclear translocation, a well-known mechanism of resistance to chemotherapy. Indeed, VPA impaired the transcription of genes induced by non-canonical activity of nuclear EGFR, such as cyclin D1 and thymidylate synthase. Finally, we confirmed the synergistic antitumor effect also in vivo in both heterotopic and orthotopic models, demonstrating that the combined treatment completely blocked HNSCC xenograft tumors growth in nude mice. Overall, the introduction of a safe and generic drug such as VPA into the conventional treatment for R/M HNSCC represents an innovative and feasible antitumor strategy that warrants further clinical evaluation. A phase II clinical trial exploring the combination of VPA and CDDP/CX in R/M HNSCC patients is currently ongoing in our institute.

Background Recurrence with distant metastases has become the predominant pattern of failure in locally advanced rectal cancer (LARC), thus the integration of new antineoplastic agents into preoperative fluoropyrimidine-based chemo-radiotherapy represents a clinical challenge to implement an intensified therapeutic strategy. The present study examined the combination of the histone deacetylase inhibitor (HDACi) valproic acid (VPA) with fluoropyrimidine-based chemo-radiotherapy on colorectal cancer (CRC) cells. Methods HCT-116 (p53-wild type), HCT-116 p53−/− (p53-null), SW620 and HT29 (p53-mutant) CRC cell lines were used to assess the antitumor interaction between VPA and capecitabine metabolite 5′-deoxy-5-fluorouridine (5′-DFUR) in combination with radiotherapy and to evaluate the role of p53 in the combination treatment. Effects on proliferation, clonogenicity and apoptosis were evaluated, along with γH2AX foci formation as an indicator for DNA damage. Results Combined treatment with equipotent doses of VPA and 5′-DFUR resulted in synergistic effects in CRC lines expressing p53 (wild-type or mutant). In HCT-116 p53−/− cells we observed antagonist effects. Radiotherapy further potentiated the antiproliferative, pro-apoptotic and DNA damage effects induced by 5′-DFUR/VPA combination in p53 expressing cells. Conclusions These results highlighted the role of VPA as valuable candidate to be added to preoperative chemo-radiotherapy in LARC. On these bases we launched the ongoing phase I/II study of VPA and short-course radiotherapy plus capecitabine as preoperative treatment in low-moderate risk rectal cancer (V-shoRT-R3). Electronic supplementary material The online version of this article (10.1186/s13046-017-0647-5) contains supplementary material, which is available to authorized users.

// Chiara Ciardiello 1, * , Maria Serena Roca 1, * , Alessia Noto 1 , Francesca Bruzzese 1 , Tania Moccia 1 , Carlo Vitagliano 1 , Elena Di Gennaro 1 , Gennaro Ciliberto 2 , Giuseppe Roscilli 3 , Luigi Aurisicchio 3 , Emanuele Marra 3 , Rita Mancini 4 , Alfredo Budillon 1 , Alessandra Leone 1 1 Experimental Pharmacology Unit, Istituto Nazionale Tumori Fondazione G. Pascale - IRCCS, 80131 Naples, Italy 2 Scientific Direction, Istituto Nazionale Tumori Fondazione G. Pascale - IRCCS, 80131 Naples, Italy 3 Takis s.r.l., University, 00161 Rome, Italy 4 Department of Surgery "P.Valdoni” and Department of Clinical and Molecular Medicine, “La Sapienza” University, 00161 Rome, Italy * These author contributed equally to this work Correspondence to: Alfredo Budillon, e-mail: a.budillon@istitutotumori.na.it Keywords: NSCLC, HDAC inhibitor, ErbB3, valproic acid, primary tumor cultures Received: September 14, 2015 Accepted: January 23, 2016 Published: February 04, 2016 ABSTRACT ErbB3, a member of the ErbB family receptors, has a key role in the development and progression of several cancers, including non-small cell lung cancer (NSCLC), and in the establishment of resistance to therapies, leading to the development of anti-ErbB3 therapies. In this study we demonstrated, in a set of malignant pleural effusion-derived cultures of NSCLC, the synergistic antitumor effect of a histone deacetylase inhibitor (HDACi), such as vorinostat or valproic acid (VPA), in combination with the anti-ErbB3 monoclonal antibody (MoAb) A3. Synergistic interaction was observed in 2D and in 3D cultures conditions, both in fully epithelial cells expressing all ErbB receptors, and in cells that had undergone epithelial to mesenchymal transition and expressed low levels of ErbB3. We provided evidences suggesting that differential modulation of ErbB receptors by vorinostat or VPA, also at low doses corresponding to plasma levels easily reached in treated patients, is responsible for the observed synergism. In details, we showed in epithelial cells that both vorinostat and VPA induced time- and dose-dependent down-regulation of all three ErbB receptors and of downstream signaling. On the contrary, in A3-resistant mesenchymal cells, we observed time- and dose-dependent increase of mRNA and protein levels as well as surface expression of ErbB3, paralleled by down-regulation of EGFR and ErbB2. Our results suggest that the combination of a HDACi plus an anti-ErbB3 MoAb represents a viable strategy that warrants further evaluation for the treatment of NSCLC patients.

INTRODUCTION: Recurrent metastatic (R/M) squamous cell carcinoma of the head and neck (HNSCC) is a devastating malignancy with a poor prognosis and the combination of cisplatin (CDDP) plus cetuximab (CX) is one of the gold standard for first-line treatment. However, this therapy is often associated with toxicity and resistance, suggesting that new combinatorial strategies are needed to improve the therapeutic index of this regimen. In our study, we evaluated the synergistic antitumor effect of valproic acid (VPA), an anticonvulsant compound with histone deacetylase inhibitor (HDACi) activity, in combination with CDDP/CX in HNSCC models in vitro and in vivo. METHODS: Synergistic anti-proliferative effects were assessed on HNSCC Cal27, FaDu and Cal33 cell lines and BJ-hTERT normal fibroblast, by calculating combination index (CI) accordingly to Chou and Talalay method. Apoptosis was measured by flow cytometry analysis and caspase assay. Tumor spheroids were obtained by low attach systems and scored with luminescence 3D-cell viability assay. In vivo experiment was performed on xenograft models in athymic mice. RESULTS: We demonstrated, in HNSCC cells, but not in normal human fibroblasts, that simultaneous exposure to equitoxic doses of VPA plus CDDP/CX results in a clear synergistic antiproliferative and proapoptotic effect. Interestingly, in order to better recapitulate tumor growth complexity compared to 2D monolayers conditions, the synergistic antitumor effect was confirmed in four different 3D-self-assembled spheroid models, as well as in in vivo Cal27 xenograft model. Mechanistically, VPA induced a dose-dependent down-regulation of EGFR expression/activation affecting its downstream canonical pathway (pAKT and pMAPK), which plays a driver role in HNSCC. Moreover, we demonstrated that VPA was able to prevent the CDDP and/or CX induced EGFR nuclear translocation, a well-known mechanism of resistance to chemotherapy. Indeed, VPA impaired the transcription of cyclin D1 and DNA repair genes, regulated by non-canonical activity of nuclear EGFR, thus increasing DNA damage induced by CDDP/CX combination. Moreover, VPA was able to enhance the sensitivity to CDDP, by upregulating, at transcriptional level, the CDDP influx channel copper transporter 1 (CTR1). CONCLUSIONS: The introduction of a safe and generic drug such as VPA into the conventional treatment for R/M HNSCC, represents an innovative and feasible antitumor strategy that warrants further clinical evaluation. Indeed, we are currently enrolling patients in a phase-2 clinical trial in order to explore whether the addition of VPA to the standard combination CDDP/CX can increase the response rate in patients with R/M HNSCC. Citation Format: Federica Iannelli, Andrea Ilaria Zotti, Maria Serena Roca, Laura Grumetti, Tania Moccia, Carlo Vitagliano, Chiara Ciardiello, Francesca Bruzzese, Alessandra Leone, Francesco Caponigro, Franco Ionna, Francesco Longo, Elena Di Gennaro, Alfredo Budillon. Valproic acid, by preventing cisplatin/cetuximab-induced EGFR nuclear translocation and increasing cisplatin uptake, potentiates the antitumor effect of the combination treatment in head and neck squamous cell carcinomas [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 5223.

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.

The 5-fluorouracil/cisplatin (5FU/CDDP) combination is one of the most widely used treatment options for several solid tumors. However, despite good anticancer responses, this regimen is often associated with high toxicity and treatment resistance. In our study, we evaluated whether the histone deacetylase inhibitor (HDACi), vorinostat, may induce synergistic antitumor and proapoptotic effects in combination with 5FU/CDDP in squamous cancer cell models. We demonstrated in cancer cell lines, including the intrinsic CDDP-resistant Cal27 cells, that simultaneous exposure to equitoxic doses of vorinostat plus 5FU/CDDP results in strong synergistic antiproliferative and proapoptotic effects related to cell-cycle perturbation and DNA damage induction. These effects were confirmed in vivo in both orthotopic and heterotopic xenograft mouse models of Cal27 cells. Mechanistically, vorinostat reverted 5FU/CDDP-induced EGFR phosphorylation and nuclear translocation, leading to the impairment of nuclear EGFR noncanonical induction of genes such as thymidylate synthase and cyclin D1. These effects were exerted by vorinostat, at least in part, by increasing lysosomal-mediated EGFR protein degradation. Moreover, vorinostat increased platinum uptake and platinated DNA levels by transcriptionally upregulating the CDDP influx channel copper transporter 1 (CTR1). Overall, to our knowledge, this study is the first to demonstrate the ability of vorinostat to inhibit two well-known mechanisms of CDDP resistance, EGFR nuclear translocation and CTR1 overexpression, adding new insight into the mechanism of the synergistic interaction between HDACi- and CDDP-based chemotherapy and providing the rationale to clinically explore this combination to overcome dose-limiting toxicity and chemotherapy resistance.

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.

The tumor stroma is vital to tumor development, progression, and metastasis. Cancer-associated fibroblasts (CAF) are among the abundant cell types in the tumor stroma, but the range of their contributions to cancer pathogenicity has yet to be fully understood. Here, we report a critical role for upregulation of the TGFβ/BMP family member GDF15 (MIC-1) in tumor stroma. GDF15 was found upregulated in situ and in primary cultures of CAF from prostate cancer. Ectopic expression of GDF15 in fibroblasts produced prominent paracrine effects on prostate cancer cell migration, invasion, and tumor growth. Notably, GDF15-expressing fibroblasts exerted systemic in vivo effects on the outgrowth of distant and otherwise indolent prostate cancer cells. Our findings identify tumor stromal cells as a novel source of GDF15 in human prostate cancer and illustrate a systemic mechanism of cancer progression driven by the tumor microenvironment. Further, they provide a functional basis to understand GDF15 as a biomarker of poor prognosis and a candidate therapeutic target in prostate cancer. Cancer Res; 74(13); 3408–17. ©2014 AACR.

// Manuela Terranova-Barberio 1 , Maria Serena Roca 1 , Andrea Ilaria Zotti 1 , Alessandra Leone 1 , Francesca Bruzzese 1 , Carlo Vitagliano 1 , Giosue Scogliamiglio 2 , Domenico Russo 3 , Giovanni D’Angelo 3 , Renato Franco 2 , Alfredo Budillon 1 , Elena Di Gennaro 1 1 Experimental Pharmacology Unit, Istituto Nazionale Tumori Fondazione G. Pascale - IRCCS, Naples, Italy 2 Pathology Unit, Istituto Nazionale Tumori Fondazione G. Pascale - IRCCS, Naples, Italy 3 Institute of Protein Biochemistry, National Research Council, Naples, Italy Correspondence to: Alfredo Budillon, e-mail: a.budillon@istitutotumori.na.it Keywords: HDAC inhibitor, valproic acid, thymidine phosphorylase, breast cancer, capecitabine Received: July 27, 2015 Accepted: December 23, 2015 Published: December 31, 2015 ABSTRACT The prognosis of patients with metastatic breast cancer remains poor, and thus novel therapeutic approaches are needed. Capecitabine, which is commonly used for metastatic breast cancer in different settings, is an inactive prodrug that takes advantage of elevated levels of thymidine phosphorylase (TP), a key enzyme that is required for its conversion to 5-fluororacil, in tumors. We demonstrated that histone deacetylase inhibitors (HDACi), including low anticonvulsant dosage of VPA, induced the dose- and time-dependent up-regulation of TP transcript and protein expression in breast cancer cells, but not in the non-tumorigenic breast MCF-10A cell line. Through the use of siRNA or isoform-specific HDACi, we demonstrated that HDAC3 is the main isoform whose inhibition is involved in the modulation of TP. The combined treatment with capecitabine and HDACi, including valproic acid (VPA), resulted in synergistic/additive antiproliferative and pro-apoptotic effects in breast cancer cells but not in TP-knockout cells, both in vitro and in vivo , highlighting the crucial role of TP in the synergism observed. Overall, this study suggests that the combination of HDACi (e.g., VPA) and capecitabine is an innovative antitumor strategy that warrants further clinical evaluation for the treatment of metastatic breast cancer.

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.

Potentiation of epidermal growth factor receptor (EGFR) inhibitors is required in squamous cell carcinoma of head and neck (SCCHN) to improve their therapeutic index. We demonstrated that the histone deacetylase inhibitor vorinostat in combination with the EGFR tyrosine kinase inhibitor gefitinib induced synergistic inhibition of proliferation, migration, and invasion as well as induction of apoptosis in SCCHN cells, including cells resistant to gefitinib. We provided evidence suggesting that differential modulation of ErbB receptors together with reversion of epithelial-to-mesenchymal transition (EMT) by vorinostat represent mechanistic bases for the observed synergism. We demonstrated in epithelial CAL27 cells expressing EGFR, ErbB2, and ErbB3 that vorinostat downregulated the expression and signaling of all three receptors. In gefitinib-resistant KB and Hep-2 cells, both of which had undergone EMT and expressed very low levels of ErbB3, vorinostat reverted the mesenchymal phenotype by inducing both E-cadherin and ErbB3 and downregulating vimentin as well as EGFR and ErbB2. Both transcriptional and post-translational mechanisms were involved in the modulation of ErbB receptors by vorinostat. Attenuation of all ErbB transcripts in CAL27 cells as well as induction of ErbB3 transcript in Hep-2 and KB cells was seen upon vorinostat treatment. We showed that vorinostat induced ubiquitination of EGFR and ErbB2 and targeted them predominantly to lysosome-degradation in all cell lines, while the induction of ErbB3-ubiquitination in CAL27 cells led to proteasomes-degradation. Overall, this study suggests that the vorinostat/gefitinib combination represents a promising therapeutic strategy that warrants further clinical evaluation in SCCHN, including tumors intrinsically resistant to EGFR-inhibitors. J. Cell. Physiol. 226: 2378–2390, 2011. © 2010 Wiley-Liss, Inc.

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 topoisomerase-I (topo-I) inhibitor topotecan, derivative of camptothecin, is the only registered drug for relapsed small cell lung cancer (SCLC). The histone deacetylase inhibitor vorinostat has shown preclinical and clinical antitumor activities in hematologic malignancies and solid tumors, including SCLC, and has recently been approved for the treatment of cutaneous T-cell lymphomas. In this study, we analyzed the antitumor effect of vorinostat combined with topotecan or camptothecin in topo-I inhibitor-sensitive H209 and inhibitor-resistant H526 SCLC cells. Simultaneous or sequential exposure (24 h delay) to either agent resulted in strong synergistic cytotoxic effect in both cell lines, as shown by calculating combination index, and confirmed by growth in soft agar. Combination treatments increased S-phase cell cycle arrest paralleled by apoptosis as measured by hypodiploid peak formation, Annexin V binding, DNA fragmentation, and mitochondria destruction. The apoptotic process was triggered by a caspase-dependent mechanism and can be ascribed to the phosphorylation of H2AX, a reporter of DNA double-strand breaks. These effects were paralleled by an increase of topo-I/DNA covalent complexes induced by combination treatment and suggest a potentiation by vorinostat of topotecan-induced DNA damage. Finally, oxidative injury played a significant functional role in the observed enhanced lethality because coadministration of the antioxidant N-acetyl-l-cysteine blocked reactive oxygen species generation, apoptosis, and mitochondria destruction induced by the vorinostat/topotecan combination. To our knowledge, this is the first demonstration of a synergistic antitumor effect between topotecan and vorinostat in SCLC. Because no well-established treatment is available for recurrent SCLC patients, our results indicate that this drug combination should be explored clinically. [Mol Cancer Ther 2009;8(11):3075–87]

In non-small-cell lung cancer (NSCLC) patients, the activation of alternative pathways contributes to the limited efficacy of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib. The present study examines a panel of EGFR wild-type, K-Ras mutated, NSCLC lines, which were all intrinsically resistant to EGFR-TKIs, and demonstrates that the histone deacetylase inhibitor vorinostat can improve the therapeutic efficacy of gefitinib or erlotinib, inducing strong synergistic antiproliferative and pro-apoptotic effects that are paralleled by reactive oxygen species accumulation and by increased DNA damage. By knockdown experiments, we suggested that the up-regulation of voltage-dependent anion-selective channel protein 1 (VDAC1), the major mitochondrial porin of the outer mitochondrial membrane, which was induced by vorinostat and further increased by the combination, could be functionally involved in oxidative stress-dependent apoptosis. Significantly, we also observed the attenuation of the expression of both the enzyme hexokinase1, a negative VDAC1 regulator, and the anti-apoptotic porin VDAC2, only in the combination setting, suggesting convergent mechanisms that enhanced mitochondria-dependent apoptosis by targeting VDAC protein functions. Furthermore, the prosurvival capacities of the cells were also inhibited by the combination treatments, as shown by complete pAKT deactivation, increased GSK3β expression, and c-Myc down-regulation. Finally, we observed that the combination treatment of vorinostat and either of the EGFR-TKIs induced the down-regulation of the c-Myc-regulated nuclear factor erythroid 2-related factor 2 (NRF2) transcription factor and the up-regulation of the NRF2 repressor Kelch-like ECH-associated protein 1 regulator (KEAP1). These two genes are crucial for the redox stress response, often dysfunctional in NSCLC, and involved in EGFR-TKI resistance. Taken together, these results are the first to demonstrate that altering redox homeostasis is a new mechanism underlying the observed synergism between vorinostat and EGFR TKIs in NSCLC.