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Abundant preclinical evidences indicate that stress-induced autophagy in tumor cells is predominantly cytoprotective and that inhibition of autophagy can enhance tumor cell death by diverse anticancer therapies. A major negative regulator of autophagy is the mammalian target of rapamycin (mTOR), activated downstream of PI3KAKT pathway. mTOR inhibitors, including rapamycin, have been shown to induce autophagy in tumor cells, while the combination of PI3K-AKT/mTOR and autophagy inhibitors shown synergistic effect on increased apoptosis and reduced autophagy (Takeuchi H. 2005). This project aimed to characterize the role of autophagy in Breast Cancer models exposed to new potent Genentech PI3K/AKT inhibitors GDC0068 (Ipatasertib) and GDC0032 (Taselisib) currently in phase III clinical trials on TNBC and ER+ patients. However, the efficacy of PI3K/AKT inhibitors may be limited by resistance mechanisms that result in minimal cell death in tumor cells. In order to investigate the role of autophagy as possible mechanisms of resistance, Ipatasertib and Taselisib have been evaluated in breast cancer cell lines characterized by different receptors profile: TNBC, HER2/c-erb-2 and luminal A cell lines. Our results showed that both drugs are able to induce G1/S cell cycle block and increase of autophagy signaling measured by p62 level and LC3 II/LC3 I ratio and by the percentage of cells exhibiting LC3 positive puncta. In addition, apoptosis increase was also evaluated by measuring positive annexin V staining, by flow cytometry technique, and apoptosis markers, such as PARP and cleaved caspase 3, by immunoblot assay. Furthermore, the supplement of pharmacologic inhibitors of autophagy, such as hydroxichloroquine, were able to reduce cell viability evaluated by both short- and long-term assays in cell lines exposed to Ipatasertib and Taselisib. 3D breast cancer models are ongoing to confirm the synergic effect of PI3K/AKT inhibitors and hydroxychloroquine, in order to provide a new therapeutic combinatorial approach potentially translatable to patients. Citation Format: Cocco S, Leone A, Buonfanti G, Sisalli MJ, Budillon A, De Laurentiis M. Role played by autophagy in breast cancer models exposed to new PI3K/AKT inhibitors, GDC-0068 and GDC-0032 [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-07-09.

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.

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 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.

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.

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.

In metastatic colorectal cancer (mCRC) patients (pts), treatment strategies integrating liver resection with more effective therapies offer better 5-year survival rates than palliative chemotherapy alone. However, resectability is established only on clinical-morphovolumetric criteria, liver resection is a complex and costly procedure and relapse occurs in almost 2/3 of pts after potentially curative resection. Therefore, prompt identification of pts at higher risk of recurrence is critical to avoid not-beneficial, expensive procedures. Aberrant metabolism is an emerging hallmark of cancer and recent observations suggest that specific metabolic changes can be used to stratify pts for prognosis and drug-response. We evaluated by 600MHz NMR spectroscopy the metabolomics profiling on sera from 30 mCRC pts, enrolled in the Obelics trial (NCT01718873), which investigated different schedules of bevacizumab in combination with oxaliplatin plus fluoropyrimidines regimens, and subdivided on the basis of outcome, in good (R) vs bad responders (NR) according to PFS: 12 months or longer (R, n = 12) and shorter than 12 months (NR, n = 18). We compared the samples of the two mCRC pts groups, collected at response evaluation when resectability was established in case of appropriate tumor reduction and PCA, sPLS-DA and loading plots evidenced metabolites with statistically different levels between the two sub-groups. ROC curves were performed to identify the cutoff levels of these significant metabolites to be correlated with patient survival. In this way we demonstrated that low levels of 3-hydroxybutyrate and of hydroxyproline as well as high levels of histidine correlated with both poor progression free survival (PFS) and overall survival (OS). Notably, either 3-hydroxybutyrate or histidine are better predictor of both PFS and OS compared to pathologic response on resected metatastases. Lipidomics analysis confirmed clear differences between R and NR pts indicating statistically significant increase of lipids in NR pts, with both higher triglycerides and phospholipids correlating with poor PFS and OS. This latter effects, may reflect, at least in part a non specific inflammatory response; indeed a significant increase of pro-inflammatory cytokines was also demonstrated in NR pts sera by cytokinomics using multiplex ELISA approach. Finally, basal serum metabolomics analysis in both NR and R pts demonstrated that on-treatment evaluation is more informative than pre-treatment evaluation to stratify patients for outcome. Overall, these data suggest that NMR-based metabolomics is a potent and affordable method that could play a role in the prediction of mCRC outcome. Citation Format: Alfredo Budillon, Susan Costantini, Angela Sorice, Francesca Capone, Silvia Marchese, Elena Di Gennaro, Carlo Vitagliano, Fabiana Tatangelo, Alfonso De Stefano, Franco Bianco, Paolo Delrio, Francesco Izzo, Antonio Avallone. Outcome prediction of metastatic colorectal cancer patients undergoing liver resection by analyzing serum metabolomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5268.

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.

This abstract was not presented at the symposium.

Recurrent metastatic (R/M) squamous cell carcinoma of the head and neck (SCCHN) is a devastating malignancy with a poor prognosis and the combination of cisplatin (CDDP) and cetuximab (CX) is one of the gold standard for treatment. However, this therapy is often associated with high toxicity and resistance, suggesting that the concomitant use of novel compounds represents a critical strategy to improve treatment results. Histone deacetylase inhibitors (HDACi) enhance the activity of several anticancer drugs including CDDP and anti-Epidermal Growth Factor Receptor (EGFR) compounds. EGFR overexpression is involved in chemotherapy resistance and recently it has been demonstrated that EGFR nuclear localization represents one of the mechanism of resistance to both CDDP and CX. Our group have demonstrated that HDACi synergize with anti-EGFR agents by modulating ErbB receptors expression including EGFR. In this study we evaluated the capability of Valproic acid (VPA), a generic low-cost anticonvulsivant drug with HDACi activity, to enhance the efficacy of CDDP-CX association in SCCHN cells with different EGFR basal expression and genetic backgrounds. We showed, by the calculation of combination indexes, an antiproliferative synergistic interaction between equipotent doses of VPA and CDDP-CX in CAL27 and FaDu SCCHIN cells using either simultaneous or sequential (with 24 h delay) schedule of administration. Notably, this effect was not observed in immortalized human fibroblasts BJ-hTERT, suggesting a selective effect toward tumor cells. Next, we confirmed synergistic interaction by testing clinically achievable concentrations of VPA/ CDDP-CX combination also on CAL27 and FaDu 3D spheroid cultures, showing inhibition of first- and second-generation spheroids formation by triple combination, compared to single agent treatments. Moreover we demonstrated that VPA was able to down-regulate EGFR and to prevent EGFR nuclear translocation induced by CDDP and CCDDP/CX combination, thus preventing the activation of DNA repair and survival pathways. Indeed, we showed that VPA was able to increase DNA damage and apoptosis induced by CDDP-CX combination, evaluated by gamma H2AX foci and annexin V-FITC assay, respectively. In vivo preclinical study is currently ongoing. All together these data represent the rationale of the ongoing V-CHANCE phase-2 clinical trial evaluating VPA/ CDDP-CX combination in R/M SCCHN patients. We are also currently investigating pharmacodynamics/predictive biomarkers of treatment efficacy/resistance on patient’s samples. Citation Format: Alfredo Budillon, Andrea I. Zotti, Carlo Vitagliano, Elena Di Gennaro, Francesco Caponigro. Preclinical and phase-2 clinical study of valproic acid administered in combination with cisplatin and cetuximab in recurrent/metastatic head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4058. doi:10.1158/1538-7445.AM2017-4058

One of the most common traits of cancer is the radical change of cellular metabolism. A distinctive aspect of this altered metabolic status is the presence of a larger pool of monounsaturated fatty acids (MUFA), the precursors of the components of cell membranes necessary to sustained the rapidly dividing cancer cells. MUFA are largely generated from saturated fatty acids by the action of the two Stearoyl-CoA desaturases isoforms, namely SCD1 and SCD5. Recent evidences suggest that the major isoform SCD1 plays a role in several cancers. Our group has previously reported, using primary adenocarcinoma cell lines from malignant pleural effusions, that SCD1 inhibition selectively kills ALDH positive cells, a marker of cancer stem cells, causes spheroid 3D cultures collapse in vitro and impairs their growth in vivo. These results suggest that SCD1 may be a critical target in lung cancer tumor-initiating cells and further studies are needed to assess the role of this enzyme in lung cancer. An increasing number of literature points to the Hippo (and their effectors YAP/ TAZ) and to the Wnt/β-catenin signaling pathways as key factors in the development of cancer. Notably, SCD1 has been shown to be involved in the secretion and maturation of active wnt ligands, contributing to the activation of the β-catenin signaling. We report here, using primary cell cultures from adenocarcinoma of the lung, that YAP and TAZ are required for the generation of 3D cultures, as silencing of both genes completely abrogated the capability of generating lung cancer spheroids. Moreover, simultaneously silencing of YAP and TAZ reduced the mRNA levels of OCT4, Nanog and ALDH. We demonstrated that both silencing of SCD1 and its pharmacological inhibition determined a decrease in the expression and nuclear localization of both YAP and TAZ. Moreover, when SCD1 was inhibited, YAP and TAZ transcriptional activity was reduced. We also demonstrated that SCD1 blockade induced a dramatic reduction of the Axin2 mRNA level, one of the β-catenin target genes and a decreased in the β-catenin transcriptional activity, without affecting β-catenin protein expression. YAP and TAZ downregulation induced by SCD1 blockade could be rescued by the addition of exogenous wnt3a ligand, thus indicating that the active β-catenin signaling is necessary for YAP and TAZ stabilization. Ultimately, we observed that the ubiquitin ligase β-TrcP is involved in the SCD1 mediated degradation of YAP and TAZ. These data, overall, demonstrate for the first time the involvement of SCD1 in the regulation of the Hippo pathway and β-catenin pathway in lung cancer 3D spheroid cultures, pointing to lipid metabolism as regulator of cancer stem features. Moreover, these results can be the starting point for further studies focused on the regulation of SCD1 in cancer stem cells, suggesting a new perspective for improving chemotherapeutic responses in cancer treatment, centered on SCD1 inhibition. Citation Format: Alessia Noto, Maria Elena Pisanu, Claudia De Vitis, Giovanni Sorrentino, Giannino Del Sal, Alfredo Budillon, Gennaro Ciliberto, Rita Mancini. Stearoyl-CoA-Desaturase (SCD1) regulates lung cancer stemness via stabilization and nuclear localization of YAP/TAZ. [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 1052.

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.

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

We have recently demonstrated that the histone deacetylase-inhibitor (HDACi) vorinostat induces synergistic antitumour effects in combination with capecitabine by up-regulating, in vitro and in vivo, in colorectal cancer cells but not in ex vivo treated peripheral blood lymphocytes, the mRNA and protein expression of thymidine phosphorylase (TP), the key enzyme converting capecitabine to 5-FU (Di Gennaro, Brit J Cancer 2010). We confirmed a time and dose-dependent induction of TP mRNA and protein expression by several other HDACi, including valproic acid (VPA). We investigated potential antitumor interaction between capecitabine metabolite 5′-deoxy-5-fluorouridine (5′-DFUR) and several HDACi showing synergistic/additive antiproliferative and pro-apoptotic effects in all cancer cells tested, with good results with VPA. Interestingly, TP protein induction is achieved also at low doses of VPA (0.5-1 mM), corresponding to a plasma level between 50 and 100 μg/ml, easily reached in patients with normal anticonvulsant doses. Although at these doses VPA did not induce growth inhibition as single agents, a significant synergistic antitumor effect was still demonstrated in combination with 5′-DFUR, suggesting a specific mechanism of interaction. TP knockdown experiments confirmed a crucial role of TP protein modulation in the observed synergism. Radiotherapy further potentiated in colorectal cancer cells the antiproliferative, pro-apoptotic and DNA damage effects induced by 5′-DFUR/VPA combination, as demonstrated by clonogenic assay, Caspase-3 cleavage and γH2AX foci formation, respectively. On these bases we launched a phase I/II clinical study (V-ShoRT-R3 trial) to explore whether the addition of both VPA and capecitabine to short-course radiotherapy (SCRT) before optimal radical surgery, might increase the pathologic complete tumor regression rate in low-moderate risk rectal cancer patients (ClinicalTrials.gov number NCT01898104). Several biomarkers will be evaluated comparing normal mucosa with tumor and on blood samples. Tumor metabolism will be measured by 18FDG-PET at baseline and 11 days after the beginning of SCRT. Currently phase I clinical study is ongoing. We have also optimized a protocol to evaluate histones and proteins acetylation in peripheral blood mononuclear cells of recruited patients by flow cytometry, as pharmacodynamic/predictive specific marker of VPA HDACi activity and preliminary results will be presented. Citation Format: Manuela Terranova Barberio, Biagio Pecori, Serena Imbimbo, Alessandra Leone, Francesca Bruzzese, Maria Carmela Piccirillo, Paolo Delrio, Franco Bianco, Luigi Aloj, Antonio Sorrentino, Fabiana Tatangelo, Antonella Petrillo, Secondo Lastoria, Paolo Muto, Francesco Perrone, Antonio Avallone, Alfredo Budillon, Elena Di Gennaro. Synergistic antitumor interaction between valproic acid, capecitabine and radiotherapy in colorectal cancer as a rationale for the innovative V-shoRT-R3 trial in locally advanced rectal cancer patients. [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 2569. doi:10.1158/1538-7445.AM2015-2569

In the last years several evidences suggested that ErbB3, a member of the HER family receptors, has a key role in the development and progression of several cancers including non-small cell lung cancer (NSCLC), and above all in the establishment of resistance to therapies, leading to major efforts towards the development of anti-ErbB3 therapies. We recently demonstrated in head and neck cancer cells that, depending on the ErbB3 expression level and on the tumor cell phenotype (epithelial vs mesenchymal), vorinostat, one of the two clinically approved histone deacetylase inhibitors (HDACi), differentially regulates HER receptors expression at the transcriptional level and/or by modulating protein degradation (Bruzzese F. et al. J Cell Physiol. 2011; 226(9):2378-90). Our group has developed a monoclonal antibody against ErbB3 called A3, that induces receptor internalization and degradation, inhibits growth and induces apoptosis only in cells overexpressing surface ErbB3 and potentiates the efficacy of EGFR TKIs (Noto A. et al. Oncotarget. 2013; 4(8):1253-65). In this study we first show, by using a set of malignant pleural effusion derived cell cultures from NSCLC patients (Mancini R. et al. PLOSone 2011; 6(7):e21320) that the combination of the anti-ErbB3 antibody A3 with HDACi such as vorinostat or valproic acid (VPA), synergistically affect cell proliferation and induce apoptosis. Interestingly synergistic interaction was observed in both fully epithelial cells expressing all HER receptors including ErbB3, as well as in NSCLC cells that had undergone EMT and expressed very low levels of ErbB3. We provide evidences suggesting that differential modulation of ErbB receptors by HDACi is responsible for the observed synergism. We show in two epithelial cells expressing EGFR, ErbB2, and ErbB3 that either vorinostat or VPA time- and dose-dependent down-regulation the of all three receptors expression and signaling. On the contrary, in two A3-resistant mesenchymal cells expressing undetectable levels of ErbB3, we observe time- and dose-dependent increase of mRNA and protein levels as well as surface expression of ErbB3, paralleled by down-regulation of EGFR, ErbB2 and the the mesenchymal marker vimentin. Interestingly, ErbB3 induction was achieved also at low doses of both vorinostat and VPA, corresponding to a plasma level easily reached in patients treated with these agents. Our results suggest that the combination treatment of antibodies against ErbB3 and HDACi represents an attractive strategy that warrant further evaluation, even in combination with other agents, for the treatment of NSCLC patients. Citation Format: Chiara Ciardiello, Alessandra Leone, Francesca Bruzzese, Maria Serena Roca, Alessia Noto, Claudia De Vitis, Luigi Aurisicchio, Gennaro Ciliberto, Rita Mancini, Alfredo Budillon. Modulation of ErbB receptors expression by histone deacetylase inhibitors increased the antitumor activity of an anti-ErbB3 monoclonal antibody in primary cultures from non-small cell lung cancer patients. [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 5444. doi:10.1158/1538-7445.AM2014-5444

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

Objectives : We have shown that pathological regression of LARC and relapse free survival can be predicted by early changes in FDG uptake during preoperative radiochemotherapy. We have attempted to improve efficacy of our neo-adjuvant treatment by adding an experimental bevacizumab (BEV) schedule. FDG PETCT was utilized to monitor response and correlate with pathological outcome. Methods : Forty six patients with poor prognosis LARC received 3 biweekly courses of oxaliplatin/ raltitrexed (day 1) and FU/ folinic acid (day 2) during pelvic RT (45 Gy). BEV (5 mg/kg) was administered on day -4 for the first 2 courses. PET-CT was performed at baseline, on day 12 and prior to surgery and lesion TLG (mean SUV x volume) values determined. Pathologic response was defined using a modified Mandard tumor regression grade (TRG) scale. Responders were defined as TRG1-2, non-responders as TRG3-4. Results : Thirty-seven patients (80%) showed pathologic response (65% with previous scheme). Eight patients (17%, previously 35%) were non-responders. Responders showed significantly higher TLG reduction on day 12 PET-CT (median -72% range -90%+31%) compared to non-responders (TRG3-4, median -38%, range -45% +25%, p < 0.05), confirming previous findings. Preoperative PET-CT studies, on the other hand, were not predictive of pathologic response (responders TLG change median -92%, range -100% +4%, non-responders median 86%, range -94%-76%, p = ns). Ongoing studies to define a correlation between FDG uptake and biomarkers of angiogenesis such as Circulating endothelial cells (CEC) will be presented. Conclusions : Our findings indicate that the current scheme significantly increases pathologic response. PET-CT confirms to be a powerful early surrogate marker of treatment efficacy. PET-CT may be used to tailor more aggressive treatment for non-responsive patient Citation Format: Antonio Avallone, Paolo Delrio, Biagio Pecori, Fabiana Tatangelo, Elena Di Gennaro, Antonella Petrillo, Vincenzo Rosario Iaffaioli, Alessandra Leone, Corradina caracò, Alfredo Budillon, Secondo Lastoria, Luigi Aloj. Predictive role of FDG PET-CT in monitoring locally advanced rectal cancer (LARC) during preoperative radiochemotherapy with an experimental bevacizumab schedule. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4695. doi:10.1158/1538-7445.AM2013-4695

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL BACKGROUND: The benefits of combining bevacizumab (BEV) plus chemotherapy have thus far been rather modest, stimulating interest in developing novel effective combination schedule as well as valid predictive biomarkers. Several evidences support the hypothesis that BEV can normalize the abnormal tumor vasculature, resulting in more efficient delivery of drugs and oxygen to cancer cells and that this effect seems to be transient and with a relatively narrow therapeutic window. In BRANCH study ([NCT01481545][1]) we assess the safety and efficacy of an experimental schedule of early (4 days before) BEV added to neoadjuvant chemotherapy (CT) and radiotherapy (RT) in poor-risk locally advanced rectal cancer (pLARC) patients (pts) and explore the potential of circulating endothelial cells (CECs) and tumor lesion glycolysis (TLG) as surrogate markers of pathological response. PATIENTS AND METHODS: 46 pts (cT4, cN+, cT3≤ 5 cm from the anal verge and/or positive circumferential margin, M1 resectable) received 3 biweekly courses of oxaliplatin (100 mg/m2)/raltitrexed (2.5 mg/m2) on day 1, and 5-FU (800 mg/m2)/folinic acid (250 mg/m2) on day 2 during pelvic RT (45 Gy). BEV (5 mg/kg) was given biweekly 4 days before beginning of CT/RT for 2 courses. Toxicity was graded with NCI-CTC v.3. Pathological response was defined using a modified Mandard tumor regression grade (TRG). According to the Simon's two-stage design, assuming an hypothesis of a 50% TRG1 (complete tumor regression) (α error=0.05, β error=0.20), at least 6/16 TRG1 should be obtained to continue accrual to 46 pts. CECs were quantified at baseline (BL) before BEV, at several time points thereafter during treatment and before surgery, by flow cytometry. TLG, was evaluated at BL, on day 10 and before surgery, by FDG-PET. Statistical analysis was by Mann-Whitney test. RESULTS: TRG1 required by statistical design was reached in the first 16 pts and in the final 46 pts: 23 TRG1 (50%), 14 TRG2 (30%) and 8 TRG3-4 (17%). One pt refused surgery. Grade ¾ neutropenia was the most common adverse event (13/46 pts, 28%). TLG reduction on day 10 vs BL was significantly higher in responders TRG1-2 compared to non-responders TRG3-4 pts (median -72%, range -90%+31% vs -38%, range -45%+25%; p < 0.05). Median CECs at BL were higher in TRG1-2 vs TRG3-4 pts (median 0.22/µl, range 0-3.98 vs 0/µl, range 0-0.174; p=0.009). Moreover, in TRG1-2 pts CECs were significantly reduced on day 10 vs BL (median 0.014/µl, range 0-2.29; p=0.002). This pattern was not seen in TRG3-4 pts with a tendency toward increased levels (median 0.316/µl, range 0-2.64; p=0.097). CONCLUSIONS: Early schedule of BEV plus CT and RT appears safe and active yielding high rate of TRG1 and TRG2 responses in pLARC. Early FDG-PET and CECs evaluation emerged as potential biomarkers for treatment selection to be incorporated in design of future studies with this regimen. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-219. doi:1538-7445.AM2012-LB-219 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01481545&atom=%2Fcanres%2F72%2F8_Supplement%2FLB-219.atom

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

Capecitabine is a pro-drug which was designed to take advantage of the increased levels of thymidine phosphorylase (TP), a key enzyme for its conversion to 5-florouracil (5-FU), observed in tumours as opposed to normal tissues, potentially allowing for selective toxicity. Capecitabine is a valuable substitute for bolus or infusion 5-FU either as monotherapy or in combination with other cytotoxic drugs in the treatment of several tumor types including breast cancer. We have recently demonstrated for the first time, that the histone deacetylase-inhibitor (HDAC-I) vorinostat induces synergistic antitumour effects in combination with capecitabine by up-regulating, in vitro and in vivo, in colorectal cancer cells but not in ex vivo treated peripheral blood lymphocytes, the mRNA and protein expression of TP. We have also demonstrated that TP knockdown by a specific siRNA significantly impairs the synergistic apoptotic cell death induced by vorinostat/capecitabine combination. In this study we demonstrated a time and dose-dependent induction of TP mRNA and protein expression by several HDAC-Is in different breast cancer cell lines such MCF-7, SKBR3, MDA-MB-231and MDA-MB-468 cells. Interestingly, this effect was shared by pan-HDAC-Is such as panobinostat (LBH589), trichostatin A (TSA) or vorinostat and by class I HDAC-Is such as valproic acid (VPA) and entinostat (MS275), but not by the specific inhibitor of HDAC-6 tubacin, indicating that targeting class I HDACs is crucial for TP induction. Moreover, the mechanism of TP induction is dependent on a transcriptional effect since preliminary data showed that HDAC-Is did not interfere with TP protein stability. On these bases we investigated potential antitumor interaction between capecitabine and either vorinostat or panobinostat or VPA, demonstrating synergistic antiproliferative effects in all breast cancer cells independently of p53, ER or Her2 status. Overall, this study suggests that HDAC-Is, by upregulating TP expression, may improve the therapeutic index of capecitabine representing an innovative antitumour strategy for the treatment of breast cancer that warrants further clinical evaluation. 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 3519. doi:10.1158/1538-7445.AM2011-3519

Background: Resistance of tumors to anti-angiogenic therapies is becoming an increasingly relevant issue. Moreover, recent studies provided evidences that VEGF-targeted drugs could make tumor cells more aggressive and metastatic. Methods: To identify the tumor-derived factors responsible for the evasive resistance of pancreatic cancer to anti-VEGF treatment we established and validated two models of human pancreatic cancer cell lines resistant in vivo to bevacizumab. We compared gene expression profiles from sensitive versus resistant cell lines using Ingenuity Pathways Analysis software. Results: The cellular functions significantly associated to the anti-VEGF- resistance gene expression profiles concerned with the chemoattraction of leucocytes, especially granulocytes, and with the mobilization of myeloid progenitor cells. Beside the minor over-expression of well-recognized direct pro-angiogenic factors, we identified several pro-inflammatory factors overexpressed by the anti-VEGF resistant cells in comparison to their sensitive controls. In this regard, the anti-VEGF- resistant tumors demonstrated a significantly higher infiltration by CD11b+ pro-angiogenic myeloid cells in comparison to the anti-VEGF- sensitive tumors. Anti-VEGF resistant cell lines demonstrated in vitro and in vivo a more aggressive phenotype. Expression of transcription factors Zeb1, Zeb2, and SMAD3, which are involved in Epithelial- to- Mesenchymal Transition (EMT), were significantly up-regulated in both anti-VEGF- resistant cell lines in comparison to the respective controls Consistently, anti-VEGF- resistant cell lines expressed significantly lower levels of E-Cadherin, and higher levels of Vimentin proteins in comparison to the respective anti-VEGF- sensitive cell lines when cultured in vitro. In vivo, anti-VEGF- sensitive tumors showed a strong expression of E-cadherin and a moderate expression of Vimentin. On the contrary, resistant tumors demonstrated no expression of E-cadherin, a strong expression of Vimentin, and a significant desmoplastic reaction. In co-colture assays, secreted factors over-expressed by the anti-VEGF- resistant cells induced EMT and a more aggressive phenotype in control sensitive cells. Conclusion: anti-VEGF therapy induces a selection of pancreatic cancer cells over-expressing secreted mediators of vascular inflammation. These factors could act in a paracrine manner recruiting pro-angiogenic myeloid cells and thus inducing an indirect angiogenesis. In addition, they could act in an autocrine manner to induce EMT and in turn a more aggressive behavior of pancreatic cancer. 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 3287. doi:10.1158/1538-7445.AM2011-3287

Enhanced expression of the RI alpha subunit of cyclic AMP-dependent protein kinase type I has been correlated with cancer cell growth. We provide evidence that RI alpha is a growth-inducing protein that may be essential for neoplastic cell growth. Human colon, breast, and gastric carcinoma and neuroblastoma cell lines exposed to a 21-mer human RI alpha antisense phosphorothioate oligodeoxynucleotide (S-oligodeoxynucleotide) exhibited growth inhibition with no sign of cytotoxicity. Mismatched sequence (random) S-oligodeoxynucleotides of the same length exhibited no effect. The growth inhibitory effect of RI alpha antisense oligomer correlated with a decrease in the RI alpha mRNA and protein levels and with an increase in RII beta (the regulatory subunit of protein kinase type II) expression. The growth inhibition was abolished, however, when cells were exposed simultaneously to both RI alpha and RII beta antisense S-oligodeoxynucleotides. The RII beta antisense S-oligodeoxynucleotide alone, exhibiting suppression of RII beta along with enhancement of RI alpha expression, led to slight stimulation of cell growth. These results demonstrate that two isoforms of cyclic AMP receptor proteins, RI alpha and RII beta, are reciprocally related in the growth control of cancer cells and that the RI alpha antisense oligodeoxynucleotide, which efficiently depletes the growth stimulatory RI alpha, is a powerful biological tool toward suppression of malignancy.

Vascular endothelial growth factor (VEGF) has a crucial role in tumor angiogenesis, and its inhibition by bevacizumab (BEV) leads to normalization of tumor vessels increasing tumor oxygenation and drug delivery. However, the clinical benefits of current anti-VEGF treatments have thus far been rather modest, stimulating interest in developing novel effective ways to combine anti-VEGF drugs and chemotherapy (CT) and in the identification of valid predictive biomarkers of clinical benefit. We have previously shown that pre-operative oxaliplatin (OXA), raltitrexed (RTX), fluorouracil (5FU), and folinic acid (LFA) during pelvic radiotherapy (RT) have high rate of complete (TRG1) or subtotal (TRG2) tumor regression in locally advanced rectal cancer. In this study in order to demonstrate the relevance of the timing of BEV added to primary CT and RT we evaluate two different schedules of treatment. Changes of CECs and glucose metabolism, evaluated by flow cytometry and FDG-PET respectively, were used as early surrogate markers of tumor response. Thirty-two patients (inclusion criteria: cT4, cN+, cT3≤ 5 cm from the anal verge and/or +ve CRM, M1 resectable/initially unresectable) received 3 biweekly courses of OXA (100 mg/m2)/RTX (2.5 mg/m2) on day 1, and 5FU (800 mg/m2)/LFA (250 mg/m2) on day 2 during pelvic RT (45 Gy). In schedule A (16 pts) BEV (5 mg/kg) was given biweekly from day −14 for 4 courses, while in schedule B (16 pts) it was given from day −4 for 2 courses. Toxicity was graded with NCI-CTC version 3. According to the Simon's two-stage design, assuming an hypothesis of a 50% TRG1 (α error=0.05, β error=0.20), at least 6/16 TRG1 should be obtained (first stage) to continue pts accrual. Grade ¾ neutropenia was the most common adverse event with the schedule A (7 pts, 44%), but it was considerably lower with the schedule B (2 pts, 13%). Pre-treatment basal CEC amounts showed increased levels in the pts samples compared to the healthy controls. Notably, a significant difference of CEC levels, compared to basal levels, was observed 2 days after CT-RT between the two schedules of treatment (median +6% and −84% in schedule A and B, respectively, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3721.

Background: Cetuximab, the chimeric monoclonal antibody (mAb) directed against the human epidermal growth factor receptor (EGFR) is widely used for the treatment of colorectal cancer and is also effective in pancreatic cancer preclinical models. Acquired resistance to cetuximab is common and the IGF-1R signaling pathway is critically involved in this process. We hypothesized that the combined activation of EGFR and IGF1-R is responsible for the constitutive activation of the transcriptional factor STAT-3 which in turn results in acquired resistance to cetuximab in colorectal and pancreatic tumors. Targeting this activation of EGFR and IGF1-R using the combination of cetuximab and R1507 could potentially reverse this resistance. Methods: A model of pancreatic cancer (PC) cell line resistant in vivo to cetuximab was established by injecting orthotopically, luciferase- and GFP- expressing AsPC-1 human PC cells into nude mice. Once established these tumors in vivo, the mice were treated with cetuximab 500 µg ip bi-weekly until the 34th week, when tumor growth occurred despite cetuximab due to acquired resistance. The tumors were rapidly excised and the GFP- positive cells were selected to establish the cetuximab- resistant clone AsPC-1-CR. Two models of colon cancer (CRC) cell line with acquired resistance in vivo to cetuximab, SW480-CR and GEO-CR, were established as previously described (Bianco et al. Clin Cancer Res. 2008 14(16):5069-80). MTT assay was used to assess the antitumor activity of cetuximab, R1507, and the combination in vitro. Synergy was assessed according the combination-index method of Chou and Talalay. The expression of IGF1-R was studied by Western blot. STAT-3 activation was analyzed by EMSA. Inhibition of directional cell migration was studied in an in vitro wound-healing model. Results: The cetuximab- resistant PC and CRC cell lines, showed a significantly lower in vitro sensitivity to cetuximab when compared to the respective control cell lines. AsPC-1-CR and SW480-CR cells demonstrated a higher expression of IGF1-R. STAT-3 DNA binding activity was significantly increased in all of the cetuximab- resistant cells as compared with control cell lines. The addition of R1507 did not improve the antitumor activity of cetuximab in the control cell lines. However, the combination of cetuximab + R1507 reversed the resistance to cetuximab in the AsPC-1-CR and SW480-CR cell lines, thereby demonstrating synergistic antitumor activity in the setting of acquired resistance. R1507+cetuximab also significantly inhibited the migration of cetuximab- resistant CRC cells. Conclusion: IGF-1R inhibition using R1507 is a promising approach to restore the sensitivity to cetuximab in PC and CRC cells that have developed acquired resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 616.

The oral fluoropyrimidine capecitabine is converted to the active compound 5-florouracil (5-FU) in several steps, the last of which is the conversion of 5′-deoxy-5-fluorouridine (5′DFUR) to 5-FU by thymidine phosphorylase (TP). Capecitabine was designed to take advantage of the increased levels of TP observed in tumors as opposed to normal tissues, potentially allowing for selective toxicity in tumors. In this study we found that the antiproliferative effect induced by the histone deacetylase inhibitor vorinostat in colorectal cancer cells in vitro and in vivo xenograft models, was paralleled by downregulation of thymidilate synthase, an essential enzyme for DNA synthesis and the target of 5-FU, and by upregulation of TP. These effects were evident at the mRNA and at the protein level and were not observed in peripheral blood lymphocyte (PBL) from healthy donors treated ex vivo by vorinostat. On the basis of these results, we have evaluated in vitro the effects of the combination between vorinostat and the capecitabine metabolite 5′-DFUR on the growth of human LoVo, LS174T and SW620 colon cancer cell lines, showing that simultaneous exposure of equipotent doses of the two agents, for 96 hours, resulted in synergistic antiproliferative effect in all cell lines as demonstrated by calculating combination indexes. We also demonstrated that vorinostat and 5′-DFUR in combination, compared to single agents treatment, increased apoptotic cell death, demonstrated by flow cytometry analysis of DNA content after propidium iodide staining, by the induction of BAX protein expression and by the cleavage of PARP. The observed apoptotic effect could be, at least in part, related to the increase in the ROS content induced by vorinostat and 5′-DFUR combination compared to single agents treatment. In order to verify in vivo the synergistic effects on tumor cell growth demonstrated in vitro, we evaluated vorinostat in combination with capecitabine in SW620 colon cancer cells xenograft model. A marked inhibition of tumor growth was observed in mice group treated with vorinostat (100 mg/kg p.o. daily) plus capecitabine (359 mg/Kg p.o. daily) as compared to single agents treatment, with a consequent increase by 2.4-fold of tumor growth delay and a substantial increase in survival. Analysis of xenografts tumor sections by immunohistochemistry showed a significant increase in apoptotic cells in vorinostat plus capecitabine treated group compared with control, or single agent treated groups, as well as the down-regulation TS and upregulation TP proteins expression in mice treated with vorinostat alone or in combination with capecitabine. Overall these data suggested that the association of vorinostat plus capecitabine could be clinically explored in colon cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5442.

Unregulated or increased expression of epidermal growth factor receptor (EGF-R) is a common event in neoplastic transformation; modulation of such a receptor by physiological agents could be, therefore, of clinical interest. We have studied the binding ability, the availability at cell surface, and the synthesis of EGF-R in the A431 and KB human epidermoid cancer cell lines after treatment with recombinant alpha-interferon (IFN-alpha). After 48 h of treatment, IFN-alpha induces, in both cell lines, growth inhibition and enhances class I major histocompatibility HLA complex expression, which is a common marker of IFN action. [125I]EGF total binding assessed after 48 h of treatment with IFN-alpha shows a dose-dependent upregulation of EGF-R binding capacity. Saturation plots of the binding data show that IFN-alpha treatment does not dramatically alter the affinity of the EGF-R and indicate that IFN-alpha only increases the number of low affinity receptors. We show that this effect is due to a specific increase in the synthesis of the receptor protein, as assessed by immunoprecipitation of [35S]methionine-labeled cell extracts. Electron microscopy analysis has confirmed an increase of EGF-R proteins at cell surface without major changes in the morphology of the cells. Taken together, these results indicate that IFN-alpha consistently induces both the binding capacity and the synthesis of EGF-R in human epidermoid cancer cells and suggest the use of such a mechanism for new anticancer therapies.