The proof of principle that a drug targeting mTOR can improve survival has been obtained recently from a large randomised trial using temsirolimus as a first-line therapy in patients with advanced poor prognostic renal cell carcinoma. Consistent data have recently shown the important role of the PI3K/AKT/mTOR signalling pathway in the regulation of crucial metabolic and mitotic functions of cancer cells and endothelial cells allowing a better understanding of the role of mTOR in controlling cancer cell proliferation and survival as well as tumour angiogenesis. As a result, rapamycin derivatives (rapalogues) that block mTOR/Raptor complex 1 were shown to exert direct antiproliferative effects against endometrial cancers, in which cancer cells frequently lose PTEN function as well as mantle cell lymphomas, in which cancer cell proliferation appears to be driven primarily by cyclin D1 overexpression. The overall antitumour effects of rapalogues in renal cell carcinoma appear to be more complex with tumour growth inhibition resulting from direct G1/S cell cycle blockage and/or apoptotic effects in carcinoma cells along with the inhibition of downstream signalling of the HIF1a-induced VEGF/VEGFR autocrine loop in endothelial cells shutting down the maintenance of tumour angiogenesis. Despite extensive cognitive researches, it is difficult to appraise which of those mechanisms is predominant in patients. This review focuses on mechanisms of action of rapalogues focusing on antitumour effects in patients with renal cell carcinoma.

PEP005 is a novel ingenol angelate that modulates protein kinases C (PKC) functions by activating PKCd and inhibiting PKCa. This study assessed the antiproliferative effects of PEP005 alone and in combination with several other anticancer agents in a panel of 10 human cancer cell lines characterised for expression of several PKC isoforms. PEP005 displayed antiproliferative effects at clinically relevant concentrations with a unique cytotoxicity profile that differs from that of most other investigated cytotoxic agents, including staurosporine. In a subset of colon cancer cells, the IC 50 of PEP005 ranged from 0.01 -140 mM. The antiproliferative effects of PEP005 were shown to be concentration-and time-dependent. In Colo205 cells, apoptosis induction was observed at concentrations ranging from 0.03 to 3 mM. Exposure to PEP005 also induced accumulation of cells in the G1 phase of the cell cycle. In addition, PEP005 increased the phosphorylation of PKCd and p38. In Colo205 cells, combinations of PEP005 with several cytotoxic agents including oxaliplatin, SN38, 5FU, gemcitabine, doxorubicin, vinorelbine, and docetaxel yielded sequence-dependent antiproliferative effects. Cell cycle blockage induced by PEP005 in late G1 lasted for up to 24 h and therefore a 24 h lag-time between PEP005 and subsequent exposure to cytotoxics was required to optimise PEP005 combinations with several anticancer agents. These data support further evaluation of PEP005 as an anticancer agent and may help to optimise clinical trials with PEP005-based combinations in patients with solid tumours.

Background: Therapeutic options for patients with advanced hepatocarcinomas (HCC) are still limited. TGF-β1 and α-fetoprotein (AFP) have been shown to be independent prognostic factors for disease progression and recurrence. LY2157299, a selective ATP-mimetic inhibitor of TGF-β receptor (TβR)-I activation, is currently under clinical investigation in HCC patients. Our study aimed at exploring the effects of LY2157299 in HCC cell lines and patient samples with various AFP expression levels. Materials and Methods: Antiproliferative effects of LY2157299 were evaluated in a panel of human HCC cells by MTT assay. Baseline and phosphorylated protein levels were detected by Western blot analysis and mRNA expressions by qRT-PCR. Invasion assays were done on matrigel and in OptiCell devices. Tumor samples from HCC patients were surgically resected, cut in 300 μm thick slices using a Tissue Slicer. Each slice was randomly selected and exposed to LY2157299 (1μM and 10μM) and sorafenib (5μM) for 48h. Tumor samples were analyzed by immunohistochemistry (IHC) or immunofluorescence (IF). Results: LY2157299 was evaluated in HEPG2, HEP3B and SK-HEP1 cells as well as SK-HEP1-derived cells tolerant to sorafenib (SK-Sora) and sunitinib (SK-Suni). Exogenous stimulation of all HCC cell lines with TGF-β yielded downstream activation of p-Smad2 and p-Smad3 that was potently inhibited with LY2157299 treatment at micromolar concentrations. Low concentrations of LY2157299 displayed antiproliferative effects in HEPG2 cells when stimulated by TGF-β but not in SK-HEP1, SK-Sora, SK-Suni and HEP3B cells. Interestingly, HEPG2 cells were the only cell line displaying high levels of AFP as along with an epithelial phenotype. LY2157299 yielded potent anti-migratory and anti-invasive properties in invasive SK-HEP1, SK-Suni and SK-Sora cells. Tumor slices from surgically resected tumor samples from 3 patients with advanced HCC, were exposed ex vivo to 1 μM and 10 μM LY2157299 or 5 μM sorafenib for 48h. This method allows evaluating the effects of novel anticancer agents in whole tumors containing cancer cells and stroma cells. LY2157299 but not sorafenib decreased p-Smad2/3 downstream TGF-β signaling as well as AFP levels. IHC analysis of LY2157299 and sorafenib-exposed samples showed a significant decrease of the proliferative marker Ki67 and increase of the apoptotic marker caspase-3. Interestingly these effects were independent of AFP expression. Conclusion: TGF-β/TβR-I inactivation using LY2157299 inhibits TGF-β-dependent cell signaling in HCC cell lines with either anti-proliferative or anti-invasive effects depending on the model. In tumor samples from patients, inhibition of TGF-β signaling was associated with decreased AFP levels, inhibition of proliferation and apoptosis induction. Our data suggest that LY2157299 may be useful for patients with HCC. Citation Format: Marie Serova, Annemilaï Tijeras-Raballand, Celia Dos Santos, Nelly Muller, Karim A. Benhadji, Valerie Paradis, Sandrine Faivre, Eric Raymond, Armand de Gramont. Effects of TGF-beta signaling inhibition with LY2157299 in hepatocarcinoma models and in ex vivo whole tumor tissue samples from patient specimen. [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 2094. doi:10.1158/1538-7445.AM2013-2094

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Background: Resistance to radiotherapy and chemotherapy-induced apoptosis is a hallmark of cancer. Inhibitors of apoptosis proteins (IAPs) are negative modulators of apoptosis frequently expressed in various cancers, and, as such, attractive targets to overcome resistance to cancer therapy. The oral SMAC mimetic Debio 1143 (a.k.a AT-406), an antagonist of multiple IAPs (cIAP1/2 and XIAP), is currently investigated in a Phase I oncology clinical trial. This study evaluated Debio 1143 activity as a single agent, and in combination with TNF-α, TRAIL, cisplatin or carboplatin, in various tumor models of squamous cell carcinoma of the head and neck (SCCHN). Materials and Methods: The antiproliferative effect of Debio 1143 was evaluated by MTT assay in human SCCHN cell lines SQ20B, SCC61, Hep2 and Detroit 562. The extent of apoptotic cell death was characterized by Western blot for cleaved caspase 3 and PARP. Tumor samples from SCCHN patients were surgically resected and cut into 300 µm thick slices using a tissue slicer (TIPCAN®). Each slice was exposed to 10 µM Debio 1143 and/or 1 µM of platinum-based drug for 48 hours. Tumor explants from 9 SCCHN patients were analyzed by immunohistochemistry or immunofluorescence to visualize the effects of treatment on various biomarkers of cell apoptosis, proliferation and drug target engagement. Results: Debio 1143 alone displayed limited antiproliferative activity in SCCHN cell lines. The addition of TRAIL or TNF-α potentiated the antiproliferative effects of Debio 1143 in 1 cell line (SQ20B) and in 3 cell lines (SQ20B, SCC61 and Detroit 562), respectively. Combining Debio 1143 with TNF-α induced cleavage of caspase 3 and PARP, suggesting induction of apoptosis. Exposure of fresh SCCHN tumor explants to Debio 1143 reduced c-IAP1 staining. Combination of Debio 1143 with cisplatin or carboplatin induced caspase 3 activation, suggesting apoptosis. Large necrotic areas were also found on tissue samples after combination therapy. Conclusion: In 3 out of 4 SCCHN cell lines, Debio 1143 potentiated TNF-α or TRAIL-induced antiproliferative effects. Debio 1143 combined with carboplatin and cisplatin induced caspase 3-dependent apoptosis in SCCHN patient tumor samples. Debio 1143 in combination with conventional chemotherapies could be a potential treatment for SCCHN patients. Citation Format: Marie Serova, Annemilai Tijeras-Raballand, Sebastien Albert, Sandrine Faivre, Eric Raymond, Anne Vaslin, Claudio Zanna, Grégoire Vuagniaux, Armand de Gramont. Effects of Debio 1143, a novel oral IAP inhibitor, in monotherapy and in combination with platinum drugs in human SCCHN tumor specimens. [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 2752. doi:10.1158/1538-7445.AM2014-2752

Background: Current management of advanced HNSCC is limited to surgery, radiotherapy, and chemotherapy. The frequent activation of the PI3K/AKT/mTOR signaling pathway in HNSCC has suggested that inhibition of PI3K/mTOR may have potential therapeutic activity. Preliminary results of the phase I/II CAPRA clinical trial combining the mTOR inhibitor everolimus with paclitaxel and carboplatin have shown promising antitumor activity. Experimental evidences have suggested that mTOR inhibition using rapalogues may be hampered by a negative feedback resulting in PI3K activation. The aim of our study was to evaluate the antiproliferative effects of novel PI3K/mTOR inhibitors in comparison to everolimus in a panel of three HNSCC cell lines. Materials and Methods: HNSCC cell lines (SQ20B, HEP2, and SCC61) were treated with mTORC1 specific inhibitor everolimus, the dual PI3K/mTOR inhibitor BEZ235, and the specific PI3K inhibitor BKM120. Antiproliferative effects were assessed using MTT assay. Protein expression and phoshorylation were assessed by western blot. Results: HNSCC cancer cells were characterized for predictive factors of mTOR inhibitors. SQ20B and SCC61 cells displayed an epithelial phenotype whereas HEP2 cells displayed mesenchymal characteristics. SQ20B and SCC61 cells also had a constitutively activated AKT pathway in contrast to HEP2 cells. Others potentials predictive factors of mTOR inhibitors (Bcl2, Cyclin D1, p27, c-Myc) were differentially expressed in HEP2, SCC61, and SQ20B cells. In all cell lines, everolimus displayed IC50 over 6 μM. Novel mTOR inhibitors were 2-30 times more potent with IC50 ranging from 0.2 to 0.8 μM for BEZ235 and from 2.1 to 2.4 μM for BKM120. Everolimus and BEZ235 were less potent in HEP2 compared to SQ20B and SCC61 cells. In SQ20B and SCC61 cell lines everolimus displayed low IC25 (0.02μM) close to BEZ235 (0.1μM) and lower than BKM120 (1.4μM). All inhibitors displayed potent inhibition of S6 phosphorylation in all cells. Everolimus and the novel mTOR inhibitors inhibited the constitutive AKT activation observed in SQ20B and SCC61. In contrast, everolimus but not the novel mTOR inhibitors, significantly increased AKT activation in HEP2 cells. In combinations with carboplatin or paclitaxel, everolimus and BEZ235 displayed synergistic antiproliferative effects in HEP2 and additive effects in SQ20B, whereas BKM120 displayed mostly additive effects in both cell lines. Conclusion: Novel generation of PI3K/mTOR inhibitors displayed more potent antiproliferative effects than everolimus in HNSCC cell lines. Everolimus and BEZ235 were more active in epithelial cells with constitutionally activated AKT pathway whereas BKM120 potently inhibited cell proliferation in all HNSCC models. Several combinations with chemotherapies were shown to be synergistic and may be regarded as an attractive strategy in HNSCC patients. Citation Format: Aurelie Hesbert, Marie Serova, Armand de Gramont, Khemaies Slimane, Eric Raymond, Sandrine Faivre. Effects of novel PI3K/mTOR inhibitors BKM120 and BEZ235 in comparison to everolimus in Head & Neck Squamous cell carcinomas (HNSCC). [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 4411. doi:10.1158/1538-7445.AM2013-4411

Background: Resistance to radiotherapy and chemotherapy-induced apoptosis is a hallmark of cancer. Inhibitors of apoptosis proteins (IAPs) are negative modulators of apoptosis frequently expressed in various cancers, and, as such, attractive targets to overcome resistance to cancer therapy. The oral SMAC mimetic Debio 1143 (D1143, a.k.a AT-406), an antagonist of multiple IAPs (cIAP1/2 and XIAP), is currently investigated in a Phase I oncology clinical trial. This study aimed at evaluating D1143 activity as a single agent and in combination with TNF-α, TRAIL, cisplatin or carboplatin, in various SCCHN models. Materials and Methods: The antiproliferative effects of D1143 were evaluated in a panel of 5 human SCCHN cell lines by MTT assay. Baseline and phosphorylated protein levels were detected by Western blot analysis. Tumor samples from SCCHN patients were surgically resected and cut into 300 µm thick slices using a tissue slicer (TIPCAN®). Each slice was exposed to 10 µM D1143 and/or 1 µM of platinum-based drug for 48 hours. Tumor samples were analyzed by immunohistochemistry (IHC) or immunofluorescence to visualize the effects of the treatment on various biomarkers of cell apoptosis, proliferation, and drug target engagement. Results: A panel of 5 SCCHN cell lines was characterized for the expression of c-IAP1/2, XIAP, Bcl2, LRIG1, and other proteins implicated in resistance to cell death. D1143 alone displayed limited antiproliferative activity in only one cell line (Detroit 562). However, two SCCHN cell lines were sensitive to D1143 and TRAIL combined (SQ20B and SCC15), three were sensitive to D1143 and TNF-α combined (SQ20B, SCC61, and Detroit 562), and one was resistant to both combinations (HEP2). In Detroit 562 sensitive cells, 10 µM D1143 induced sustained cIAP1/2 degradation after 15 minutes of exposure. In contrast, in the HEP2 insensitive cell line, D1143 induced slight and transient inhibition of cIAP1/2. IHC analyses revealed that ex vivo exposure to D1143 of tumor explants freshly resected from SCCHN patients decreased cIAP1 staining. Treatment of tumor samples with D1143 combined with cisplatin or carboplatin augmented the cleavage of caspase 3 compared to controls suggesting induction of apoptosis. Conclusion: In 4 out of 5 SCCHN cell lines, D1143 induced cIAP1/2 degradation and potentiated TNF-α or TRAIL-induced antiproliferative effects. D1143 combined with carboplatin and cisplatin in SCCHN patient samples induced caspase 3-dependent apoptosis. D1143 in combination with conventional chemotherapies may be considered as a potential treatment for SCCHN patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C20. Citation Format: Marie Serova, Annemilaï Tijeras-Raballand, Sebastien Albert, Sandrine Faivre, Eric Raymond, Anne Vaslin, Claudio Zanna, Gregoire Vuagniaux, Armand de Gramont. In vitro effects of Debio 1143, a novel oral IAP inhibitor, in human SCCHN cell lines and tumor specimens. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C20.

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