Upregulation of ERK-EGR1-heparanase axis by HDAC inhibitors provides targets for rational therapeutic intervention in synovial sarcoma.

BACKGROUND: Synovial sarcoma (SS) is an aggressive soft tissue tumor with limited therapeutic options in advanced stage. SS18-SSX fusion oncogenes, which are the hallmarks of SS, cause epigenetic rewiring involving histone deacetylases (HDACs). Promising preclinical studies supporting HDAC targeting for SS treatment were not reflected in clinical trials with HDAC inhibitor (HDACi) monotherapies. We investigated pathways implicated in SS cell response to HDACi to identify vulnerabilities exploitable in combination treatments and improve the therapeutic efficacy of HDACi-based regimens. METHODS: Antiproliferative and proapoptotic effects of the HDACi SAHA and FK228 were examined in SS cell lines in parallel with biochemical and molecular analyses to bring out cytoprotective pathways. Treatments combining HDACi with drugs targeting HDACi-activated prosurvival pathways were tested in functional assays in vitro and in a SS orthotopic xenograft model. Molecular mechanisms underlying synergisms were investigated in SS cells through pharmacological and gene silencing approaches and validated by qRT-PCR and Western blotting. RESULTS: SS cell response to HDACi was consistently characterized by activation of a cytoprotective and auto-sustaining axis involving ERKs, EGR1, and the ß-endoglycosidase heparanase, a well recognized pleiotropic player in tumorigenesis and disease progression. HDAC inhibition was shown to upregulate heparanase by inducing expression of the positive regulator EGR1 and by hampering negative regulation by p53 through its acetylation. Interception of HDACi-induced ERK-EGR1-heparanase pathway by cell co-treatment with a MEK inhibitor (trametinib) or a heparanase inhibitor (SST0001/roneparstat) enhanced antiproliferative and pro-apoptotic effects. HDAC and heparanase inhibitors had opposite effects on histone acetylation and nuclear heparanase levels. The combination of SAHA with SST0001 prevented the upregulation of ERK-EGR1-heparanase induced by the HDACi and promoted caspase-dependent cell death. In vivo, the combined treatment with SAHA and SST0001 potentiated the antitumor efficacy against the CME-1 orthotopic SS model as compared to single agent administration. CONCLUSIONS: The present study provides preclinical rationale and mechanistic insights into drug combinatory strategies based on the use of ERK pathway and heparanase inhibitors to improve the efficacy of HDACi-based antitumor therapies in SS. The involvement of classes of agents already clinically available, or under clinical evaluation, indicates the transferability potential of the proposed approaches.

Overactive IGF1/Insulin Receptors and NRASQ61R Mutation Drive Mechanisms of Resistance to Pazopanib and Define Rational Combination Strategies to Treat Synovial Sarcoma.

Pazopanib is approved for treatment of advanced soft tissue sarcomas, but primary and secondary drug resistance limits its clinical utility. We investigated the molecular mechanisms mediating pazopanib resistance in human synovial sarcoma (SS) models. We found reduced cell sensitivity to pazopanib associated with inefficient inhibition of the two critical signaling nodes, AKT and ERKs, despite strong inhibition of the main drug target, PDGFRα. In the CME-1 cell line, overactivation of IGF1 and Insulin receptors (IGF1R/InsR) sustained AKT activation and pazopanib resistance, which was overcome by a combination treatment with the double IGF1R/InsR inhibitor BMS754807. In the highly pazopanib resistant MoJo cell line, NRASQ61R mutation sustained constitutive ERK activation. Transfection of the NRAS mutant in the pazopanib sensitive SYO-1 cell line increased the drug IC50. MoJo cells treatment with pazopanib in combination with the MEK inhibitor trametinib restored ERK inhibition, synergistically inhibited cell growth, and induced apoptosis. The combination significantly enhanced the antitumor efficacy against MoJo orthotopic xenograft abrogating growth in 38% of mice. These findings identified two different mechanisms of intrinsic pazopanib resistance in SS cells, supporting molecular/immunohistochemical profiling of tumor specimens as a valuable approach to selecting patients who may benefit from rational drug combinations.

Supersulfated low-molecular weight heparin synergizes with IGF1R/IR inhibitor to suppress synovial sarcoma growth and metastases.

Synovial sarcoma (SS) is an aggressive tumor with propensity for lung metastases which significantly impact patients' prognosis. New therapeutic approaches are needed to improve treatment outcome. Targeting the heparanase/heparan sulfate proteoglycan system by heparin derivatives which act as heparanase inhibitors/heparan sulfate mimetics is emerging as a therapeutic approach that can sensitize the tumor response to chemotherapy. We investigated the therapeutic potential of a supersulfated low molecular weight heparin (ssLMWH) in preclinical models of SS. ssLMWH showed a potent anti-heparanase activity, dose-dependently inhibited SS colony growth and cell invasion, and downregulated the activation of receptor tyrosine kinases including IGF1R and IR. The combination of ssLMWH and the IGF1R/IR inhibitor BMS754807 synergistically inhibited proliferation of cells exhibiting IGF1R hyperactivation, also abrogating cell motility and promoting apoptosis in association with PI3K/AKT pathway inhibition. The drug combination strongly enhanced the antitumor effect against the CME-1 model, as compared to single agent treatment, abrogating orthotopic tumor growth and significantly repressing spontaneous lung metastatic dissemination in treated mice. These findings provide a strong preclinical rationale for developing drug regimens combining heparanase inhibitors/HS mimetics with IGF1R antagonists for treatment of metastatic SS.

Antitumor efficacy of the heparan sulfate mimic roneparstat (SST0001) against sarcoma models involves multi-target inhibition of receptor tyrosine kinases.

The heparan sulfate (HS) mimic/heparanase inhibitor roneparstat (SST0001) shows antitumor activity in preclinical sarcoma models. We hypothesized that this 100% N-acetylated and glycol-split heparin could interfere with the functions of several receptor tyrosine kinases (RTK) coexpressed in sarcomas and activated by heparin-binding growth factors. Using a phospho-proteomic approach, we investigated the drug effects on RTK activation in human cell lines representative of different sarcoma subtypes. Inhibition of FGF, IGF, ERBB and PDGF receptors by the drug was biochemically and functionally validated. Roneparstat counteracted the autocrine loop induced by the COL1A1/PDGFB fusion oncogene, expressed in a human dermatofibrosarcoma protuberans primary culture and in NIH3T3COL1A1/PDGFB transfectants, inhibiting cell anchorage-independent growth and invasion. In addition, roneparstat inhibited the activation of cell surface PDGFR and PDGFR-associated FAK, likely contributing to the reversion of NIH3T3COL1A1/PDGFB cell transformed and pro-invasive phenotype. Biochemical and histological/immunohistochemical ex vivo analyses confirmed a reduced activation of ERBB4, EGFR, INSR, IGF1R, associated with apoptosis induction and angiogenesis inhibition in a drug-treated Ewing's sarcoma family tumor xenograft. The combination of roneparstat with irinotecan significantly improved the antitumor effect against A204 rhabdoid xenografts resulting in a high rate of complete responses and cures. These findings reveal that roneparstat exerts a multi-target inhibition of RTKs relevant in the pathobiology of different sarcoma subtypes. These effects, likely cooperating with heparanase inhibition, contribute to the antitumor efficacy of the drug. The study supports heparanase/HS axis targeting as a valuable approach in combination therapies of different sarcoma subtypes providing a preclinical rationale for clinical investigation.

Synergistic cooperation between sunitinib and cisplatin promotes apoptotic cell death in human medullary thyroid cancer.

CONTEXT: Tyrosine kinase inhibitors represent a new treatment option for patients with advanced medullary thyroid cancer (MTC). However, cures have not been achieved with current available agents used in monotherapy. OBJECTIVE: Because RET has been shown to negatively regulate CD95 death receptor activation in preclinical models of RET-dependent MTC, we investigated the potential of the combination approach with the RET-targeting tyrosine kinase inhibitor sunitinib and cisplatin to enhance apoptosis activation through the extrinsic pathway. DESIGN: The effects of sunitinib and cisplatin were examined in human MTC cell lines harboring oncogenic RET mutations. Experiments were designed to determine drug effects on RET signaling, cell growth, apoptosis, autophagy, and tumor growth in mice and to investigate the mechanisms of the drug interaction. RESULTS: Sunitinib and cisplatin synergistically inhibited the growth of MZ-CRC-1 cells harboring the RET M918T activating mutation. The combination enhanced apoptosis activation through CD95-mediated, caspase-8-dependent pathway. Moreover, sunitinib induced a severe perturbation of the autophagic flux characterized by autophagosome accumulation and a remarkable lysosomal dysfunction, which was further enhanced, with lysosomal leakage induction, by cisplatin. Administration of the drug combination to mice xenografted with MZ-CRC-1 cells improved the antitumor efficacy, as compared with single-agent treatments, inducing complete responses in 30% of the treated mice, a significant increase in caspase-3 activation (P < .01 vs cisplatin, and P < .0005 vs sunitinib) and apoptosis in tumor cells. CONCLUSIONS: Addition of cisplatin to sunitinib potentiates apoptotic cell death and has promising preclinical activity in MTCs harboring the RET M918T oncogene.

Design, synthesis and anticancer activities of stilbene-coumarin hybrid compounds: Identification of novel proapoptotic agents.

The naturally occurring coumarins and resveratrol, attract great attention due to their wide range of biological properties, including anticancer, antileukemic, antibacterial and anti-inflammatory activities; moreover, their cancer chemopreventive property have been recently emphasized. A novel class of hybrid compounds, obtained by introducing a substituted trans-vinylbenzene moiety on a coumarin backbone, was synthesized and evaluated for the antitumor profile. A number of derivatives showed a good antiproliferative activity, in some cases higher to that of the reference compound resveratrol. The most promising compounds in this series were 14 and 17, endowed with excellent antiproliferative and proapoptotic activities. The present study suggests that the 7-methoxycoumarin nucleus, together with the 3,5-disubstitution pattern of the trans-vinylbenzene moiety, are likely promising structural features to obtain excellent antitumor compounds endowed with a apoptosis-inducing capability.

Biological effects of a new vacuolar-H,-ATPase inhibitor in colon carcinoma cell lines.

Vacuolar-H(+)-ATPase plays a critical role in the cellular balance of protons, thus regulating intracellular pH and contributing to apoptosis resistance, drug resistance, and invasive and metastatic behavior of cells. NiK-12192, a vacuolar-H(+)-ATPase inhibitor, caused a reduction in the volume and/or acidity of lysosomes, a polarization of alphavbeta5 integrin distribution, and a number of floating live cells, whereas signs of apoptosis appeared only after 72 h of treatment. In conclusion, NiK-12192, by affecting vacuolar- H(+)-ATPase activity (and intracellular pH), causes a modification of structures crucial for cell adhesion and induces cell death, likely by a modality involving an anoikis-mediated apoptosis.

Effect of a novel vacuolar-H+-ATPase inhibitor on cell and tumor response to camptothecins.

The vacuolar-H(+)-ATPase, functionally expressed in cell membranes, is known to play a relevant role in intracellular pH regulatory mechanisms, because it is implicated in pumping protons into the extracellular environment or in sequestrating excess protons into acidic vacuolar compartments. Because tumor cells exist in a hypoxic microenvironment and produce acidic metabolites, this regulatory mechanism is recognized as a protective function. This study was designed to investigate the effect of NiK-12192 [4-(5,6-dichloro-1H-indol-2-yl)-3-ethoxy-N-(2,2,6,6-tetramethyl-piperidin-4-yl)-benzamide], an indole derivative identified as an effective inhibitor of vacuolar-H(+)-ATPase, on the cytotoxic activity of two camptothecins, i.e., topotecan and SN-38 (7-ethyl-10-hydroxycamptothecin, the active metabolite of irinotecan). The cellular studies performed in two pairs of human colon carcinoma cell lines, i.e., LoVo and LoVo/DX (overexpressing P-glycoprotein) and HT29 and HT29/Mit (overexpressing breast cancer resistant protein), indicated an enhancement of the antiproliferative effect of camptothecins by concomitant exposure to subtoxic concentrations of NiK-12192. Studies of subcellular distribution indicated that whereas topotecan alone localized mainly in mitochondria and endoplasmic compartment, the simultaneous presence of NiK-12192 caused a cytoplasmic redistribution. In HT29/Mit cells, NiK-12192 reverted the pattern of acidification induced by topotecan. The potentiation of topotecan efficacy by NiK-12192 was documented by an increased efficacy of the combination in both the HT29 tumor xenografts, being more evident in the topotecan-resistant HT29/Mit tumor. In conclusion, the vacuolar-H(+)-ATPase inhibitor NiK-12192 was able to potentiate the cytotoxic/antitumor effects of camptothecins, either in in vitro or in in vivo systems. Such findings support a potential interest for the use of vacuolar-H(+)-ATPase inhibitors in combination therapy to improve camptothecin efficacy.

Molecular alterations of cells resistant to platinum drugs: role of PKCalpha.

Development of resistance to platinum compounds may involve not only overexpression of defence mechanisms but also alterations in cellular response to the drug-induced genotoxic stress. To investigate the cellular bases of response to platinum compounds, we examined the profile of gene expression of ovarian carcinoma cells exhibiting sensitivity (A2780) or resistance (A2780/BBR3464) to platinum compounds. Using display PCR, we found that acquisition of resistance to the multinuclear platinum complex BBR3464 was associated with modulation of several transcripts, including up-regulation of the major substrate of protein kinase C (PKC), the myristoylated alanine-rich C kinase substrate (MARCKS). This feature was associated with PKCalpha down-regulation. To explore the role of PKCalpha in cellular sensitivity to platinum compounds, resistant cells were transfected with a PKCalpha-containing vector. PKCalpha-overexpressing resistant cells exhibited a decrease in sensitivity to cisplatin, whereas no significant change in sensitivity to BBR3464 was observed. A number of approaches designed to modulate the function or expression of PKCalpha support that the isoenzyme may play a role in determining resistance only to cisplatin but not to BBR3464, which is known to activate a different pathway of cell response. In conclusion, in spite of PKCalpha down-regulation in our model, its regulatory function was not apparently implicated in the development of resistance to platinum compounds and the present results do not support a general role of PKCalpha as a determinant of the resistance status.

Development of resistance to a trinuclear platinum complex in ovarian carcinoma cells.

BBR3464 is a trinuclear platinum complex that exhibits a potent cytotoxicity and efficacy against cisplatin-resistant tumors. To better understand the determinants of cellular resistance to BBR3464, we selected a resistant ovarian carcinoma cell line after exposure to the complex. The resistant cells (A2780/BBR3464) exhibited a high level of resistance to the selecting agent, but a marginal cross-resistance to cisplatin. Although cellular accumulation of BBR3464 was similar in parental and in resistant cells, DNA platination was decreased in A2780/BBR3464 cells, suggesting a reduced drug accessibility to DNA. This behavior reflected a partial drug inactivation at cytoplasmic level, as a consequence of increased levels of nucleophilic molecules including metallothioneins and human neurofilament low, but not glutathione. A2780/BBR3464 cells also exhibited a reduced susceptibility to apoptosis, which was consistent with reduced expression of Bax, and an alteration of DNA mismatch repair system, as reflected by lack of expression of MLH1 and PMS2, which could impair the recognition/repair of DNA lesions. Whereas both platinum drugs induced G2/M arrest in the parental cells, BBR3464, but not cisplatin, caused a late G1 arrest of resistant cells. Cisplatin induced an appreciable increase of p21(WAF1) levels in both models, in contrast to BBR3464 that produced a substantial upregulation of p21(WAF1) only in parental cells. An inverse relationship with p21(WAF1) modulation was found for CHK1 in parental cells treated with both agents and in resistant cells treated with cisplatin. This pattern of response is consistent with a regulatory loop involving p53 and p21(WAF1) at G2 checkpoint. In contrast, no modulation of CHK1 was found in A2780/BBR3464 treated with the triplatinum compound. These findings, indicating a different activation of regulatory pathways at DNA damage checkpoints in response to cisplatin and BBR3464, support an altered ability of resistant cells to recognize or tolerate sublethal lesions induced by BBR3464.