Inhibitors Of Survival Pathways Research Articles

Dual Inhibition of Histone Deacetylases and the Mechanistic Target of Rapamycin Promotes Apoptosis in Cell Line Models of Uveal Melanoma

PurposeOver 90% of uveal melanomas harbor pathogenic variants of the GNAQ or GNA11 genes that activate survival pathways. As previous studies found that Ras-mutated cell lines were vulnerable to a combination of survival pathway inhibitors and the histone-deacetylase inhibitor romidepsin, we investigated whether this combination would be effective in models of uveal melanoma.MethodsA small-scale screen of inhibitors of bromodomain-containing protein 4 (BRD4; OTX-015), extracellular signal-related kinase (ERK; ulixertinib), mechanistic target of rapamycin (mTOR; AZD-8055), or phosphoinositide 3-kinase (PI3K; GDC-0941) combined with a clinically relevant administration of romidepsin was performed on a panel of uveal melanoma cell lines (92.1, Mel202, MP38, and MP41) and apoptosis was quantified by flow cytometry after 48 hours. RNA sequencing analysis was performed on Mel202 cells treated with romidepsin alone, AZD-8055 alone, or the combination, and protein changes were validated by immunoblot.ResultsAZD-8055 with romidepsin was the most effective combination in inducing apoptosis in the cell lines. Increased caspase-3 and PARP cleavage were noted in the cell lines when they were treated with romidepsin and mTOR inhibitors. RNA sequencing analysis of Mel202 cells revealed that apoptosis was the most affected pathway in the romidepsin/AZD-8055-treated cells. Increases in pro-apoptotic BCL2L11 and decreases in anti-apoptotic BIRC5 and BCL2L1 transcripts noted in the sequencing analysis were confirmed at the protein level in Mel202 cells.ConclusionsOur data suggest that romidepsin in combination with mTOR inhibition could be an effective treatment strategy against uveal melanoma due in part to changes in apoptotic proteins.

Decorin Protects Cardiac Myocytes against Simulated Ischemia/Reperfusion Injury.

Search for new cardioprotective therapies is of great importance since no cardioprotective drugs are available on the market. In line with this need, several natural biomolecules have been extensively tested for their potential cardioprotective effects. Previously, we have shown that biglycan, a member of a diverse group of small leucine-rich proteoglycans, enhanced the expression of cardioprotective genes and decreased ischemia/reperfusion-induced cardiomyocyte death via a TLR-4 dependent mechanism. Therefore, in the present study we aimed to test whether decorin, a small leucine-rich proteoglycan closely related to biglycan, could exert cardiocytoprotection and to reveal possible downstream signaling pathways. Methods: Primary cardiomyocytes isolated from neonatal and adult rat hearts were treated with 0 (Vehicle), 1, 3, 10, 30 and 100 nM decorin as 20 h pretreatment and maintained throughout simulated ischemia and reperfusion (SI/R). In separate experiments, to test the mechanism of decorin-induced cardio protection, 3 nM decorin was applied in combination with inhibitors of known survival pathways, that is, the NOS inhibitor L-NAME, the PKG inhibitor KT-5823 and the TLR-4 inhibitor TAK-242, respectively. mRNA expression changes were measured after SI/R injury. Results: Cell viability of both neonatal and adult cardiomyocytes was significantly decreased due to SI/R injury. Decorin at 1, 3 and 10 nM concentrations significantly increased the survival of both neonatal and adult myocytes after SI/R. At 3nM (the most pronounced protective concentration), it had no effect on apoptotic rate of neonatal cardiac myocytes. No one of the inhibitors of survival pathways (L-NAME, KT-5823, TAK-242) influenced the cardiocytoprotective effect of decorin. MYND-type containing 19 (Zmynd19) and eukaryotic translation initiation factor 4E nuclear import factor 1 (Eif4enif1) were significantly upregulated due to the decorin treatment. In conclusion, this is the first demonstration that decorin exerts a direct cardiocytoprotective effect possibly independent of NO-cGMP-PKG and TLR-4 dependent survival signaling.

Countering TRAIL Resistance in Melanoma.

Melanoma of the skin has become a prime example for demonstrating the success of targeted cancer therapy. Nevertheless, high mortality has remained, mainly related to tumor heterogeneity and inducible therapy resistance. But the development of new therapeutic strategies and combinations has raised hope of finally defeating this deadly disease. TNF-related apoptosis-inducing ligand (TRAIL) represents a promising antitumor strategy. The principal sensitivity of melanoma cells for TRAIL was demonstrated in previous studies; however, inducible resistance appeared as a major problem. To address this issue, combination strategies were tested, and survival pathway inhibitors were shown to sensitize melanoma cells for TRAIL-induced apoptosis. Finally, cell cycle inhibition was identified as a common principle of TRAIL sensitization in melanoma cells. Mitochondrial apoptosis pathways, pro- and antiapoptotic Bcl-2 proteins as well as the rheostat consisted of Smac (Second mitochondria-derived activator of caspase) and XIAP (X-linked inhibitor of apoptosis protein) appeared to be of particular importance. Furthermore, the role of reactive oxygen species (ROS) was recognized in this setting. Inducible TRAIL resistance in melanoma can be explained by (i) high levels of antiapoptotic Bcl-2 proteins, (ii) high levels of XIAP, and (iii) suppressed Bax activity. These hurdles have to be overcome to enable the use of TRAIL in melanoma therapy. Several strategies appear as particularly promising, including new TRAIL receptor agonists, Smac and BH3 mimetics, as well as selective kinase inhibitors.

Low-power photodynamic therapy induces survival signaling in perihilar cholangiocarcinoma cells.

BackgroundPhotodynamic therapy (PDT) of solid cancers comprises the administration of a photosensitizer followed by illumination of the photosensitizer-replete tumor with laser light. This induces a state of local oxidative stress, culminating in the destruction of tumor tissue and microvasculature and induction of an anti-tumor immune response. However, some tumor types, including perihilar cholangiocarcinoma, are relatively refractory to PDT, which may be attributable to the activation of survival pathways in tumor cells following PDT (i.e., activator protein 1 (AP-1)-, nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB)-, hypoxia-inducible factor 1-alpha (HIF-1α)-, nuclear factor (erythroid-derived 2)-like 2 (NFE2L2)-, and unfolded protein response-mediated pathways).MethodsTo assess the activation of survival pathways after PDT, human perihilar cholangiocarcinoma (SK-ChA-1) cells were subjected to PDT with zinc phthalocyanine (ZnPC)-encapsulating liposomes. Following 30-minute incubation with liposomes, the cells were either left untreated or treated at low (50 mW) or high (500 mW) laser power (cumulative light dose of 15 J/cm2). Cells were harvested 90 min post-PDT and whole genome expression analysis was performed using Illumina HumanHT-12 v4 expression beadchips. The data were interpreted in the context of the survival pathways. In addition, the safety of ZnPC-encapsulating liposomes was tested both in vitro and in vivo.ResultsPDT-treated SK-ChA-1 cells exhibited activation of the hypoxia-induced stress response via HIF-1α and initiation of the pro-inflammatory response via NF-кB. PDT at low laser power in particular caused extensive survival signaling, as evidenced by the significant upregulation of HIF-1- (P < 0.001) and NF-кB-related (P < 0.001) genes. Low-power PDT was less lethal to SK-ChA-1 cells 90 min post-PDT, confirmed by annexin V/propidium iodide staining. In vitro toxicogenomics and toxicological testing in chicken embryos and mice revealed that the ZnPC-encapsulating liposomes are non-toxic.ConclusionsPDT-treated perihilar cholangiocarcinoma cells exhibit extensive survival signaling that may translate to a suboptimal therapeutic response and possibly tumor recurrence. These findings encourage the development of photosensitizer delivery systems with co-encapsulated inhibitors of survival pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1994-2) contains supplementary material, which is available to authorized users.

Abstract IA11: Monoclonal antibodies and in situ vaccination.

Abstract Traditional therapies for lymphoma have targeted features of the tumor, itself, such as cell surface molecules and intracellular signaling pathways. Increasingly, we are gaining therapeutic approaches that target the host microenvironment, and especially the host immune system. An exciting challenge is to combine these therapies in ways that are at least additive, and hopefully synergistic. We will present recent examples that combine therapies that target both the tumor and the host. Small molecule inhibitors of survival pathways in lymphoma cells, such as B cell receptor signaling, also have effects on the immune system. A case is point is Ibrutinib, a covalent inhibitor of Bruton's tyrosine kinase (Btk), recently approved for the treatment of CLL and MCL and with significant clinical activity in other lymphoid malignancies. Ibrutinib inhibits other members of the Tec kinase family, such as Itk, an important signaling molecule in subsets of T cells. By doing so, it can sculpt the T cell immune response and direct it against the tumor. Recent preclinical experiments indicate that Ibrutinib can enhance the T cell immune response induced by in situ vaccination as well as other immunotherapeutic maneuvers. These results imply a greater role for Ibrutinib than currently envisioned, one where it may be employed as an immune enhancer in addition to a targeted anti-tumor agent. Abscopal tumor responses can be induced in patients with lymphoma by local in situ vaccination. CpG ologonucleotide, a ligand for a Toll-like receptor, is injected into one site of tumor and low dose radiation is directed to the same site (Brody et. al. J Clin Oncol. 2010;28(28):4324). In preclinical animal models this treatment generates an anti-tumor T cell immune response. Such T cell responses can be enhanced by additional intra-tumoral injections of antibodies against immune checkpoints, such as CTLA4 and OX40 (Marabelle et al. J Clin Invest. 2013;123(6):2447). These antibodies work by depleting the negative T regulatory cells (Tregs) in the local tumor microenvironment, unleasing T effector cells to attack tumor at distant sites. Very low doses of checkpoint antibodies can be used to avoid autoimmune adverse effects. Such combinations of low dose radiation with immune enhancing agents may change the way we employ radiotherapy in the future- to help trigger immune responses rather than to sterilize the local tumor site. The therapeutic power of antibodies can be enhanced by stimulating ADCC, a primary mechanism of anti-tumor effect. One way to accomplish this is to target CD137, an activation molecule that appears on host NK cells after they encounter tumor cells coated by an anti-tumor antibody. In preclinical models the combination of an anti-CD20 with an anti-CD137 antibody is impressively synergistic (Kohrt et. al. Blood. 2011,117(8):2423). CD137 is also expressed on activated T cells and therefore the antibody against CD137 might also enhance an underlying host anti tumor T cell immune response. Two clinical trials are now under way to test the effect of an antibody against CD137 as a single agent and in combination with rituximab in patients with relapsed/refractory lymphoma. Citation Format: Ronald Levy. Monoclonal antibodies and in situ vaccination. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr IA11.

Sensitization of melanoma cells for TRAIL-induced apoptosis by activation of mitochondrial pathways via Bax

The death ligand TRAIL (TNF-related apoptosis-inducing ligand) represents a promising therapeutic strategy for metastatic melanoma, however prevalent and inducible resistance limits its applicability and therapeutic use. Recent work has revealed that combinations with survival pathway inhibitors could efficiently sensitize melanoma cells for TRAIL. Here, a particular role was attributed to the activation of Bax, which is regulated by phosphorylation. Thus, TRAIL resistance in melanoma is explained by three major steps, namely high levels of antiapoptotic Bcl-2 proteins, high levels of inhibitor of apoptosis proteins (cIAPs) and suppressed Bax activity. Importantly, Bid was activated in response to TRAIL alone also in resistant cells to antagonize Bcl-2, and Bax was activated in response to pathway inhibitors. However, only in combinations, mitochondrial apoptosis pathways were opened to result in release of Smac/DIABLO, which functions as antagonist of cIAPs. Opening the caspase cascade by Smac then allowed efficient induction of apoptosis. Thus, direct or indirect targeting of Bax represents a suitable strategy to overcome TRAIL resistance in melanoma and may allow the establishment of TRAIL-based therapeutic approaches.

Emerging therapeutic approaches in the management of metastatic castration-resistant prostate cancer.

Although treatment options for men with castration-resistant prostate cancer (CRPC) have improved with the recent and anticipated approvals of novel immunotherapeutic, hormonal, chemotherapeutic and bone-targeted agents, clinical benefit with these systemic therapies is transient and survival times remain unacceptably short. Thus, we devote the second section of this two-part review to discussing emerging therapeutic paradigms and research strategies that are entering phase II and III clinical testing for men with metastatic CRPC. We will discuss a range of emerging hormonal, immunomodulatory, antiangiogenic, epigenetic and cell survival pathway inhibitors in current clinical trials, with an emphasis on how these therapies may complement our existing treatment options.

Amacrine Cell Gene Expression and Survival Signaling: Differences from Neighboring Retinal Ganglion Cells

PURPOSE. To describe how developing amacrine cells and retinal ganglion cells (RGCs) differ in survival signaling and global gene expression. METHODS. Amacrine cells were immunopurified and processed for gene microarray analysis. For survival studies, purified amacrine cells were cultured at low density in serum-free medium, with and without peptide trophic factors and survival pathway inhibitors. The differences in gene expression between amacrine cells and RGCs were analyzed by comparing the transcriptomes of these two cell types at the same developmental ages. RESULTS. The amacrine cell transcriptome was very dynamic during development. Amacrine cell gene expression was remarkably similar to that of RGCs, but differed in several gene ontologies, including polarity- and neurotransmission-associated genes. Unlike RGCs, amacrine cell survival in vitro was independent of cell density and the presence of exogenous trophic factors, but necessitated Erk activation via MEK1/2 and AKT signaling. Finally, comparison of the gene expression profile of amacrine cells and RGCs provided a list of polarity-associated candidate genes that may explain the inability of amacrine cells to differentiate axons and dendrites as RGCs do. CONCLUSIONS. Comparison of the gene expression profile between amacrine cells and RGCs may improve our understanding of why amacrine cells fail to differentiate axons and dendrites during retinal development and of what makes amacrine cells differ in their resistance to neurodegeneration. Switching RGCs to an amacrine cell-like state could help preserve their survival in neurodegenerative diseases like glaucoma, and amacrine cells could provide a ready source of replacement RGCs in such optic neuropathies.

Signalling pathways involved in clinical responses to chemotherapy

Chemotherapeutic agents and also radiotherapy trigger a series of signalling pathways in the cells that activate not only the apoptotic machinery but also cell survival pathways. Some of these pathways are also altered by genetic changes in specific type of tumours, and are different even between patients with the same tumour. Among these pathways, the majority of survival signals involve the ERK, AKT and nuclear factor-kappaB pathways and those related to cell death, which are driven mainly either by inhibition of such survival networks or by upregulation of the JNK/p38 MAP-kinases. Thus, the efficacy of a given chemotherapy appears as a result of the balance between cell death and survival pathways elicited in each individual tumour. Modulation of such survival pathways would help to increase the efficacy of chemotherapy. Different strategies based on conventional chemotherapy have been used in the past with modest success. The availability of new molecules such as inhibitors of survival pathways and the use of new technologies for the study of individual tumours would have a positive impact on patient survival.

Crosstalk between EGFR and TrkB enhances ovarian cancer cell migration and proliferation

Ovarian cancer remains the leading cause of fatality among all gynecologic cancers, although promising therapies are in the making. It has been speculated that metastasis is critical for ovarian cancer, and yet the molecular mechanisms of metastasis in ovarian cancer are poorly understood. Growth factors have been proven to play important roles in cell migration associated with metastasis, and inhibition of growth factor receptors and their distinct cell signaling pathways has been intensively studied, and yet the uncovered interaction or crosstalk among various growth factor receptors complicates this otherwise promising approach. We investigated the crosstalk between EGFR and TrkB, both of which have been known to be important in cell survival and migration in response to EGF and BDNF. Our results showed that both EGF and BDNF induced cell migration and cell proliferation in cultured human ovarian cancer cells (Caov3 cell line). EGF and BDNF transactivated TrkB and EGFR respectively, and activated downstream cell survival components such as Akt. EGFR and TrkB kinase inhibitors inhibited EGF- and BDNF-induced TrkB and EGFR activation and Akt phosphorylation, and cell proliferation and migration. Using EGFR knockout cells, we further demonstrated that EGFR is required for EGF-induced cell migration. Collectively, our data indicate that EGFR and TrkB crosstalk each other in response to EGF and BDNF, leading to cell survival pathway activation in ovarian cancer cells. Our data suggest that a combination of inhibitors of both receptors with cell survival pathway inhibitors would provide a better outcome in the clinical treatment of ovarian cancer.

Inhibition of EGFR/PI3K/AKT cell survival pathway promotes TSA's effect on cell death and migration in human ovarian cancer cells

Trichostatin A (TSA), a hydroxamate-type inhibitor of mammalian histone deacetylases, is emerging as one of a potentially new class of anticancer agents. TSA is known to act by promoting the acetylation of histones, leading to uncoiling of chromatin and activation of a variety of genes implicated in the regulation of cell survival, proliferation, differentiation, and apoptosis. In addition, there is an increasing appreciation of the fact that TSA may act through mechanisms other than induction of histone acetylation. Accumulated experimental data indicate that TSA activates phosphatidyl inositol-3-kinase (PI3K)/AKT signaling. Using human ovarian cancer cell line Caov3 cells, we observed that TSA induced cell death in a time- and dose-dependent manner and also inhibited cell migration. TSA transiently activated EGFR tyrosine phosphorylation and AKT activation in a time- and dose-dependent manner, which had been inhibited by EGFR inhibitor PD153035 and PI3 kinase inhibitor LY294002. We also observed that TSA transiently induced survivin expression that had been inhibited by PD153035 and LY294002, suggesting that TSA-induced survivin expression is mediated by EGFR/PI3 kinase pathway. Combination of EGFR inhibitor 153035 or PI3 kinase inhibitor LY294002 with TSA enhanced TSA-induced cell death and TSA reduction of cell migration. Collectively, our data demonstrate that TSA transiently activated EGFR/PI3K/AKT cell survival pathway, leading to expression of survivin. Inhibition of this pathway enhanced TSA-induced cell death and inhibited cell migration. Our data suggest that combination of EGFR/PI3K/AKT cell survival pathway inhibitors with TSA be a better approach to ovarian cancer treatment.

Combined treatment with EGFR inhibitors and arsenite upregulated apoptosis in human EGFR-positive melanomas: a role of suppression of the PI3K-AKT pathway

Epidermal growth factor receptor (EGFR) is expressed, albeit at low or intermediate levels, in human melanomas at the different stages of tumor progression. Coexpression of EGFR with its ligand TGFalpha indicates their role in paracrine and autocrine growth regulation of melanomas. As it was previously observed for several types of cancer, specific inhibitors of EGFR-mediated signaling may reduce antiapoptotic properties of cancer cells and sensitize them to cytotoxic drugs. We recently reported that arsenite, particularly in combination with inhibitors of the PI3K-AKT and mitogen-activated protein kinase (MAPK) kinase (MEK)-extracellular signal-regulated kinase (ERK) pathways, induces high levels of apoptosis in different melanomas. Since EGFR signaling operates via activation of the PI3K-AKT and MEK-ERK pathways, we suggested that the combination of arsenite and EGFR inhibitors might also effectively induce apoptosis in melanoma. Here, we demonstrate that a moderate concentration of arsenite (5-10 muM) indeed upregulates apoptosis induced by EGFR inhibitors in EGFR-positive melanomas. In contrast, induction of apoptosis in melanomas with negligible surface expression of EGFR or with defective EGFR signaling requires direct suppression of the PI3K-AKT and MAPK pathways by specific pharmacological inhibitors in the presence of arsenite. Under these conditions, metastatic melanoma cell lines undergo TNF-related apoptosis-inducing ligand (TRAIL)- and tumor necrosis factor alpha (TNFalpha)-mediated apoptosis. Taken together, these data provide additional approaches in sensitizing melanomas to the cytotoxic effects of specific inhibitors of survival pathways.