Renata de Freitas Saito

A novel proteasome inhibitor acting in mitochondrial dysfunction, ER stress and ROS production

SummaryIn cancer-treatment, potentially therapeutic drugs trigger their effects through apoptotic mechanisms. Generally, cell response is manifested by Bcl-2 family protein regulation, the impairment of mitochondrial functions, and ROS production. Notwithstanding, several drugs operate through proteasome inhibition, which, by inducing the accumulation and aggregation of misfolded or unfolded proteins, can lead to endoplasmic reticulum (ER) stress. Accordingly, it was shown that Amblyomin-X, a Kunitz-type inhibitor identified in the transcriptome of the Amblyomma cajennense tick by ESTs sequence analysis of a cDNA library, obtained in recombinant protein form, induces apoptosis in murine renal adenocarcinoma (RENCA) cells by: inducing imbalance between pro- and anti-apoptotic Bcl-2 family proteins, dysfunction/mitochondrial damage, production of reactive oxygen species (ROS), caspase cascade activation, and proteasome inhibition, all ER-stress inductive. Moreover, there was no manifest action on normal mouse-fibroblast cells (NHI3T3), suggesting an Amblyomin-X tumor-cell selectivity. Taken together, these evidences indicate that Amblyomin-X could be a promising candidate for cancer therapy.

Expression of PAFR as part of a prosurvival response to chemotherapy: a novel target for combination therapy in melanoma.

Melanoma cells express the platelet-activating factor receptor (PAFR) and, thus, respond to PAF, a bioactive lipid produced by both tumour cells and those in the tumour microenvironment such as macrophages. Here, we show that treatment of a human melanoma SKmel37 cell line with cisplatin led to increased expression of PAFR and its accumulation. In the presence of exogenous PAF, melanoma cells were significantly more resistant to cisplatin-induced cell death. Inhibition of PAFR-dependent signalling pathways by a PAFR antagonist (WEB2086) showed chemosensitisation of melanoma cells in vitro. Nude mice were inoculated with SKmel37 cells and treated with cisplatin and WEB2086. Animals treated with both agents showed significantly decreased tumour growth compared to the control group and groups treated with only one agent. PAFR accumulation and signalling are part of a prosurvival program of melanoma cells, therefore constituting a promising target for combination therapy for melanomas.

Emerging targets for combination therapy in melanomas

Cutaneous melanomas are often difficult to treat when diagnosed in advanced stages. Melanoma cells adapt to survive in extreme environmental conditions and are among the tumors with larger genomic instability. Here we discuss some intrinsic and extrinsic mechanisms of resistance of melanoma cells to both conventional and target therapies, such as autophagy, adaptation to endoplasmic reticulum stress, metabolic reprogramming, mechanisms of tumor repopulation and the role of extracellular vesicles in this later phenomenon. These biological processes are potentially targetable and thus provide a platform for research and discovery of new drugs for combination therapy to manage melanoma patient treatment.

Analysis of the insulin-like growth factor 1 receptor gene in children born small for gestational age: in vitro characterization of a novel mutation (p.Arg511Trp).

Insulin‐like growth factor 1 insensitivity caused by IGF1R mutations has been previously identified as one of the causes of growth impairment in children born small for gestational age (SGA).

Polymorphisms in the p27kip-1 and prohibitin genes denote novel genes associated with melanoma risk in Brazil, a high ultraviolet index region.

Ultraviolet (UV) radiation is a major environmental risk factor to the development of cutaneous melanoma as it induces pyrimidine dimers in DNA. Genes that exert their function by arresting the cell cycle are critical to avoid carcinogenic mutations, allowing the processing of DNA repair systems. This study was carried out to evaluate the role of polymorphisms in cell cycle genes such as TP53, p27kip-1, CDKN2A, prohibitin, and GADD153 in melanoma risk as well as their influence on known risk factors in a high UV index region. A hospital-based case–control study was carried out in Brazil to evaluate the contribution of polymorphisms in cell cycle genes toward melanoma risk. The study comprised 202 melanoma patients and 210 controls. The polymorphisms analyzed were TP53 Arg72Pro, p27kip-1 Val109Gly, GADD153 Phe10Phe (rs697221), CDKN2A 3′UTR C540G, and prohibitin 3′UTR C1703T. As regards, p27kip-1 Val109Gly, both heterozygous and homozygous Gly genotypes were shown to be protective genotypes on calculating both crude and adjusted odds ratios (ORs) for age, sex, and educational level [OR 0.37; 95% confidence interval (CI) 0.16–0.87; P<0.05]. Similarly, the prohibitin TT genotype increased melanoma risk in the crude and adjusted analyses (OR 2.40; 95% CI 1.10–5.26; P<0.05). The p27kip-1 Gly protective genotype decreased the risk for melanoma in a stratified analysis of the known risk factors such as hair and eye color, sunburns, pigmented lesions, and European ancestry. The prohibitin TT genotype increased the risk of melanoma by such host factors. Our results showed for the first time that polymorphisms in p27kip-1 Val109Gly and in prohibitin 3′UTR C1703T genotypes modulate the risk to melanoma in a high UV index region.

Accumulation of prohibitin is a common cellular response to different stressing stimuli and protects melanoma cells from ER stress and chemotherapy-induced cell death

Melanoma is responsible for most deaths among skin cancers and conventional and palliative care chemotherapy are limited due to the development of chemoresistance. We used proteomic analysis to identify cellular responses that lead to chemoresistance of human melanoma cell lines to cisplatin. A systems approach to the proteomic data indicated the participation of specific cellular processes such as oxidative phosphorylation, mitochondrial organization and homeostasis, as well as the unfolded protein response (UPR) to be required for the survival of cells treated with cisplatin. Prohibitin (PHB) was among the proteins consistently accumulated, interacting with the functional clusters associated with resistance to cisplatin. We showed PHB accumulated at different levels in melanoma cell lines under stressing stimuli, such as (i) treatment with temozolomide (TMZ), dacarbazine (DTIC) and cisplatin; (ii) serum deprivation; (iii) tunicamycin, an UPR inducer. Prohibitin accumulated in the mitochondria of melanoma cells after cisplatin and tunicamycin treatment and its de novo accumulation led to chemoresistance melanoma cell lines. In contrast, PHB knock-down sensitized melanoma cells to cisplatin and tunicamycin treatment. We conclude that PHB participates in the survival of cells exposed to different stress stimuli, and can therefore serve as a target for the sensitization of melanoma cells to chemotherapy.

Galectin-3 sensitized melanoma cell lines to vemurafenib (PLX4032) induced cell death through prevention of autophagy

Melanoma is a current worldwide problem, as its incidence is increasing. In the last years, several studies have shown that melanoma cells display high levels of autophagy, a self-degradative process that can promote survival leading to drug resistance. Consequently, autophagy regulation represents a challenge for cancer therapy. Herein, we showed that galectin-3 (Gal-3), a β-galactoside binding lectin which is often lost along melanoma progression, is a negative regulator of autophagy in melanoma cells. Our data demonstrated that Gal-3low/negative cells were more resistant to the inhibition of the activity of the cancer driver gene BRAFV600E by vemurafenib (PLX4032). Interestingly, in these cells, starvation caused further LC3-II accumulation in cells exposed to chloroquine, which inhibits the degradative step in autophagy. In addition, Gal-3 low/negative tumor cells accumulated more LC3-II than Gal-3 high tumor cells in vivo. Resistance of Gal-3low/negative cells was associated with increased production of superoxide and activation of the Endoplasmic Reticulum (ER) stress response, as evaluated by accumulation of GRP78. Pharmacological inhibition of autophagy with bafilomycin A reversed the relative resistance of Gal-3low/negative cells to vemurafenib treatment. Taken together, these results show that the autophagic flux is dependent on Gal-3 levels, which attenuate the prosurvival role of autophagy.

Abstract A67: Jarid1b protects from metformin-induced chemosensitization to cisplatin through p53 downregulation in NSCLC

Background: Instead of nontransformed cells, tumor cells use the glycolytic pathway as their main energetic source, even when oxygen is fully available. Recently, a new tumor subpopulation that is characterized by the overexpression of the histone demethylase Jarid1b has been found in melanomas and other tumors. Jarid1b-high cells use the mitochondria as the main energy source and are more resistant to chemotherapy. Also, Jarid1b-high cells have downregulation of p53. Changes in the metabolic profile of chemoresistant tumor cells could restore chemosensitization to drugs that induce high levels of oxidative stress, such as cisplatin. A possible strategy to modify the metabolism of tumor cells that uses the mitochondria as the main energy source is to use metformin. Many reports shows that patients who use metformin as treatment for type II diabetes have less incidence of cancer in general. However, many other reports show that metformin is unable to improve cancer treatment when used as an adjuvant therapy. Results from our group show that the use of metformin as a chemosensitizing agent depends on p53 status. When p53 is present, metformin can sensitize cells to cisplatin. Cells that lose p53 expression, like Jarid1b-high cells, or p53 KO cells are resistant to the combination of metformin and cisplatin. Aim: To evaluate when metformin can be used as a chemosensitizing agent to cisplatin. Methods: The human NSCLC A549 (p53 WT) and H1299 (p53 KO) and the colon carcinoma cells HCT 116 (p53 WT) and HCT 116 (CRISPR to p53) were used in this study. A549 cells were pretreated with low dose of cisplatin (PT-Pop) to induce Jarid1b overexpression and p53 downregulation. Results: Metformin sensitized A549 cells to cisplatin, leading to high levels of cell death. On the PT-Pop, metformin was not able to induce chemosensitization to cisplatin due to Jarid1b overexpression and p53 downregulation. Also, on the p53 KO cells H1299, metformin was not able to induce chemosensitization to cisplatin. Jarid1b inhibition during pretreatment with low dose of cisplatin on A549 cells restores p53 expression, allowing metformin to chemosensitize the A549 PT-Pop to cisplatin. Overexpression of p53 on the Jarid1b-high cells in the PT-Pop also restores the ability of metformin to sensitize these cells to cisplatin. Metformin sensitized the p53 WT HCT 116 cells to cisplatin, but not the HCT 116 p53 KO cells. Metformin translocates p53 to the mitochondria in A549 cells and the inhibition of the p53 compartmentalization to the mitochondria protects from metformin-induced chemosensitization to cisplatin. Metformin does not lead to ROS increase on A549 cells that were not pretreated with low dose of cisplatin. Only on PT-Pop, metformin was able to increase ROS levels. In the PT-Pop, where Jarid1b was inhibited by PBIT, metformin was able to increase even more ROS levels on A549 cells. Also, in NSCLC, metformin was able to increase lactate production only on A549 cells that was not pretreated with low dose of cisplatin. In the PT-Pop of A549 cells and in H1299 cells, metformin could not increase lactate production. Conclusion: Metformin needs the presence of p53 in NSCLC and colon cancer cells to lead to chemosensitization to cisplatin. Citation Format: Tharcisio Citrangulo Tortelli, Jr., Rodrigo Esaki Tamura, Michele Tatiana Pereira Tomitao, Silvina Odete Bustos, Renata de Freitas Saito, Sarah Milani de Moraes Leandrini, Mayara D9Aurea Jacomassi, Ulysses Ribeiro, Jr., Bryan Erik Strauss, Roger Chammas. Jarid1b protects from metformin-induced chemosensitization to cisplatin through p53 downregulation in NSCLC [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; Sao Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A67.

G D3 ganglioside-enriched extracellular vesicles stimulate melanocyte migration

Melanomas often accumulate gangliosides, sialic acid-containing glycosphingolipids found in the outer leaflet of plasma membranes, as disialoganglioside GD3 and its derivatives. Here, we have transfected the GD3 synthase gene (ST8Sia I) in a normal melanocyte cell line in order to evaluate changes in the biological behavior of non-transformed cells. GD3-synthase expressing cells converted GM3 into GD3 and accumulated both GD3 and its acetylated form, 9-O-acetyl-GD3. Melanocytes were rendered more migratory on laminin-1 surfaces. Cell migration studies using the different transfectants, either treated or not with the glucosylceramide synthase inhibitor d-1-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (PPPP), allowed us to show that while GM3 is a negative regulator of melanocyte migration, GD3 increases it. We showed that gangliosides were shed to the matrix by migrating cells and that GD3 synthase transfected cells shed extracellular vesicles (EVs) enriched in GD3. EVs enriched in GD3 stimulated cell migration of GD3 negative cells, as observed in time lapse microscopy studies. Otherwise, EVs shed by GM3+veGD3-ve cells impaired migration and diminished cell velocity in cells overexpressing GD3. The balance of antimigratory GM3 and promigratory GD3 gangliosides in melanocytes could be altered not only by the overexpression of enzymes such as ST8Sia I, but also by the horizontal transfer of ganglioside enriched extracellular vesicles. This study highlights that extracellular vesicles transfer biological information also through their membrane components, which include a variety of glycosphingolipids remodeled in disease states such as cancer.

Abstract B21: Endoplasmic reticulum stress conditioned melanoma cell lines to chemotherapy-induced cell death.

Melanoma is among the most aggressive malignancies with increasing worldwide incidence and there is no effective treatment for the metastatic disease. The absence of an effective therapy may be due to adaptation and selection of melanoma cells to endoplasmic reticulum (ER) stress (Hersey P and Zhang XD). We showed that GADD153, one of the components of the ER stress-mediated apoptosis pathway, was mostly excluded from the nucleus of primary and metastatic melanoma cells compared with nevus cells. These data suggest that the unexpected GADD153 cellular localization could be involved in melanoma cell adaption to ER stress, since GADD153 accumulates in the nucleus during ER stress. Unfolded protein response (UPR) signaling induced in response to ER stress is a dual process that acts inducing a protective response to restore ER homeostasis or cell death in a severe or persistent ER stress induction. We investigated if induction of ER stress was a potential strategy to chemosensitize melanoma cells to genotoxic drugs by surpassing the adaptive levels to ER stress. We first treated human melanoma cells (SBCl2, Skmel28 and Mel85) with tunicamycin (TUN), an ER stress inducer before cis-diamminedichloroplatinum(II)(CDDP) treatment. CDDP is a low cost chemotherapeutic drug currently used in Brazil as a second line for melanoma treatment, especially in youngsters. We demonstrated an >50% increase in the percentage of cells in cell death process with TUN/CDDP treatment when compared to CDDP only. UPR markers, GRP78 and GADD153, were induced by TUN. One of the cellular mechanisms that are regulated by ER stress is autophagy. Accordingly, we observed an increase in the acidic vesicular organelles, assessed by acridine orange staining, in response to combined treatment. Autophagy inhibition with 3-Methyladenine increased TUN/CDDP induced-cell death, suggesting that autophagy plays a protective role in this response. Reactive oxygen species were involved in TUN and CDDP response, but more prominently in combined treatment response. However, the oxidative stress inhibition with N-acetylcysteine did not alter the sensitization effect of TUN pretreatment. We also tested temozolomide (TMZ), an equivalent drug to the active form of dacarbazine, the first line treatment of melanoma. The same sensitization capacity was observed in cells pretreated with TUN and further treated with TMZ. These results indicate that tumor cells could be preconditioned to cell death if exposed to a first ER stressor, such as TUN, which would compromise an effective adaptive response to a cell death inducer, as CDDP and TMZ. This combined approach may be a promising strategy for melanoma therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B21. Citation Format: Renata F. Saito, Andreia H. Otake, Margarita M. Cortes, Roger Chammas. Endoplasmic reticulum stress conditioned melanoma cell lines to chemotherapy-induced cell death. [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 B21.