Maria L. Rodriguez

PP2A Inhibition Is a Common Event in Colorectal Cancer and Its Restoration Using FTY720 Shows Promising Therapeutic Potential

Protein phosphatase 2A (PP2A) is a tumor suppressor that regulates many signaling pathways crucial for cell transformation. In fact, decreased activity of PP2A has been reported as a recurrent alteration in many types of cancer. Here, we show that PP2A is frequently inactivated in patients with colorectal cancer, indicating that PP2A represents a potential therapeutic target for this disease. We identified overexpression of the endogenous PP2A inhibitors SET and CIP2A, and downregulation of regulatory PP2A such as PPP2R2A and PPP2R5E, as contributing mechanisms to PP2A inhibition in colorectal cancer. Moreover, we observed that its restoration using FTY720 impairs proliferation and clonogenic potential of colorectal cancer cells, induces caspase-dependent apoptosis, and affects AKT and extracellular signal-regulated kinase-1/2 activation status. Interestingly, treatment with FTY720 showed an additive effect with 5-fluorouracil, SN-38, and oxaliplatin, drugs used in standard chemotherapy in patients with colorectal cancer. These results suggest that PP2A activity is commonly decreased in colorectal cancer cells, and that the use of PP2A activators, such as FTY720, might represent a potential novel therapeutic strategy in colorectal cancer. Mol Cancer Ther; 13(4); 938–47. ©2014 AACR.

Pterostilbene: Biomedical applications.

Abstract Resveratrol and its naturally dimethylated analog, pterostilbene, show similar biological activities. However, the higher in vivo bioavailability of pterostilbene represents a fundamental advantage. The main focus of this review is on biomedical applications of pterostilbene. The metabolism and pharmacokinetics of this stilbene in inflammatory dermatoses and photoprotection, cancer prevention and therapy, insulin sensitivity, blood glycemia and lipid levels, cardiovascular diseases, aging, and memory and cognition are addressed. Safety and toxicity, as well as recommendations for future research and biomedical uses, are discussed. This review includes comparisons between pterostilbene and other polyphenols, with particular emphasis on resveratrol. Potential benefits of using combinations of different polyphenols are considered. Based on present evidences we conclude that pterostilbene is an active phytonutrient and also a potential drug with multiple biomedical applications.

Pterostilbene-induced tumor cytotoxicity: a lysosomal membrane permeabilization-dependent mechanism.

The phenolic phytoalexin resveratrol is well known for its health-promoting and anticancer properties. Its potential benefits are, however, limited due to its low bioavailability. Pterostilbene, a natural dimethoxylated analog of resveratrol, presents higher anticancer activity than resveratrol. The mechanisms by which this polyphenol acts against cancer cells are, however, unclear. Here, we show that pterostilbene effectively inhibits cancer cell growth and stimulates apoptosis and autophagosome accumulation in cancer cells of various origins. However, these mechanisms are not determinant in cell demise. Pterostilbene promotes cancer cell death via a mechanism involving lysosomal membrane permeabilization. Different grades of susceptibility were observed among the different cancer cells depending on their lysosomal heat shock protein 70 (HSP70) content, a known stabilizer of lysosomal membranes. A375 melanoma and A549 lung cancer cells with low levels of HSP70 showed high susceptibility to pterostilbene, whereas HT29 colon and MCF7 breast cancer cells with higher levels of HSP70 were more resistant. Inhibition of HSP70 expression increased susceptibility of HT29 colon and MCF7 breast cancer cells to pterostilbene. Our data indicate that lysosomal membrane permeabilization is the main cell death pathway triggered by pterostilbene.

Stress hormones promote growth of B16-F10 melanoma metastases: an interleukin 6- and glutathione-dependent mechanism

BackgroundInterleukin (IL)-6 (mainly of tumor origin) activates glutathione (GSH) release from hepatocytes and its interorgan transport to B16-F10 melanoma metastatic foci. We studied if this capacity to overproduce IL-6 is regulated by cancer cell-independent mechanisms.MethodsMurine B16-F10 melanoma cells were cultured, transfected with red fluorescent protein, injected i.v. into syngenic C57BL/6J mice to generate lung and liver metastases, and isolated from metastatic foci using high-performance cell sorting. Stress hormones and IL-6 levels were measured by ELISA, and CRH expression in the brain by in situ hybridization. DNA binding activity of NF-κB, CREB, AP-1, and NF-IL-6 was measured using specific transcription factor assay kits. IL-6 expression was measured by RT-PCR, and silencing was achieved by transfection of anti-IL-6 small interfering RNA. GSH was determined by HPLC. Cell death analysis was distinguished using fluorescence microscopy, TUNEL labeling, and flow cytometry techniques. Statistical analyses were performed using Student’s t test.ResultsPlasma levels of stress-related hormones (adrenocorticotropin hormone, corticosterone, and noradrenaline) increased, following a circadian pattern and as compared to non-tumor controls, in mice bearing B16-F10 lung or liver metastases. Corticosterone and noradrenaline, at pathophysiological levels, increased expression and secretion of IL-6 in B16-F10 cells in vitro. Corticosterone- and noradrenaline-induced transcriptional up-regulation of IL-6 gene involves changes in the DNA binding activity of nuclear factor-κB, cAMP response element-binding protein, activator protein-1, and nuclear factor for IL-6. In vivo inoculation of B16-F10 cells transfected with anti-IL-6-siRNA, treatment with a glucocorticoid receptor blocker (RU-486) or with a β-adrenoceptor blocker (propranolol), increased hepatic GSH whereas decreased plasma IL-6 levels and metastatic growth. Corticosterone, but not NORA, also induced apoptotic cell death in metastatic cells with low GSH content.ConclusionsOur results describe an interorgan system where stress-related hormones, IL-6, and GSH coordinately regulate metastases growth.

Role of Natural Stilbenes in the Prevention of Cancer

Natural stilbenes are an important group of nonflavonoid phytochemicals of polyphenolic structure characterized by the presence of a 1,2-diphenylethylene nucleus. Stilbenes have an extraordinary potential for the prevention and treatment of different diseases, including cancer, due to their antioxidant, cell death activation, and anti-inflammatory properties which associate with low toxicity under in vivo conditions. This review aims to discuss various approaches related to their mechanisms of action, pharmacological activities in animal models and humans, and potential chemoprevention in clinical studies. The biological activity of natural stilbenes is still incompletely understood. Furthermore, after administration to animals or humans, these molecules are rapidly metabolized. Thus pharmacokinetics and/or activities of the natural structures and their metabolites may be very different. Novel drug formulations have been postulated in order to improve stability and bioavailability, to minimize side effects, and to facilitate interaction with their domains in target proteins. These pharmacological improvements should lead stilbenes to become effective candidates as anticancer drugs.

Abstract 2810: Pterostilbene, a natural phytoalexin, weakens the antioxidant defenses of aggressive cancer cells in vivo: a pituitary gland- and Nrf2-dependent mechanism

Polyphenolic phytochemicals have anticancer properties. However, in mechanistic studies lack of correlation to the bioavailable concentrations is a critical issue. We studied the underlying mechanisms using different human melanomas (A2058, MeWo and MelJuso) and pancreatic cancers (AsPC-1 and BxPC-3) (with genetic backgrounds correlating with most tumors in patients), growing in nude mice as xenografts, and pterostilbene (Pter, 3’,5’-dimethoxy-4-stilbenol; abundant in e.g. blueberries and a natural dimethoxylated analog of resveratrol). RESULTS: Intravenous administration of Pter decreased human melanoma and pancreatic cancer growth (an effect associated with lower rates of tumor cell proliferation and increased apoptosis) in vivo. However Pter, at levels measured within the tumors, did not affect cancer growth or viability in vitro. Pter inhibited pituitary production of the adrenocorticotropin hormone (ACTH), decreased plasma levels of corticosterone and, thereby, down regulated the glucocorticoid receptor- and nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-dependent antioxidant defense system in growing cancers (i.e. the glutathione and thioredoxin systems, superoxide dismutases, catalase, and NADPH supplying dehydrogenases). Exogenous corticosterone or genetically-induced Nrf2 overexpression in the cancer cells prevented the inhibition of tumor growth and antioxidant defenses in these malignant cells. Glutathione depletion (selected as a potential anti-cancer strategy) facilitated the complete elimination by chemotherapy of cancer cells isolated from mice treated with Pter. CONCLUSIONS: This report shows a novel link between a neuroendocrine system- and stress response-dependent mechanism and the regulation of cancer growth in vivo. Natural polyphenols can interfere with the growth and defense of cancer cells by down-regulating the pituitary gland-dependent ACTH synthesis. Lower levels of plasma ACTH cause a decrease in the suprarenal glands-dependent glucocorticoid production, thus decreasing the glucocorticoid receptor and Nrf2-dependent signaling/transcription and the antioxidant protection of melanoma and pancreatic cancer cells. Hence facilitating identification of molecular targets to sensitize aggressive cancers to oncotherapy. Citation Format: Maria Benlloch, Soraya L. Valles, Maria L. Rodriguez, J. Antoni Sirerol, Javier Alcacer, Jose Pellicer, Rosario Salvador, Concha Cerda, Guillermo T. Saez, Jose M. Estrela. Pterostilbene, a natural phytoalexin, weakens the antioxidant defenses of aggressive cancer cells in vivo: a pituitary gland- and Nrf2-dependent mechanism. [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 2810.

Abstract 753: Genomic alterations of autophagy genes disrupts autophagic flux in human lung adenocarcinomas

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Targeted therapy using EGFR tyrosine kinase inhibitor (TKI) is a standard therapy for a subset of non-small cell lung cancer (NSCLC) patients with lung adenocarcinomas (LADs) harboring EGFR kinase domain mutations; however, EGFR TKI therapy shows limited efficacy due to de novo and acquired resistance. Consequently, formulating strategies to potentiate the efficacy of EGFR TKI is of great interest. In EGFR TKI sensitive cells harboring EGFR mutation, it has been shown that EGFR inhibition induces autophagy to protect the cells from metabolic stress. Hydroxychloroquine (HQ), an inhibitor of autophagy, has been shown to potentiate EGFR TKIs in preclinical models, however, preliminary results from recent phase II clinical trials shows no added benefits to the development of resistance to erlotinib in patients with EGFR mutant NSCLC. The result suggests additional layers of mechanisms in controlling autophagy in EGFR mutated NSCLC. Furthermore, little is known about genomic alterations affecting these autophagy genes in lung cancer samples. The meta-analysis of The Cancer Genome Atlas (TCGA) database shows that essential genes for autophagic flux such as ATG5 and ATG7 tend to reduce their expression levels and/or are deleted in a significant proportion of LAD patients, suggesting a more complex scenario where the presence of these genetic alterations may lead to a structurally deficient autophagy. Besides, the detailed CNV analysis of the Cancer Cell Line Encyclopedia (CCLE) dataset suggests that a small subset of NSCLC cell lines may have lost ATG7 locus at chromosome 3. Our validation study showed that, as expected, EGFR-mutant H1650 cells exhibit an intragenic deletion at ATG7 and lack of protein expression. Moreover, functional assays of autophagic flow by western blot and confocal microscopy demonstrated that ATG7-deficient H1650 cells are not able to activate autophagy, whereas infection with lentivirus expressing ectopic ATG7 reconstituted autophagic flux and sensitivity to autophagy inhibitors including HQ. Taken together, our results suggest that genomic biomarkers based on autophagy genes could allow stratification of tumors, and selection of those candidates who could benefit from anti-autophagy therapy. Citation Format: Ines Pulido, Juan L. Pascual, Margaret Soucheray, Maria L. Rodriguez, Daniel T. Crespo, Salvador Aparisi, Joan A. Sirerol, Salvador Mena, Javier Pereda, Fatima Al-shahrour, Angel L. Ortega, Takeshi Shimamura, Julian Carretero. Genomic alterations of autophagy genes disrupts autophagic flux in human lung adenocarcinomas. [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 753. doi:10.1158/1538-7445.AM2015-753

Abstract A292: Salinomycin, an anti-cancer stem cell antibiotic, overcomes acquired resistance to BRAF inhibitors in BRAF-mutant human melanoma cell lines.

Advanced malignant melanoma is one of the most lethal cancers, because it is highly metastatic and refractory to conventional chemotherapy. About 60% of melanomas harbor oncogenic BRAF mutations which aberrantly activate MEK/ERK signaling pathway. BRAF and MEK inhibitors have been shown efficacious in patients with BRAF-mutant melanoma, but there is not effective target therapy for BRAF wild type melanomas. Unfortunately acquired resistance to BRAF targeted therapies is a common event: 50% of treated patients progressed within 6 to 7 months after the initiation of treatment. Resistance is associated with reactivation of the MAPK pathway (through development of de novo NRAS, NF1 or MEK mutations) or activation of parallel pathways, such as the PI3K/AKT pathway. Cancer stem cells (CSCs) represent a subpopulation of tumor cells that possess self-renewal and tumor initiation capacity. It has been postulated that CSC could be linked to acquired resistance to both conventional and targeted therapies. Salinomycin, an ionophore antibiotic, has been shown to selectively kill CSCs in several types of human cancers, most likely by interfering with ABC drug transporters, the Wnt/β-catenin signaling pathway, and other CSC pathways. In this study, we have analyzed a wide panel of human melanoma cell lines with a known mutational status of BRAF, tumor suppressors, and other frequently mutated oncogenes in melanoma, and their sensibility to several protein kinase inhibitors (PKIs) in vitro, including the BRAF inhibitor vemurafenib. We have combined these PKIs with the CSC inhibitor, salinomycin, in order to find therapeutically relevant synergies. To better characterize CSC phenotype, we carried out tumorsphere formation assays in vitro, FACS analysis of CSC canonical markers, to find that salinomycin sensitizes BRAF-wild type melanoma cell lines to the cytotoxic effect of particular PKIs. Importantly, we demonstrated that salinomycin-PKI combination therapy supresses the emergence of vemurafenib-resistant subpopulations in BRAF-mutant melanoma cell lines. Overall, we find that targeting CSC property of wild-type BRAF inhibitor with salinomycin significantly potentiates therapeutic activity of PKIs. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A292. Citation Format: Daniel Crespo, Ines Pulido, Maria L. Rodriguez, Salvador Aparisi, Alejandro Lopez, Margaret Soucheray, Fatima Al-Shahrour, Takeshi Shimamura, Angel Ortega, Julian Carretero. Salinomycin, an anti-cancer stem cell antibiotic, overcomes acquired resistance to BRAF inhibitors in BRAF-mutant human melanoma cell lines. [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 A292.

Abstract 1280: Pterostilbene exerts full protection against UVB-induced skin carcinogenesis.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Epidemiological, clinical and laboratory studies have demonstrated that solar ultraviolet (UV) radiation is the main cause of nonmelanoma skin cancer (i.e., basal cell and squamous cell carcinoma) and it is also a prime factor in the etiology of cutaneous melanoma. The cancer-causing effects of solar exposure on the skin are mainly produced by UV-B radiation in the 290- to 320-nm range, the same range that produces burning in human skin (erythema), inflammation, oxidative stress, DNA damage, etc. Thus, and considering the alarming numbers of skin cancers being diagnosed around the world, it is increasingly evident the need of an effective protection against UV radiation. Polyphenols (PFs) are abundant in many fruits and vegetables, wine, tea, and various dietary supplements. The role of these natural molecules as potential anticarcinogens and photoprotectors has been postulated (e.g. Clifford JL & DiGiovanni J. Cancer Prev Res 3: 132-5, 2010). Resveratrol (trans-3,5,4′-trihydroxystilbene; RES) is a phytoalexin present in a wide variety of plant species, where its synthesis is induced by stress conditions. The cancer chemopreventive activity of RES was first reported by Jang et al [Science 275, 218 - 220 (1997)] in a model of skin carcinogenesis where topic administration of this polyphenol inhibited multistage mouse skin carcinogenesis. Equally promising action is exerted by resveratrol analogues, mainly pterostilbene (3,5-dimethoxy-4′-hydroxy-transstilbene: PTER), which shows a higher half-life and more potent anticancer effects in vivo than RES. The long-term UV-B administration (180 mJ/cm2; 3 doses/week; for a total of 30 weeks) reproduces, in an animal model, which are the consequences in humans of receiving chronic UV-B radiations. We observed that pretreatment of the skin with PTER prevented UV-B-induced skin tumorigenesis (∼90 % of tumor free-mice at the end of treatment, n=20; P<0.01). Our aim was to study the key molecular mechanisms involved in this strong anticancer effect elicited by PTER. Our results indicate that PTER efficiently avoid skin carcinogenesis induced by solar radiation by decreasing DNA mutagenesis, and protein and lipid oxidation. Citation Format: Angel Ortega, Maria L. Rodriguez, Joan A. Sirerol, Ines Pulido, Daniel Crespo, Julian Carretero, Miguel A. Asensi, Salvador Mena, Jose M. Estrela. Pterostilbene exerts full protection against UVB-induced skin carcinogenesis. [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 1280. doi:10.1158/1538-7445.AM2013-1280

Abstract 1690: Differential autophagy activation in KRAS and EGFR mutant lung adenocarcinomas.

Lung cancer is the leading cause of cancer deaths in western countries, and adenocarcinomas (LADs) are the most frequent histological subtype. The aberrant activation of the kinases promotes plethora of tumorigenic processes, mainly through PI3K and MAPK oncogenic pathways leading to oncogene addiction. The activation of PI3K pathway deregulates mTOR, a master kinase for cell growth and autophagy. Autophagy can be pro- or anti- tumorigenic, however its roles in protecting tumors exposed to metabolic stress under chemotherapy are considered as a survival mechanism for the tumors leading to acquired resistance. Consequently, the inhibition of autophagy is an attractive therapy to prevent the emergence of acquired resistance. Activating mutations in EGFR and KRAS are mutually exclusive and are the most frequent oncogene activation in LAD. There are considerable differences in the control of signalling pathways including PI3K-mTOR axis. Therefore, it is hypothesized that the susceptibility to autophagy differs depending on EGFR or KRAS mutational status. To test the hypothesis, we applied nutrient-starvation conditions, the most well-known inducer of autophagy, to a panel of LAD cell lines bearing known EGFR or KRAS mutations. As a positive control, cells were treated with rapamycin, which also induced autophagy inhibiting MTOR activity. Autophagy activation was analyzed by: i) Western blot of LC3-II and p62/SQSTM1 protein levels, that measures autophagy flux and autophagic protein degradation, respectively; and ii) autophagolysosome detection and lysosomal activity by fluorescent microscopy. Our data suggest that KRAS-mutant LAD cell lines are able to activate autophagy, whereas EGFR-mutant are not, under starvation stress. Furthermore, we found a potential interaction between mutant EGFR and Beclin 1 (BECN1, a protein required for the initiation of the autophagosome) at the cell membrane proximity, using co-immunolocalization by confocal microscopy and immunoprecipitation followed by Western blot. Together, these data provide a compelling rationale to investigate anti-autophagy therapy in mutant KRAS LADs and warrant further investigation in the regulation of autophagy via a novel link between Beclin1 and mutant EGFR. Citation Format: Maria L. Rodriguez, Ines Pulido, Margaret Soucheray, Daniel Crespo, Fatima Al-shahrour, Takeshi Shimamura, Angel Ortega, Julian Carretero. Differential autophagy activation in KRAS and EGFR mutant lung adenocarcinomas. [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 1690. doi:10.1158/1538-7445.AM2013-1690