Erdene Baljinnyam

Store-Operated Ca2+ Entry (SOCE) Regulates Melanoma Proliferation and Cell Migration

Store-operated Ca2+ entry (SOCE) is a major mechanism of Ca2 + import from extracellular to intracellular space, involving detection of Ca2+ store depletion in endoplasmic reticulum (ER) by stromal interaction molecule (STIM) proteins, which then translocate to plasma membrane and activate Orai Ca2+ channels there. We found that STIM1 and Orai1 isoforms were abundantly expressed in human melanoma tissues and multiple melanoma/melanocyte cell lines. We confirmed that these cell lines exhibited SOCE, which was inhibited by knockdown of STIM1 or Orai1, or by a pharmacological SOCE inhibitor. Inhibition of SOCE suppressed melanoma cell proliferation and migration/metastasis. Induction of SOCE was associated with activation of extracellular-signal-regulated kinase (ERK), and was inhibited by inhibitors of calmodulin kinase II (CaMKII) or Raf-1, suggesting that SOCE-mediated cellular functions are controlled via the CaMKII/Raf-1/ERK signaling pathway. Our findings indicate that SOCE contributes to melanoma progression, and therefore may be a new potential target for treatment of melanoma, irrespective of whether or not Braf mutation is present.

Exchange Protein Directly Activated by Cyclic AMP Increases Melanoma Cell Migration by a Ca2+-Dependent Mechanism

Melanoma has a poor prognosis due to its strong metastatic ability. Although Ca(2+) plays a major role in cell migration, little is known about the role of Ca(2+) in melanoma cell migration. We recently found that the exchange protein directly activated by cyclic AMP (Epac) increases melanoma cell migration via a heparan sulfate-related mechanism. In addition to this mechanism, we also found that Epac regulates melanoma cell migration by a Ca(2+)-dependent mechanism. An Epac agonist increased Ca(2+) in several different melanoma cell lines but not in melanocytes. Ablation of Epac1 with short hairpin RNA inhibited the Epac agonist-induced Ca(2+) elevation, suggesting the critical role of Epac1 in Ca(2+) homeostasis in melanoma cells. Epac-induced Ca(2+) elevation was negated by the inhibition of phospholipase C (PLC) and inositol triphosphate (IP(3)) receptor. Furthermore, Epac-induced cell migration was reduced by the inhibition of PLC or IP(3) receptor. These data suggest that Epac activates Ca(2+) release from the endoplasmic reticulum via the PLC/IP(3) receptor pathway, and this Ca(2+) elevation is involved in Epac-induced cell migration. Actin assembly was increased by Epac-induced Ca(2+), suggesting the involvement of actin in Epac-induced cell migration. In human melanoma specimens, mRNA expression of Epac1 was higher in metastatic melanoma than in primary melanoma, suggesting a role for Epac1 in melanoma metastasis. In conclusion, our findings reveal that Epac is a potential target for the suppression of melanoma cell migration, and, thus, the development of metastasis.

Prevention of heart failure in mice by an antiviral agent that inhibits type 5 cardiac adenylyl cyclase

Despite numerous discoveries from genetically engineered mice, relatively few have been translated to the bedside, mainly because it is difficult to translate from genes to drugs. This investigation examines an antiviral drug, which also has an action to selectively inhibit type 5 adenylyl cyclase (AC5), a pharmaceutical correlate of the AC5 knockout (KO) model, which exhibits longevity and stress resistance. Our objective was to examine the extent to which pretreatment with this drug, adenine 9-β-d-arabinofuranoside (Ara-A), favorably ameliorates the development of heart failure (HF). Ara-A exhibited selective inhibition for AC5 compared with the other major cardiac AC isoform, AC6, i.e., it reduced AC activity significantly in AC5 transgenic (Tg) mice, but not in AC5KO mice and had little effect in either wild-type or AC6Tg mice. Permanent coronary artery occlusion for 3 wk in C57Bl/6 mice increased mortality and induced HF in survivors, as reflected by reduced cardiac function, while increasing cardiac fibrosis. The AC5 inhibitor Ara-A significantly improved all of these end points and also ameliorated chronic isoproterenol-induced cardiomyopathy. As with the AC5KO mice, Ara-A increased mitogen/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) phosphorylation. A MEK inhibitor abolished the beneficial effects of the AC5 inhibitor in the HF model, indicating the involvement of the downstream MEK-ERK pathway of AC5. Our data suggest that pharmacological AC5 inhibition may serve as a new therapeutic approach for HF.

Epac increases melanoma cell migration by a heparan sulfate-related mechanism

Melanoma, the most malignant form of human skin cancer, has a poor prognosis due to its strong metastatic ability. It was recently demonstrated that Epac, an effector molecule of cAMP, is involved in regulating cell migration; however, the role of Epac in melanoma cell migration remains unclear. We thus examined whether Epac regulates cell migration and metastasis of melanoma. Epac activation, by either specific agonist or overexpression of Epac, increased melanoma cell migration. Deletion of endogenous Epac with small interfering RNA decreased basal melanoma cell migration. These data suggested a major role of Epac in melanoma cell migration. Epac-induced cell migration was mediated by translocation of syndecan-2, a cell-surface heparan sulfate proteoglycan, to lipid rafts. This syndecan-2 translocation was regulated by tubulin polymerization via the Epac/phosphoinositol-3 kinase pathway. Epac-induced cell migration was also regulated by the production of heparan sulfate, a major extracellular matrix. Epac-induced heparan sulfate production was attributable to the increased expression of N-deacetylase/N-sulfotransferase-1 (NDST-1) accompanied by an increased NDST-1 translation rate. Finally, Epac overexpression enhanced lung colonization of melanoma cells in mice. Taken together, these data indicate that Epac regulates melanoma cell migration/metastasis mostly via syndecan-2 translocation and heparan sulfate production.

Epac1 increases migration of endothelial cells and melanoma cells via FGF2-mediated paracrine signaling.

Fibroblast growth factor (FGF2) regulates endothelial and melanoma cell migration. The binding of FGF2 to its receptor requires N‐sulfated heparan sulfate (HS) glycosamine. We have previously reported that Epac1, an exchange protein activated by cAMP, increases N‐sulfation of HS in melanoma. Therefore, we examined whether Epac1 regulates FGF2‐mediated cell–cell communication. Conditioned medium (CM) of melanoma cells with abundant expression of Epac1 increased migration of human umbilical endothelial cells (HUVEC) and melanoma cells with poor expression of Epac1. CM‐induced increase in migration was inhibited by antagonizing FGF2, by the removal of HS and by the knockdown of Epac1. In addition, knockdown of Epac1 suppressed the binding of FGF2 to FGF receptor in HUVEC, and in vivo angiogenesis in melanoma. Furthermore, knockdown of Epac1 reduced N‐sulfation of HS chains attached to perlecan, a major secreted type of HS proteoglycan that mediates the binding of FGF2 to FGF receptor. These data suggested that Epac1 in melanoma cells regulates melanoma progression via the HS–FGF2‐mediated cell–cell communication.

Reduced malignancy as a mechanism for longevity in mice with adenylyl cyclase type 5 disruption

Disruption of adenylyl cyclase type 5 (AC5) knockout (KO) is a novel model for longevity. Because malignancy is a major cause of death and reduced lifespan in mice, the goal of this investigation was to examine the role of AC5KO in protecting against cancer. There have been numerous discoveries in genetically engineered mice over the past several decades, but few have been translated to the bedside. One major reason is that it is difficult to alter a gene in patients, but rather a pharmacological approach is more appropriate. The current investigation employs a parallel construction to examine the extent to which inhibiting AC5, either in a genetic knockout (KO) or by a specific pharmacological inhibitor protects against cancer. This study is unique, not only because a combined genetic and pharmacological approach is rare, but also there are no prior studies on the extent to which AC5 affects cancer. We found that AC5KO delayed age‐related tumor incidence significantly, as well as protecting against mammary tumor development in AC5KO × MMTV‐HER‐2 neu mice, and B16F10 melanoma tumor growth, which can explain why AC5KO is a model of longevity. In addition, a Food and Drug Administration approved antiviral agent, adenine 9‐β‐D‐arabinofuranoside (Vidarabine or AraAde), which specifically inhibits AC5, reduces LP07 lung and B16F10 melanoma tumor growth in syngeneic mice. Thus, inhibition of AC5 is a previously unreported mechanism for prevention of cancers associated with aging and that can be targeted by an available pharmacologic inhibitor, with potential consequent extension of lifespan.

Abstract 1439: Combination of metformin plus orlistat prevents tumor progression: novel role of the metabolic hormone fibroblast growth factor 21 (FGF21)

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Caloric Restriction (CR) is known to be the only model to consistently increase the lifespan and postpone age-related diseases in all species. CR increases glycogenolysis, lipolysis with fatty acid and ketone body utilization and accelerates protein catabolism. We previously reported that CR induces metabolic and signaling changes that affect the tumor microenvironment preventing the growth of 4T1 mammary tumors and their metastases. 4T1 is a highly metastatic mouse breast cancer model which resembles breast cancer in patients. Obesity causes subclinical inflammation in adipose tissue that contributes to insulin resistance and cancer progression. Chronic inflammation predisposes to different forms of cancer and diabetes, and is correlated with increased risk of breast cancer. CR reduces insulin resistance, adiposity and inflammation and the anti-diabetic drug metformin (MET) is a CR mimetic agent. In this study, we evaluated whether the combination of MET with the anti-obesity drug orlistat (OR) enhances the CR mimetic and anti-cancer properties of MET on 4T1 mammary tumor cells. Female 8 week-old BALB/c mice received: vehicle, MET (in drinking water, 3mg/ml), OR i.p. injection (240mg/kg/day) or MET + OR. After 3 weeks on drugs, 4T1 cells (105) were injected into mice and drugs continued till the end of the experiment. Treated-mice had a reduced adiposity (p<0.05) without any signs of toxicity. At 30 days, MET + OR co-treatment reduced tumor volume (p<0.01) and displayed a range of cellular alterations such as decreased proliferative index (p<0.05); increased apoptotic rate (p<0.01) and altered intra-tumor collagen deposit. MET + OR treatment reduced total vessel length (p<0.01) and the number of spontaneous lung metastases (p<0.01). In addition, combinations of MET (1-2.5mM) + OR (2.5-5µM) decreased in vitro 4T1 mammary cell proliferation (p<0.01), adhesion (p<0.05) and migration (p<0.01) compared to vehicle and drugs alone. Interestingly, we found that levels of fibroblast growth factor 21 (FGF21), an endocrine factor that regulates glucose and lipid metabolism, were significantly increased with MET + OR treatment (p<0.05). It has been shown that increased circulating levels of FGF21 correlated with decreased obesity and increased sensitivity to insulin; but up to date the role of FGF21 in tumor progression was not elucidated. We found that treatment of 4T1 cells with MET + OR increased the secretion of FGF21 (p<0.05) measured by ELISA assays. To elucidate the role of FGF21; we initially demonstrated that addition of recombinant protein FGF21 (0-10nM) did not change 4T1 cell proliferation. In contrast, the reduced cell proliferation and adhesion induced by MET + OR, were abolished by the pre- and co-treatment with anti-FGF21 neutralizing antibody; indicating that FGF21 may play an important role. Our results provide a new rationale basis for the use of MET plus OR as cancer therapy. Citation Format: Shobika Sivaram, Erdene Baljinnyam, Kousaku Iwatsubo, Lydia I. Puricelli, Mariana S. De Lorenzo. Combination of metformin plus orlistat prevents tumor progression: novel role of the metabolic hormone fibroblast growth factor 21 (FGF21). [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 1439. doi:10.1158/1538-7445.AM2014-1439

Abstract 5280: Exchange protein activated by cAMP (Epac) accelerates melanoma cell migration through Ca 2+-dependent actin assembly

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: We previously reported that Epac, a guanine nucleotide exchange factor directly activated by cAMP, increases melanoma cell migration through Ca2+ elevation. Both Epac-induced cell migration and Ca2+ elevation were reduced by pharmacological inhibition of phospholipase C (PLC) and inositol triphosphate (IP3) receptor. Accordingly, we studied the involvement of PLC/IP3 receptor in Epac-induced Ca2+ elevation by knockdown of these molecules. However, the mechanism how Epac-induced Ca2+ elevation mediates melanoma cell migration is unknown. Here, we investigate the effect of Epac induced-Ca2+ elevation on actin assembly, which plays a major role in cell migration. Methods: SK-Mel-2, a cell line from human metastatic melanoma, was used in this study. Intracellular Ca2+ was measured with Fura-2AM fluorescent dye. Cell migration was examined with the Boyden chambers. IP3 production was examined by IP3 radioimmunoassay. Actin assembly was examined by immunocytochemistry. In some experiments, ablation of a target protein with lentivirus-based shRNA induction system was performed. Results: 8-pMeOPT-2′-O-Me-cAMP (8-pMeOPT), an Epac-specific agonist, increased Ca2+ and cell migration in SK-Mel-2. Such Ca2+ elevation and cell migration was negated by ablation of PLCe or IP3 receptor 1. In addition, Epac-induced IP3 production was inhibited by U73122, a PLC inhibitor, and ablation of PLCe. These data suggest that Epac increases Ca2+, and thus cell migration, via the PLCe/IP3 receptor 1 signaling pathway. Cytochalasin D, an actin assembly inhibitor, reduced 8-pMeOPT-induced cell migration. In addition, immunocytochemistry showed that 8-pMeOPT increases actin assembly. When U73122 or Xestopongin C, an IP3 receptor inhibitor, was used, Epac-induced actin assembly was decreased. These data suggest that Epac-induced Ca2+ elevation mediates actin assembly. Further, immunoprecipitation showed that 8-pMeOPT increased physical interaction of myosin heavy chain IIA (MHCIIA), which enhances actin assembly, with S100A4, a Ca2+-binding protein which activates MHCIIA. These data suggest that Epac-induced Ca2+ elevation mediates actin assembly via the S100A4/MHCIIA signaling pathway. Conclusion: Epac increases Ca2+ via the PLCe/IP3 receptor 1 pathway. Also, Epac-induced Ca2+ elevation mediates melanoma cell migration via actin assembly. These data suggested that Epac could be a target for treating patients with melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5280.