Abscisic acid signaling through LANCL2 and PPARγ induces activation of p38MAPK resulting in dormancy of prostate cancer metastatic cells.

Prostate cancer (PCa) is one the most common malignancies in men. The high incidence of bone metastasis years after primary therapy suggests that disseminated tumor cells must become dormant, but maintain their ability to proliferate in the bone marrow. Abscisic acid (ABA) is a stress response molecule best known for its regulation of seed germination, stomal opening, root shoot growth and other stress responses in plants. ABA is also synthesized by mammalian cells and has been linked to human disease. The aim of the present study was to examine the role of ABA in regulating tumor dormancy via signaling through lanthionine synthetase C­like protein 2 (LANCL2) and peroxisome proliferator activated receptor γ (PPARγ) receptors. ABA signaling in human PCa cell lines was studied using targeted gene knockdown (KD), western blotting, quantitative PCR, cell proliferation, migration, invasion and soft agar assays, as well as co­culture assays with bone marrow stromal cells. The data demonstrated that ABA signaling increased the expression of p21, p27 and p16, while inhibiting viability, migration, invasion and colony size in a reversable manner without toxicity. ABA also induced p38MAPK activation and NR2F1 signaling. Targeted gene KD of LANCL2 and PPARγ abrogated the cellular responses to ABA. Taken together, these data demonstrate that ABA may induce dormancy in PCa cell lines through LANCL2 and PPARγ signaling, and suggest novel targets to manage metastatic PCa growth.

Role of Death Receptors-associated Lipid Rafts in Oxaliplatin-induced Death Mode Regulation of HepG2 Cells.

BACKGROUND/AIM: We previously showed that oxaliplatin induces necrotic-like cell death in hepatocarcinomas, and combination with ursodexoycholic acid (UDCA) significantly shifts the necrotic-like death to apoptosis. Since cell death mode is crucial on inflammatory responses and chemotherapeutic efficacy, the mechanism underlying determination of cell death mode by UDCA was investigated in this study. MATERIALS AND METHODS: Apoptosis or necrosis was determined by apoptotic body formation, caspase-8 activity, LDH release and PI inclusion. The involvement of lipid rafts and death receptors was examined by rafts fractionation, confocal microscopy and gene silencing assays. RESULTS: UDCA combination enhanced recruitment of death receptors and adaptors into cholesterol-enriched lipid rafts, and induced a stronger raft clustering. Lipid raft disruption decreased the UDCA/oxaliplatin-mediated apoptosis and increased necrotic-like death. CONCLUSION: UDCA promotes lipid raft localization of multiple death receptors, thereby contributing to a shift of cell death mode from oxaliplatin-induced necrotic death to apoptosis in HepG2 cells.

KLF7 promotes pancreatic cancer growth and metastasis by up-regulating ISG expression and maintaining Golgi complex integrity.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a dismal prognosis. Currently, there is no effective therapy for PDAC, and a detailed molecular and functional evaluation of PDACs is needed to identify and develop better therapeutic strategies. Here we show that the transcription factor Krüppel-like factor 7 (KLF7) is overexpressed in PDACs, and that inhibition of KLF7 blocks PDAC tumor growth and metastasis in cell culture and in mice. KLF7 expression in PDACs can be up-regulated due to activation of a MAP kinase pathway or inactivation of the tumor suppressor p53, two alterations that occur in a large majority of PDACs. ShRNA-mediated knockdown of KLF7 inhibits the expression of IFN-stimulated genes (ISGs), which are necessary for KLF7-mediated PDAC tumor growth and metastasis. KLF7 knockdown also results in the down-regulation of Discs Large MAGUK Scaffold Protein 3 (DLG3), resulting in Golgi complex fragmentation, and reduced protein glycosylation, leading to reduced secretion of cancer-promoting growth factors, such as chemokines. Genetic or pharmacologic activation of Golgi complex fragmentation blocks PDAC growth and metastasis similar to KLF7 inhibition. Our results demonstrate a therapeutically amenable, KLF7-driven pathway that promotes PDAC growth and metastasis by activating ISGs and maintaining Golgi complex integrity.

Loss of HAT1 expression confers BRAFV600E inhibitor resistance to melanoma cells by activating MAPK signaling via IGF1R.

BRAF inhibitors (BRAFi) have been approved for the clinical treatment of BRAF-mutant metastatic melanoma. Although initial responses to BRAFi are generally favorable, acquired BRAFi resistance emerges rapidly, resulting in treatment failure. Only some of the underlying mechanisms responsible for BRAFi resistance are currently understood. Here, we showed that the genetic inhibition of histone acetyltransferase 1 (HAT1) in BRAF-mutant melanoma cells resulted in BRAFi resistance. Using quantitative immunofluorescence analysis of patient sample pairs, consisting of pre-treatment along with matched progressed BRAFi + MEKi-treated melanoma samples, HAT1 downregulation was observed in 7/11 progressed samples (~63%) in comparison with pre-treated samples. Employing NanoString-based nCounter PanCancer Pathway Panel-based gene expression analysis, we identified increased MAPK, Ras, transforming growth factor (TGF)-ß, and Wnt pathway activation in HAT1 expression inhibited cells. We further found that MAPK pathway activation following the loss of HAT1 expression was partially driven by increased insulin growth factor 1 receptor (IGF1R) signaling. We showed that both MAPK and IGF1R pathway inhibition, using the ERK inhibitor SCH772984 and the IGF1R inhibitor BMS-754807, respectively, restored BRAFi sensitivity in melanoma cells lacking HAT1. Collectively, we show that the loss of HAT1 expression confers acquired BRAFi resistance by activating the MAPK signaling pathway via IGF1R.

Keratin 6, induced by chronic cisplatin exposure, confers chemoresistance in human gastric carcinoma cells.

Currently, various types of keratins have been reported to be highly expressed in cancer cells and to be associated with a malignant phenotype. In the present study, it was found that expression levels of keratin 6 (K6), keratin 16 (K16), and keratin 17 (K17) were highly elevated in SNU601 cells resistant to cisplatin (SNU601­cis2 and SNU601­cis10), but not in the parental SNU601 cells as confirmed by quantitative PCR, immunoblotting, and immunofluorescence assays. K6 is a type II keratin and is known to form a keratin filament in conjugation with type I keratin, K16 or K17. Thus, we attempted to understand the role of the overexpression of K6/K16 or K6/K17 keratin filaments by regulating the expression of K6. Silencing of K6 by siRNA in SNU601­cis2 cells promoted oxaliplatin­induced apoptosis in the resistant cells as shown by increased apoptotic body formation, caspase­8 and caspase­3 cleavage, and cytochrome c release. In addition, induction of K6 levels in wild­type SNU601 cells, by transfection with pCMV6­K6A and pCMV6­K6B overexpression vectors, resulted in decreased apoptosis in response to cisplatin and L­OHP. Platinum drugs, such as oxaliplatin, were shown to induce the extrinsic apoptotic pathway by inducing lipid raft formation and death receptor recruitment into lipid rafts. However, in the resistant cells, the oxaliplatin­triggered extrinsic apoptotic pathway appeared to be suppressed by decreased lipid raft formation, and recruitment of death receptor 5 and FADD into lipid rafts. Therefore, the increase in the levels of the K6 filament may be associated with the regulation of lipid raft formation and may contribute, at least in part, to resistance to anticancer drugs.

Aminomethylphosphonic acid inhibits growth and metastasis of human prostate cancer in an orthotopic xenograft mouse model.

Aminomethylphosphonic acid (AMPA) has been shown to inhibit prostate cancer cell growth in vitro. The purpose of the present study was to determine if AMPA could inhibit growth and metastasis of prostate cancer in vivo. Human prostate cancer PC-3-LacZ-luciferase cells were implanted into the ventral lateral lobes of the prostate in 39 athymic Nu/Nu nude male mice. Seven days later, mice were randomized into the control group (n = 14, treated intraperitoneally with phosphate buffered saline), low dose group (n = 10, treated intraperitoneally with AMPA at 400 mg/kg body weight/day), and high dose group (n = 15, treated intraperitoneally with AMPA at 800 mg/kg body weight/day). Tumor growth and metastasis were examined every 4-7 days by bioluminescence imaging of live mice. We found that AMPA treatment significantly inhibited growth and metastasis of orthotopic xenograft prostate tumors and prolonged the survival time of the mice. AMPA treatment decreased expression of BIRC2 and activated caspase 3, leading to increased apoptosis in the prostate tumors. AMPA treatment decreased expression of cyclin D1. AMPA treatment also reduced angiogenesis in the prostate tumors. Taken together, these results demonstrate that AMPA can inhibit prostate cancer growth and metastasis, suggesting that AMPA may be developed into a therapeutic agent for the treatment of prostate cancer.

The alkyllysophospholipid edelfosine enhances TRAIL-mediated apoptosis in gastric cancer cells through death receptor 5 and the mitochondrial pathway.

The ether phospholipid edelfosine is the prototype of a group of synthetic antitumor alkyllysophospholipid (ALP) compounds that exert pro-apoptotic effects in various types of cancer cells through cell type-dependent mechanisms. In this study, we examined the antitumor effect of edelfosine in human gastric cancer cells. Edelfosine decreased cell viability and induced autophagic death at a moderate concentration (~30 µM), whereas it induced apoptotic cell death at concentrations over 30 µM. Interestingly, low concentrations of edelfosine (5-10 µM) effectively enhanced recombinant human tumor necrosis factor (TNF)-related apoptosis-inducing ligand (rhTRAIL/TNFSF10)-induced apoptosis and clonogenicity in gastric cancer cells, including TRAIL-resistant AGS cells. Edelfosine upregulated the protein level of death receptor 5 (DR5/TNFRSF10B) and/or increased DR5 upregulation in lipid rafts. In addition, edelfosine-mediated rhTRAIL sensitization was regulated by the DR5 pathway. Edelfosine also activated p38MAPK (MAPK14), and edelfosine-mediated rhTRAIL sensitization was partially regulated by a p38-mediated decrease in mitochondrial membrane potential. This study suggests a novel therapeutic strategy targeting gastric cancer cells by using the combination of edelfosine and TRAIL.

Cholesterol induces autophagic and apoptotic death in gastric carcinoma cells.

Despite conflicting results, there is evidence to suggest an inverse link between total body cholesterol levels and the risk of certain malignancies. Based on previous reports, this phenomenon appears to vary with cancer site, and, in particular, more consistent data on inverse relations was reported in the risk of gastric cancer. In the current study, the effect of cholesterol on gastric cancer cell viability was examined using an in vitro cell culture system. Addition of cholesterol in culture medium resulted in reduced viability and clonogenicity of SNU601, SNU638 and SNU216 gastric cancer cells by induction of both autophagic and apoptotic death. Transient inactivation of ERK1/2 was linked to reduction of cholesterol-mediated cell viability, and tumor necrosis factor­related apoptosis-inducing ligand receptor 2 (TRAIL­R2/DR5) was also involved in cell death signaling. In conclusion, these results imply that cholesterol can act as a signal regulator to modulate cell viability and that proper cellular cholesterol levels may be advantageous to suppress growth of gastric carcinomas.

Pro-apoptotic role of the MEK/ERK pathway in ursodeoxycholic acid-induced apoptosis in SNU601 gastric cancer cells.

Ursodeoxycholic acid (UDCA) has been regarded as a suppressor of gastrointestinal cancer, but the mechanisms underlying its antitumor effects are not fully understood. Previously, we reported the antitumor effect of UDCA by demonstrating that UDCA induces apoptosis of gastric cancer cells. Bile acids are known to activate the ERK pathway and ERK is a representative oncogenic kinase in cancer cells. Here, we investigated the role of ERK in UDCA-induced gastric cancer cell apoptosis. We found that UDCA enhanced the phosphorylation of ERK1/2 and MEK1/2. The prevention of MEK by the pharmacologic inhibitors PD98059 and U0126, resulted in decreased UDCA-induced apoptosis as shown by the reduction of apoptotic body formation, caspase-8 activity, and caspase-3, -6 and PARP cleavage, indicating that ERK exerts pro-apoptotic activity upon exposure to UDCA. In addition, U0126 reduced UDCA-triggered TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2/DR5) expression. In gene silencing studies, we observed that RNA interference of ERK2 decreased apoptosis and reduced DR5 overexpression. Lipid raft disrupting agent, methyl-ß-cyclodextrin, blunted the phosphorylation of ERK1/2, indicating that ERK activation is regulated in a lipid raft-dependent manner. On the other hand, tumor-promoting bile acid, deoxycholic acid (DCA), also phosphorylated ERK in SNU601 cells. However, the DCA-triggered ERK pathway exerted anti-apoptotic function in the cells. Suppression of the ERK pathway enhanced DCA-induced apoptosis, and ERK activation was observed to be lipid raft-independently controlled. These results indicated that UDCA and DCA may cause differential responses in gastric cancer cells through the ERK signaling molecule. Thus, ERK activation may be a possible mechanism by which UDCA and DCA represent differential activities in gastrointestinal cancer.

Implication of PI3K-dependent HSP27 and p53 expression in mild heat shock-triggered switch of metabolic stress-induced necrosis to apoptosis in A549 cells.

Previously, we showed that mild heat shock modulates patterns of cell death in response to glucose deprivation (GD), a common characteristic of the tumor microenvironment, by switching necrosis to apoptosis through ERK-dependent suppression of reactive oxygen species production in A549 cells. In the present study, we further examined the molecular mechanism underlying mild heat shock-induced necrosis-to-apoptosis switch. We examined the possible implication of p53 and heat shock proteins (HSPs) in the mechanism. Inhibition of p53 by pifithrin-alpha or p53 siRNA markedly suppressed apoptosis induced by heat shock/GD. On the other hand, silencing of HSP27, but not of HSP70, reversed heat shock/GD-induced apoptosis to necrosis, and HSP27 overexpression suppressed GD-induced necrosis. We further demonstrate that mild heat shock activated AKT and ERK1/2 through phosphorylation. Prevention of PI3K by LY294002 blocked heat shock/GD-induced apoptosis without reversing the cell death mode to necrosis, while inhibition of MEK1/2 by U0126 reversed heat shock/GD-induced apoptosis to necrosis, indicating a different role(s) of PI3K and ERK1/2 in heat shock/GD-induced cell death mode determination. We also found that mild heat shock increased HSP27 and p53 protein levels dependent on PI3K and suppressed the GD-induced increase in RIPA-insoluble HSP27 and p53 protein levels dependent on PI3K and ERK1/2. In conclusion, these results indicate that PI3K-dependent HSP27 and p53 induction and PI3K- and ERK1/2-dependent inhibition of the GD-induced increase in RIPA-insoluble HSP27 and p53 protein levels by heat play a key role(s) in heat shock-mediated switch of GD-induced necrosis to apoptosis.