The Growth and Tumor Suppressors NORE1A and RASSF1A Are Targets for Calpain-Mediated Proteolysis

Background: NORE1A and RASSF1A are growth and tumour suppressors inactivated in a variety of cancers. Methylation of NORE1A and RASSF1A promoters is the predominant mechanism for downregulation of these proteins; however, other mechanisms are likely to exist. Methodology/Principal Findings: Here we describe a proteolysis of NORE1A and RASSF1A by calpains as alternative mechanism of their downregulation. Extracts of H358 cell line, a human bronchoalveolar carcinoma, and H460, a large cell carcinoma, were capable of proteolysis of NORE1A protein in the calpain-dependent manner. Likewise, RASSF1A tumor suppressor was proteolyzed by the H358 cell extract. Addition of calpain inhibitor to H358 and H460 cells growing in tissue culture resulted in re-expression of endogenous NORE1A. A survey of 10 human lung tumours revealed that three of them contain an activity capable of inducing NORE1A degradation. Conclusions/Significance: Thus, degradation by calpains is a novel mechanism for downregulation of NORE1A and RASSF1A proteins and might be the mechanism allowing cancer cells to escape growth suppression

Induction of interleukin-8 preserves the angiogenic response in HIF-1 alpha-deficient colon cancer cells

authorHypoxia inducible factor-1 (HIF-1) is considered a crucial mediator of the cellular response to hypoxia through its regulation of genes that control angiogenesis^1, ^2, ^3, ^4. It represents an attractive therapeutic target^5, ^6 in colon cancer, one of the few tumor types that shows a clinical response to antiangiogenic therapy^7. But it is unclear whether inhibition of HIF-1 alone is sufficient to block tumor angiogenesis^8, ^9. In HIF-1_α knockdown DLD-1 colon cancer cells (DLD-1^HIF-kd), the hypoxic induction of vascular endothelial growth factor (VEGF) was only partially blocked. Xenografts remained highly vascularized with microvessel densities identical to DLD-1 tumors that had wild-type HIF-1_α (DLD-1^HIF-wt). In addition to the preserved expression of VEGF, the proangiogenic cytokine interleukin (IL)-8 was induced by hypoxia in DLD-1^HIF-kd but not DLD-1^HIF-wt cells. This induction was mediated by the production of hydrogen peroxide and subsequent activation of NF-_KB. Furthermore, the KRAS oncogene, which is commonly mutated in colon cancer, enhanced the hypoxic induction of IL-8. A neutralizing antibody to IL-8 substantially inhibited angiogenesis and tumor growth in DLD-1^HIF-kd but not DLD-1^HIF-wt xenografts, verifying the functional significance of this IL-8 response. Thus, compensatory pathways can be activated to preserve the tumor angiogenic response, and strategies that inhibit HIF-1α may be most effective when IL-8 is simultaneously targeted

Nuclear Entry of Activated MAPK Is Restricted in Primary Ovarian and Mammary Epithelial Cells

The MAPK/ERK1/2 serine kinases are primary mediators of the Ras mitogenic signaling pathway. Phosphorylation by MEK activates MAPK/ERK in the cytoplasm, and phospho-ERK is thought to enter the nucleus readily to modulate transcription.Here, however, we observe that in primary cultures of breast and ovarian epithelial cells, phosphorylation and activation of ERK1/2 are disassociated from nuclear translocalization and transcription of downstream targets, such as c-Fos, suggesting that nuclear translocation is limited in primary cells. Accordingly, in import assays in vitro, primary cells showed a lower import activity for ERK1/2 than cancer cells, in which activated MAPK readily translocated into the nucleus and activated c-Fos expression. Primary cells express lower levels of nuclear pore complex proteins and the nuclear transport factors, importin B1 and importin 7, which may explain the limiting ERK1/2 import found in primary cells. Additionally, reduction in expression of nucleoporin 153 by siRNA targeting reduced ERK1/2 nuclear activity in cancer cells.ERK1/2 activation is dissociated from nuclear entry, which is a rate limiting step in primary cells and in vivo, and the restriction of nuclear entry is disrupted in transformed cells by the increased expression of nuclear pores and/or nuclear transport factors

Oncogenic KRAS sensitises colorectal tumour cells to chemotherapy by p53-dependent induction of Noxa

BACKGROUND: Oxaliplatin and 5-fluorouracil (5-FU) currently form the backbone of conservative treatment in patients with metastatic colorectal cancer. Tumour responses to these agents are highly variable, but the underlying mechanisms are poorly understood. Our previous results have indicated that oncogenic KRAS in colorectal tumour cells sensitises these cells to chemotherapy. METHODS: FACS analysis was used to determine cell-cycle distribution and the percentage of apoptotic and mitotic cells. A multiplexed RT-PCR assay was used to identify KRAS-controlled apoptosis regulators after exposure to 5-FU or oxaliplatin. Lentiviral expression of short-hairpin RNAs was used to suppress p53 or Noxa. RESULTS: Oncogenic KRAS sensitised colorectal tumour cells to oxaliplatin and 5-FU in a p53-dependent manner and promoted p53 phosphorylation at Ser37 and Ser392, without affecting p53 stabilisation, p21 induction, or cell-cycle arrest. Chemotherapy-induced expression of the p53 target gene Noxa was selectively enhanced by oncogenic KRAS. Suppression of Noxa did not affect p21 induction or cell-cycle arrest, but reduced KRAS/p53-dependent apoptosis after exposure to chemotherapy in vitro and in tumour xenografts. Noxa suppression did not affect tumour growth per se, but strongly reduced the response of these tumours to chemotherapy. CONCLUSION: Oncogenic KRAS determines the cellular response to p53 activation by oxaliplatin or 5-FU, by facilitating apoptosis induction through Noxa. British Journal of Cancer (2010) 102, 1254-1264. doi: 10.1038/sj.bjc.6605633 www.bjcancer.com Published online 30 March 2010 (C) 2010 Cancer Research U

Mst1/2 signalling to Yap: gatekeeper for liver size and tumour development

The mechanisms controlling mammalian organ size have long been a source of fascination for biologists. These controls are needed to both ensure the integrity of the body plan and to restrict inappropriate proliferation that could lead to cancer. Regulation of liver size is of particular interest inasmuch as this organ maintains the capacity for regeneration throughout life, and is able to regain precisely its original mass after partial surgical resection. Recent studies using genetically engineered mouse strains have shed new light on this problem; the Hippo signalling pathway, first elucidated as a regulator of organ size in Drosophila, has been identified as dominant determinant of liver growth. Defects in this pathway in mouse liver lead to sustained liver overgrowth and the eventual development of both major types of liver cancer, hepatocellular carcinoma and cholangiocarcinoma. In this review, we discuss the role of Hippo signalling in liver biology and the contribution of this pathway to liver cancer in humans

SSeCKS/Gravin/AKAP12 attenuates expression of proliferative and angiogenic genes during suppression of v-Src-induced oncogenesis

BACKGROUND: SSeCKS is a major protein kinase C substrate with kinase scaffolding and metastasis-suppressor activity whose expression is severely downregulated in Src- and Ras-transformed fibroblast and epithelial cells and in human prostate, breast, and gastric cancers. We previously used NIH3T3 cells with tetracycline-regulated SSeCKS expression plus a temperature-sensitive v-Src allele to show that SSeCKS re-expression inhibited parameters of v-Src-induced oncogenic growth without attenuating in vivo Src kinase activity. METHODS: We use cDNA microarrays and semi-quantitative RT-PCR analysis to identify changes in gene expression correlating with i) SSeCKS expression in the absence of v-Src activity, ii) activation of v-Src activity alone, and iii) SSeCKS re-expression in the presence of active v-Src. RESULTS: SSeCKS re-expression resulted in the attenuation of critical Src-induced proliferative and pro-angiogenic gene expression including Afp, Hif-1α, Cdc20a and Pdgfr-β, and conversely, SSeCKS induced several cell cycle regulatory genes such as Ptpn11, Gadd45a, Ptplad1, Cdkn2d (p19), and Rbbp7. CONCLUSION: Our data provide further evidence that SSeCKS can suppress Src-induced oncogenesis by modulating gene expression downstream of Src kinase activity

Tumor Cell Phenotype Is Sustained by Selective MAPK Oxidation in Mitochondria

Mitochondria are major cellular sources of hydrogen peroxide (H2O2), the production of which is modulated by oxygen availability and the mitochondrial energy state. An increase of steady-state cell H2O2 concentration is able to control the transition from proliferating to quiescent phenotypes and to signal the end of proliferation; in tumor cells thereby, low H2O2 due to defective mitochondrial metabolism can contribute to sustain proliferation. Mitogen-activated protein kinases (MAPKs) orchestrate signal transduction and recent data indicate that are present in mitochondria and regulated by the redox state. On these bases, we investigated the mechanistic connection of tumor mitochondrial dysfunction, H2O2 yield, and activation of MAPKs in LP07 murine tumor cells with confocal microscopy, in vivo imaging and directed mutagenesis. Two redox conditions were examined: low 1 µM H2O2 increased cell proliferation in ERK1/2-dependent manner whereas high 50 µM H2O2 arrested cell cycle by p38 and JNK1/2 activation. Regarding the experimental conditions as a three-compartment model (mitochondria, cytosol, and nuclei), the different responses depended on MAPKs preferential traffic to mitochondria, where a selective activation of either ERK1/2 or p38-JNK1/2 by co-localized upstream kinases (MAPKKs) facilitated their further passage to nuclei. As assessed by mass spectra, MAPKs activation and efficient binding to cognate MAPKKs resulted from oxidation of conserved ERK1/2 or p38-JNK1/2 cysteine domains to sulfinic and sulfonic acids at a definite H2O2 level. Like this, high H2O2 or directed mutation of redox-sensitive ERK2 Cys214 impeded binding to MEK1/2, caused ERK2 retention in mitochondria and restricted shuttle to nuclei. It is surmised that selective cysteine oxidations adjust the electrostatic forces that participate in a particular MAPK-MAPKK interaction. Considering that tumor mitochondria are dysfunctional, their inability to increase H2O2 yield should disrupt synchronized MAPK oxidations and the regulation of cell cycle leading cells to remain in a proliferating phenotype

RAB3C IS A SYNAPTIC VESICLE PROTEIN THAT DISSOCIATES FROM SYNAPTIC VESICLES AFTER STIMULATION OF EXOCYTOSIS

Fischer von Mollard G, STAHL B, KHOKHLATCHEV A, SUDHOF TC, JAHN R. RAB3C IS A SYNAPTIC VESICLE PROTEIN THAT DISSOCIATES FROM SYNAPTIC VESICLES AFTER STIMULATION OF EXOCYTOSIS. JOURNAL OF BIOLOGICAL CHEMISTRY. 1994;269(15):10971-10974.Rab3 proteins are small GTP-binding proteins of the Ras superfamily. Four highly homologous Rab3 proteins termed Rab3A, Rab3B, Rab3C, and Rab3D have been described. Rab3A has previously been shown to be a constituent of synaptic vesicles in neurons that undergoes membrane dissociation-association cycles during synaptic vesicle recycling. Here we report that Rab3C copurifies with Rab3A during the isolation of synaptic vesicles. Organelles immunoisolated with monoclonal antibodies directed against Rab3A led to a coenrichment of Rab3A and Rab3C, demonstrating that both Rab3 proteins are colocalized on the same organelle. In isolated nerve terminals, stimulation of neurotransmitter release resulted in a dissociation of Rab3C from synaptic vesicle and/or recycling membranes. This dissociation parallels that of Rab3A observed under the same conditions. In contrast, no change was observed in the membrane-association of Rab5, a Rab protein localized on early endosomes. We conclude that in the nervous system Rab3C is localized on synaptic vesicles and, like Rab3A, cycles on and off the synaptic vesicle membrane in parallel with exocytotic release of neurotransmitter