The balance between cell death and survival, two critical aspects of oncogenic transformation, determines the outcome of tumorigenesis. Nuclear factor-kappaB (NF-kappaB) is a critical regulator of survival; it is induced by the oncogene Ras and, when inhibited, accounts for the cell death response of Ras-transformed cells. Here, we show that the signaling adaptor p62 is induced by Ras, its levels are increased in human tumors, and it is required for Ras-induced survival and transformation. p62-/- mice are resistant to Ras-induced lung adenocarcinomas. p62 is necessary for Ras to trigger IkappaB kinase (IKK) through the polyubiquitination of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), and its deficiency produces increased reactive oxygen species (ROS) levels, which account for the enhanced cell death and reduced tumorigenicity of Ras in the absence of p62.
SUMMARY Tumor cells have high-energetic and anabolic needs and are known to adapt their metabolism to be able to survive and keep proliferating under conditions of nutrient stress. We show that PKCζ deficiency promotes the plasticity necessary for cancer cells to reprogram their metabolism to utilize glutamine through the serine biosynthetic pathway in the absence of glucose. PKCζ represses the expression of two key enzymes of the pathway, PHGDH and PSAT1, and phosphorylates PHGDH at key residues to inhibit its enzymatic activity. Interestingly, the loss of PKCζ in mice results in enhanced intestinal tumorigenesis and increased levels of these two metabolic enzymes, whereas patients with low levels of PKCζ have a poor prognosis. Furthermore, PKCζ and caspase-3 activities are correlated with PHGDH levels in human intestinal tumors. Taken together, this demonstrates that PKCζ is a critical metabolic tumor suppressor in mouse and human cancer.
Abstract-Regulating the balance between synthesis and proteasomal degradation of cellular proteins is essential for tissue growth and maintenance, but the critical pathways regulating protein ubiquitination and degradation are incompletely defined. Although participation of calpain calcium-activated proteases in post-necrotic myocardial autolysis is well characterized, their importance in homeostatic turnover of normal cardiac tissue is controversial. Hence, we evaluated the consequences of physiologic calpain (calcium-activated protease) activity in cultured cardiomyocytes and unstressed mouse hearts. Comparison of in vitro proteolytic activities of cardiac-expressed calpains 1 and 2 revealed calpain 1, but not calpain 2, activity at physiological calcium concentrations. Physiological calpain 1 activation was evident in adenoviral transfected cultured cardiomyocytes as proteolysis of specific substrates, generally increased protein ubiquitination, and accelerated protein turnover, that were each inhibited by coexpression of the inhibitor protein calpastatin. Conditional forced expression of calpain 1, but not calpain 2, in mouse hearts demonstrated substratespecific proteolytic activity under basal conditions, with hyperubiquitination of cardiac proteins and increased 26S proteasome activity. Loss of myocardial calpain activity by forced expression of calpastatin diminished ubiquitination of 1 or more specific myocardial proteins, without affecting overall ubiquitination or proteasome activity, and resulted in a progressive dilated cardiomyopathy characterized by accumulation of intracellular protein aggregates, formation of autophagosomes, and degeneration of sarcomeres. Thus, calpain 1 is upstream of, and necessary for, ubiquitination and proteasomal degradation of a subset of myocardial proteins whose abnormal accumulation produces autophagosomes and degeneration of cardiomyocytes with functional decompensation. (Circ Res.
Up-regulation of myocardial Nix and BNip3 is associated with apoptosis in cardiac hypertrophy and ischemia, respectively. To identify mechanisms of gene regulation for these critical cardiac apoptosis effectors, the determinants of Nix and BNip3 promoter activation were elucidated by luciferase reporter gene expression in neonatal rat cardiac myocytes. BNip3 transcription was increased by hypoxia but not by phenylephrine (10 M), angiotensin II (100 nM), or isoproterenol (10 M). In contrast, Nix transcription was increased by phenylephrine but not by isoproterenol, angiotensin II, or hypoxia. Since phenylephrine stimulates cardiomyocyte hypertrophy via protein kinase C (PKC), the effects of phorbol myristate acetate (PMA, 10 nM for 24 h) and adenoviral PKC expression were assessed. PMA and PKC␣, but not PKC⑀ or dominant negative PKC␣, increased Nix transcription. Multiple Nix promoter GC boxes bound transcription factor Sp-1, and basal and PMA-or PKC␣-stimulated Nix promoter activity was suppressed by mithramycin inhibition of Sp1-DNA interactions. In vivo determinants of Nix expression were evaluated in Nix promoter-luciferase (NixP) transgenic mice that underwent ischemia-reperfusion (1 h/24 h), transverse aortic coarctation (TAC), or cross-breeding with the G q overexpression model of hypertrophy. Luciferase activity increased in G␣ q -NixP hearts 3.2 ؎ 0.4-fold and in TAC hearts 2.8 ؎ 0.4-fold but did not increase with infarction-reperfusion. NixP activity was proportional to the extent of TAC hypertrophy and was inhibited by mithramycin. These studies revealed distinct mechanisms of transcriptional regulation for cardiac Nix and BNip3. BNip3 is hypoxiainducible, whereas Nix expression was induced by G␣ q -mediated hypertrophic stimuli. PKC␣, a G q effector, transduced Nix transcriptional induction via Sp1.Cardiomyocyte apoptosis contributes to functional deterioration in ischemic, hypertrophic, and dilated cardiomyopathies (1-5). A critical but poorly understood feature of the cardiomyocyte cell death program is stress-mediated induction of gene expression for several pro-apoptotic factors belonging to the Bcl2 family of apoptosis-regulating proteins (4, 6). Recent studies of apoptosis gene induction in cardiac hypoxia and hypertrophy decompensation have assigned particular importance to two members of the BH3-only subgroup of Bcl2-like proteins, BNip3 and Nix (7-10). These two factors are each expressed in the heart, localized to mitochondria, and sufficient to induce apoptosis via the intrinsic, or mitochondrial, pathway (8, 10, 11). The potential for BNip3 or Nix, alone or in association with other Bcl2 family proteins (12, 13), to disrupt mitochondrial integrity by communicating with the mitochondrial permeability transition pore has suggested to some that a major function of the BH3-only proteins is to determine the on/off state of the mitochondrial permeability transition pore (14). Indeed, mitochondrial disruption may have especially profound consequences for the heart as myocardium is enriched in mit...
Gene alterations in tumor cells that confer the ability to grow under nutrient-and mitogen-deficient conditions constitute a competitive advantage that leads to more-aggressive forms of cancer. The atypical protein kinase C (PKC) isoform, PKC, has been shown to interact with the signaling adapter p62, which is important for Ras-induced lung carcinogenesis. Here we show that PKC-deficient mice display increased Ras-induced lung carcinogenesis, suggesting a new role for this kinase as a tumor suppressor in vivo. We also show that Ras-transformed PKC-deficient lungs and embryo fibroblasts produced more interleukin-6 (IL-6), which we demonstrate here plays an essential role in the ability of Ras-transformed cells to grow under nutrient-deprived conditions in vitro and in a mouse xenograft system in vivo. We also show that PKC represses histone acetylation at the C/EBP element in the IL-6 promoter. Therefore, PKC, by controlling the production of IL-6, is a critical signaling molecule in tumorigenesis.
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