Autophagy facilitates oncogene-induced senescence

Oncogenic stress triggers a range of intracellular protective responses including DNA damage checkpoints, senescence and apoptosis, depending on the cell type and the severity of the particular stress. Senescent cells are metabolically viable but are stably arrested. Senescence is a collective phenotype, however, that is comprised of various signaling pathways and effector mechanisms. Thus, to understand and manipulate the senescence phenotype, it is critical to find its effector mechanisms and determine the relationships between them. We have recently found that autophagy is activated upon acute induction of senescence and facilitates another effector mechanism: the senescence associated secretory phenotype (SASP).     

deltaNp73 facilitates cell immortalization and cooperates with oncogenic Ras in cellular transformation in vivo

TP73, despite significant homology to TP53, is not a classic tumor suppressor gene, since it exhibits upregulation of nonmutated products in human tumors and lacks a tumor phenotype in p73-deficient mice. We recently reported that an N-terminally truncated isoform, DeltaNp73, is upregulated in breast and gynecological cancers. We further showed that DeltaNp73 is a potent transdominant inhibitor of wild-type p53 and TAp73 in cultured human tumor cells by efficiently counteracting their target gene transactivations, apoptosis, and growth suppression functions (A. I. Zaika et al., J. Exp. Med. 6:765-780, 2002). Although these data strongly suggest oncogenic properties of DeltaNp73, this can only be directly shown in primary cells. We report here that DeltaNp73 confers resistance to spontaneous replicative senescence of primary mouse embryo fibroblasts (MEFs) and immortalizes MEFs at a 1,000-fold-higher frequency than occurs spontaneously. DeltaNp73 cooperates with cMyc and E1A in promoting primary cell proliferation and colony formation and compromises p53-dependent MEF apoptosis. Importantly, DeltaNp73 rescues Ras-induced senescence. Moreover, DeltaNp73 cooperates with oncogenic Ras in transforming primary fibroblasts in vitro and in inducing MEF-derived fibrosarcomas in vivo in nude mice. Wild-type p53 is likely a major target of DeltaNp73 inhibition in primary fibroblasts since deletion of p53 or its requisite upstream activator ARF abrogates the growth-promoting effect of DeltaNp73. Taken together, DeltaNp73 behaves as an oncogene that targets p53 that might explain why DeltaNp73 upregulation may be selected for during tumorigenesis of human cancers.     

Molecular weight dependence of hyaluronic acid produced during oncogenic transformation

Cultured chicken embryo fibroblasts synthesize two distinct molecular size classes of hyaluronic acid. The high molecular weight material (form I, 2.98 x 10(6) is the predominant species synthesized by transformed cells, whereas form II (1.42 x 10(5)) is the major product of non-transformed cells. A shift to synthesis of predominantly form I hyaluronic acid is an early transformation event in cells infected with LA24 Rous sarcoma virus and maintained at the permissive temperature for transformation (35 degrees C). Form I hyaluronic acid exhibits greater binding to preparations of cellular fibronectin and to both normal and transformed cells than does form II. Both forms bind more to transformed cells than to normal, uninfected cells. Hyaluronic acid (predominantly form I) isolated from transforming cells stimulates proliferation in growth-retarded, non-transformed cells.     

Aerobic glycolysis and lymphocyte transformation

1. The role of enhanced aerobic glycolysis in the transformation of rat thymocytes by concanavalin A has been investigated. Concanavalin A addition doubled [U-(14)C]glucose uptake by rat thymocytes over 3h and caused an equivalent increased incorporation into protein, lipids and RNA. A disproportionately large percentage of the extra glucose taken up was converted into lactate, but concanavalin A also caused a specific increase in pyruvate oxidation, leading to an increase in the percentage contribution of glucose to the respiratory fuel. 2. Acetoacetate metabolism, which was not affected by concanavalin A, strongly suppressed pyruvate oxidation in the presence of [U-(14)C]glucose, but did not prevent the concanavalin A-induced stimulation of this process. Glucose uptake was not affected by acetoacetate in the presence or absence of concanavalin A, but in each case acetoacetate increased the percentage of glucose uptake accounted for by lactate production. 3. [(3)H]Thymidine incorporation into DNA in concanavalin A-treated thymocyte cultures was sensitive to the glucose concentration in the medium in a biphasic manner. Very low concentrations of glucose (25mum) stimulated DNA synthesis half-maximally, but maximum [(3)H]thymidine incorporation was observed only when the glucose concentration was raised to 1mm. Lactate addition did not alter the sensitivity of [(3)H]-thymidine uptake to glucose, but inosine blocked the effect of added glucose and strongly inhibited DNA synthesis. 4. It is suggested that the major function of enhanced aerobic glycolysis in transforming lymphocytes is to maintain higher steady-state amounts of glycolytic intermediates to act as precursors for macromolecule synthesis.     

Radioresistance induced by oncogenic transformation

Rat embryo cells at various stages of oncogenic transformation are obtained by a combination of X irradiation and transfection with the ras and the myc oncogenes. Transfection with either the ras or the myc oncogenes can lead to increased radioresistance, relative to the parental cells. X-ray-transformed clones of the transfected cells do not show additional alteration in radioresponse. Incorporation of the two oncogenes appears to lead to a higher degree of radioresistance.     

Cell surface fibronectin and oncogenic transformation

Fibronectin is a large glycoprotein at the cell surface of many different cell types; a related protein is present in plasma. Fibronectin is a dimer of 230,000-dalton subunits and also occurs in larger aggregates; it forms fibrillar networks at the cell surface, between cells and substrata and between adjacent cells, and it is not a typical membrane protein. Cell surface fibronectin is reduced in amount or absent on transformed cells and in many cases its loss correlates with acquisition of tumorigenicity and, in particular, metastatic ability. Exceptions to the correlations with transformation and tumorigenicity exist. Loss of fibronectin and the resulting reduced adhesion appear to be involved in pleiotrpoic alterations in cell behavior and may be responsible for several aspects of the transformed phenotype in vitro. Fibronectin interacts with other macromolecules (collagen/gelatin, fibrin/fibrinogen, proteoglycans) and is apparently connected to microfilaments inside the cell.     

Integrins uncouple Src-induced morphological and oncogenic transformation

Expression of activated mutants of c-Src in epithelial cells can induce tumorigenicity. In addition to such oncogenic transformation, the cells undergo a dramatic morphological transformation: cell-cell contacts are disrupted, spreading on extracellular matrix proteins is suppressed, actin stress fibers and focal contacts are lost, and podosomes are formed. We have previously shown that integrin alphavbeta3 strongly supports Src-mediated oncogenic transformation through an interaction at the beta3 cytoplasmic tail. Our current findings demonstrate that this interaction does not affect Src-mediated morphological alterations, thus separating oncogenic from morphological transformation. Moreover, beta1 and beta3 integrins differently affect the various aspects of Src-induced morphological transformation. High levels of beta3, but not beta1, integrins can prevent Src-induced cell rounding although stress fiber disassembly and podosome formation still occur. Studies using chimeric integrin subunits demonstrate that this protection requires the beta3 extracellular domain. Finally, like tumor formation, podosome assembly occurs independent of beta3 phosphorylation. Instead, phosphorylation of beta1 is required to suppress Rho-mediated contractility in order to assemble podosomes. Thus, integrins regulate Src-mediated oncogenic transformation and various aspects of morphological transformation through dissociable pathways.     

Involvement of mitophagy in oncogenic K-Ras-induced transformation

Although mitochondrial impairment has often been implicated in carcinogenesis, the mechanisms of its development in cancer remain unknown. We report here that autophagy triggered by oncogenic K-Ras mediates functional loss of mitochondria during cell transformation to overcome an energy deficit resulting from glucose deficiency. When Rat2 cells were infected with a retrovirus harboring constitutively active K-Ras^V12, mitochondrial respiration significantly declined in parallel with the acquisition of transformation characteristics. Decreased respiration was not related to mitochondrial biogenesis but was inversely associated with the increased formation of acidic vesicles enclosing mitochondria, during which autophagy-related proteins such as Beclin 1, Atg5, LC3-II and vacuolar ATPases were induced. Interestingly, blocking autophagy with conventional inhibitors (bafilomycin A, 3-methyladenin) and siRNA-mediated knockdown of autophagy-related genes recovered respiratory protein expression and respiratory activity; JNK was involved in these phenomena as an upstream regulator. The cells transformed by K-Ras^V12 maintained cellular ATP level mainly through glycolytic ATP production without induction of GLUT1, the low K_m glucose transporter. Finally, K-Ras^V12-triggered LC3-II formation was modulated by extracellular glucose levels, and LC3-II formation increased only in hepatocellular carcinoma tissues exhibiting low glucose uptake and increased K-Ras expression. Taken together, our observations suggest that mitochondrial functional loss may be mediated by oncogenic K-Ras-induced mitophagy during early tumorigenesis even in the absence of hypoxia, and that this mitophagic process may be an important strategy to overcome the cellular energy deficit triggered by insufficient glucose.     

Autophagy in malignant transformation and cancer progression

Autophagy plays a key role in the maintenance of cellular homeostasis. In healthy cells, such a homeostatic activity constitutes a robust barrier against malignant transformation. Accordingly, many oncoproteins inhibit, and several oncosuppressor proteins promote, autophagy. Moreover, autophagy is required for optimal anticancer immunosurveillance. In neoplastic cells, however, autophagic responses constitute a means to cope with intracellular and environmental stress, thus favoring tumor progression. This implies that at least in some cases, oncogenesis proceeds along with a temporary inhibition of autophagy or a gain of molecular functions that antagonize its oncosuppressive activity. Here, we discuss the differential impact of autophagy on distinct phases of tumorigenesis and the implications of this concept for the use of autophagy modulators in cancer therapy.     

p27kip1 protein levels reflect a nexus of oncogenic signaling during cell transformation

SV40 small t-antigen (ST) collaborates with SV40 large T-antigen (LT) and activated rasv12 to promote transformation in a variety of immortalized human cells. A number of oncogenes or the disruption of the general serine-threonine phosphatase protein phosphatase 2A (PP2A) can replace ST in this paradigm. However, the relationship between these oncogenes and PP2A activity is not clear. To address this, we queried the connectivity of these molecules in silico. We found that p27 was connected to each of those oncogenes that could substitute for ST. We further determined that p27 loss can substitute for the expression of ST during transformation of both rodent and human cells. Conversely, knock-in cells expressing the degradation-resistant S10A and T187A mutants of p27 were resistant to the transforming activities of ST. This suggests that p27 is an important target of the tumor-suppressive effects of PP2A and likely an important target of the multitude of cellular oncoproteins that emulate the transforming function of ST.     

Dimerize RACK1 upon transformation with oncogenic ras

From our previous studies, we learned that syndecan-2/p120-GAP complex provided docking site for Src to prosecute tyrosine kinase activity upon transformation with oncogenic ras. And, RACK1 protein was reactive with syndecan-2 to keep Src inactivated, but not when Ras was overexpressed. In the present study, we characterized the reaction between RACK1 protein and Ras. RACK1 was isolated from BALB/3T3 cells transfected with plasmids pcDNA3.1-[S-ras(Q61K)] of shrimp Penaeus japonicus and RACK1 was revealed to react with GTP-K(B)-Ras(Q61K), not GDP-K(B)-Ras(Q61K). This selective interaction between RACK1 and GTP-K(B)-Ras(Q61K) was further confirmed with RACK1 of human placenta and mouse RACK1-encoded fusion protein. We found that RACK1 was dimerized upon reaction with GTP-K(B)-Ras(Q61K), as well as with 14-3-3beta and geranylgeranyl pyrophosphate, as revealed by phosphorylation with Src tyrosine kinase. We reported the complex of RACK1/GTP-K(B)-Ras(Q61K) reacted selectively with p120-GAP. This interaction was sufficient to dissemble RACK1 into monomers, a preferred form to compete for the binding of syndecan-2. These data indicate that the reaction of GTP-K(B)-Ras(Q61K) with RACK1 in dimers may operate a mechanism to deplete RACK1 from reaction with syndecan-2 upon transformation by oncogenic ras and the RACK1/GTP-Ras complex may provide a route to react with p120-GAP and recycle monomeric RACK1 to syndecan-2.     

Autophagy facilitates secretion and protects against degeneration of the Harderian gland

The epithelial derived Harderian gland consists of 2 types of secretory cells. The more numerous type A cells are responsible for the secretion of lipid droplets, while type B cells produce dark granules of multilamellar bodies. The process of autophagy is constitutively active in the Harderian gland, as confirmed by our analysis of LC3 processing in GFP-LC3 transgenic mice. This process is compromised by epithelial deletion of Atg7. Morphologically, the Atg7 mutant glands are hypotrophic and degenerated, with highly vacuolated cells and pyknotic nuclei. The mutant glands accumulate lipid droplets coated with PLIN2 (perilipin 2) and contain deposits of cholesterol, ubiquitinated proteins, SQSTM1/p62 (sequestosome 1) positive aggregates and other metabolic products such as porphyrin. Immunofluorescence stainings show that distinct cells strongly aggregate both proteins and lipids. Electron microscopy of the Harderian glands reveals that its organized structure is compromised, and the presence of large intracellular lipid droplets and heterologous aggregates. We attribute the occurrence of large vacuoles to a malfunction in the formation of multilamellar bodies found in the less abundant type B Harderian gland cells. This defect causes the formation of large tertiary lysosomes of heterologous content and is accompanied by the generation of tight lamellar stacks of endoplasmic reticulum in a pseudo-crystalline form. To test the hypothesis that lipid and protein accumulation is the cause for the degeneration in autophagy-deficient Harderian glands, epithelial cells were treated with a combination of the proteasome inhibitor and free fatty acids, to induce aggregation of misfolded proteins and lipid accumulation, respectively. The results show that lipid accumulation indeed enhanced the toxicity of misfolded proteins and that this was even more pronounced in autophagy-deficient cells. Thus, we conclude autophagy controls protein and lipid catabolism and anabolism to facilitate bulk production of secretory vesicles of the Harderian gland.     

Autophagy facilitates secretion and protects against degeneration of the Harderian gland

The epithelial derived Harderian gland consists of 2 types of secretory cells. The more numerous type A cells are responsible for the secretion of lipid droplets, while type B cells produce dark granules of multilamellar bodies. The process of autophagy is constitutively active in the Harderian gland, as confirmed by our analysis of LC3 processing in GFP-LC3 transgenic mice. This process is compromised by epithelial deletion of Atg7. Morphologically, the Atg7 mutant glands are hypotrophic and degenerated, with highly vacuolated cells and pyknotic nuclei. The mutant glands accumulate lipid droplets coated with PLIN2 (perilipin 2) and contain deposits of cholesterol, ubiquitinated proteins, SQSTM1/p62 (sequestosome 1) positive aggregates and other metabolic products such as porphyrin. Immunofluorescence stainings show that distinct cells strongly aggregate both proteins and lipids. Electron microscopy of the Harderian glands reveals that its organized structure is compromised, and the presence of large intracellular lipid droplets and heterologous aggregates. We attribute the occurrence of large vacuoles to a malfunction in the formation of multilamellar bodies found in the less abundant type B Harderian gland cells. This defect causes the formation of large tertiary lysosomes of heterologous content and is accompanied by the generation of tight lamellar stacks of endoplasmic reticulum in a pseudo-crystalline form. To test the hypothesis that lipid and protein accumulation is the cause for the degeneration in autophagy-deficient Harderian glands, epithelial cells were treated with a combination of the proteasome inhibitor and free fatty acids, to induce aggregation of misfolded proteins and lipid accumulation, respectively. The results show that lipid accumulation indeed enhanced the toxicity of misfolded proteins and that this was even more pronounced in autophagy-deficient cells. Thus, we conclude autophagy controls protein and lipid catabolism and anabolism to facilitate bulk production of secretory vesicles of the Harderian gland.     

Autophagy during beef aging

The conversion of muscle into meat is a complex process of major concern for meat scientists due to its influence on the final meat quality. The aim of this study was to investigate the occurrence of autophagic processes in the conversion of muscle into meat. Our findings demonstrated, for the first time, the occurrence of autophagic processes in the muscle tissue at early postmortem period (2 h to 24 h) in both beef breeds studied (Asturiana de los Valles and Asturiana de la Montaña) showing significant time-scale differences between breeds, which could indicate a role of this process in meat maturation. These breeds have different physiological features: while Asturiana de los Valles is a meat-specialized breed showing high growth rate, an elevated proportion of white fibers in the muscle and low intramuscular fat level, Asturiana de la Montaña is a small- to medium-sized rustic breed adapted to less-favored areas, showing more red fibers in the muscle and a high intramuscular fat content.     

Advances in Rho-dependent actin regulation and oncogenic transformation.

Cellular movement is central to invasion. The Rho family of small GTPases co-ordinate the cytoskeletal and adhesion modelling within cells that is crucial for normal migratory responses. Consequently, Rho proteins, and their regulators and effectors, are targets for subversion during oncogenic transformation and tumour development. Recent findings have thrown light on how actin regulators may be linked to oncogenesis and the development of cancer.