Attenuated phosphorylation of heat shock protein 27 correlates with tumor progression in patients with hepatocellular carcinoma

Heat shock protein 27 (HSP27) is expressed at high levels in human hepatocellular carcinoma (HCC). We examined correlations of total HSP27 and serine phosphorylated (Ser-15, Ser-78, and Ser-82) HSP27 levels in HCC tissues with clinical and pathologic characteristics in 48 resected HCC specimens. The levels of total and Ser-phosphorylated HSP27 were evaluated by Western blot analysis. Immunohistochemical analysis of HSP27 expression was also performed on some samples. Phosphorylation of HSP27 was detected in all 48 HCC tissues. Levels of phosphorylated HSP27 were correlated inversely with tumor size, microvascular invasion of HCC, and tumor stage by TNM classification. In contrast, only microvascular invasion showed an inverse correlation with total HSP27 levels. The decrease in phosphorylated HSP27 in progressed HCC was also observed by immunohistochemistry. Levels of phosphorylated HSP27 gradually decreased in parallel with HCC progression. Our findings suggest that phosphorylated HSP27 may have a suppressive role in progression of human HCC.     

Loss of Klotho during melanoma progression leads to increased filamin cleavage, increased Wnt5A expression, and enhanced melanoma cell motility

We have previously shown that Wnt5A-mediated signaling can promote melanoma metastasis. It has been shown that Wnt signaling is antagonized by the protein Klotho, which has been implicated in aging. We show here that in melanoma cells, expressions of Wnt5A and Klotho are inversely correlated. In the presence of recombinant Klotho (rKlotho), we show that Wnt5A internalization and signaling is decreased in high Wnt5A-expressing cells. Moreover, in the presence of rKlotho, we observe an increase in Wnt5A remaining in the medium, coincident with an increase in sialidase activity, and decrease in syndecan expression. These effects can be inhibited using a sialidase inhibitor. In addition to its effects on Wnt5A internalization, we also demonstrate that Klotho decreases melanoma cell invasive potential by a second mechanism that involves the inhibition of calpain and a resultant decrease in filamin cleavage, which we demonstrate is critical for melanoma cell motility.     

Loss of keratin 10 is accompanied by increased sebocyte proliferation and differentiation

Here, we present strong evidence that the targeted deletion of keratin 10 (K10) alters sebocyte differentiation in mice, mediated by an increased proliferation and differentiation of cells located in the periphery of the glands. This was not accompanied by the induction of the proliferation-associated keratins K6, K16 and K17. Sebaceous gland cells of K10-/- mice showed an accelerated turnover and secreted more sebum including wax esters, triglycerides, and cholesterol esters. The levels of the major epidermal lipids ceramides and cholesterol were also increased, whereas glycosylceramides and sphingomyelin were decreased which was not based on altered sphingolipid biosynthesis. The amount of Cer(OS), covalently bound to the cornified envelope, remained unchanged, as well as the amount of loricrin and involucrin. In agreement with the unaltered expression of beta-catenin and its targets cyclin D1 and c-Myc, we conclude that the altered composition of the suprabasal intermediate filament cytoskeleton in K10-/- mice increased the differentiation of epidermal stem cells towards the sebocyte lineage.     

Loss of tafazzin in yeast leads to increased oxidative stress during respiratory growth

The tafazzin (TAZ) gene is highly conserved from yeast to humans, and the yeast taz1 null mutant shows alterations in cardiolipin (CL) metabolism, mitochondrial dysfunction and stabilization of supercomplexes similar to those found in Barth syndrome, a human disorder resulting from loss of tafazzin. We have previously shown that the yeast tafazzin mutant taz1Delta, which cannot remodel CL, is ethanol-sensitive at elevated temperature. In the current report, we show that in response to ethanol, CL mutants taz1Delta as well as crd1Delta, which cannot synthesize CL, exhibited increased protein carbonylation, an indicator of reactive oxygen species (ROS). The increase in ROS is most likely not due to defective oxidant defence systems, as the CL mutants do not display sensitivity to paraquat, menadione or hydrogen peroxide (H2O2). Ethanol sensitivity and increased protein carbonylation in the taz1Delta mutant but not in crd1Delta can be rescued by supplementation with oleic acid, suggesting that oleoyl-CL and/or oleoyl-monolyso-CL enables growth of taz1Delta in ethanol by decreasing oxidative stress. Our findings of increased oxidative stress in the taz1Delta mutant during respiratory growth may have important implications for understanding the pathogenesis of Barth syndrome.     

Loss of TGF-beta 1 leads to increased neuronal cell death and microgliosis in mouse brain

TGF-beta1 is a key regulator of diverse biological processes in many tissues and cell types, but its exact function in the developing and adult mammalian CNS is still unknown. We report that lack of TGF-beta1 expression in neonatal Tgfb1(-/-) mice results in a widespread increase in degenerating neurons accompanied by reduced expression of synaptophysin and laminin and a prominent microgliosis. Lack of TGF-beta1 also strongly reduces survival of primary neurons cultured from Tgfb1(-/-) mice. TGF-beta1 deficiency in adult Tgfb1(-/+) mice results in increased neuronal susceptibility to excitotoxic injury, whereas astroglial overexpression of TGF-beta1 protects adult mice against neurodegeneration in acute, excitotoxic and chronic injury paradigms. This study reveals a nonredundant function for TGF-beta1 in maintaining neuronal integrity and survival of CNS neurons and in regulating microglial activation. Because individual TGF-beta1 expression levels in the brain vary considerably between humans, this finding could have important implications for susceptibility to neurodegeneration.     

Loss of lipoprotein lipase-derived fatty acids leads to increased cardiac glucose metabolism and heart dysfunction

Long-chain fatty acids (FAs) are the predominant energy substrate utilized by the adult heart. The heart can utilize unesterified FA bound to albumin or FA obtained from lipolysis of lipoprotein-bound triglyceride (TG). We used heart-specific lipoprotein lipase knock-out mice (hLpL0) to test whether these two sources of FA are interchangeable and necessary for optimal heart function. Hearts unable to obtain FA from lipoprotein TG were able to compensate by increasing glucose uptake, glycolysis, and glucose oxidation. HLpL0 hearts had decreased expression of pyruvate dehydrogenase kinase 4 and increased cardiomyocyte expression of glucose transporter 4. Conversely, FA oxidation rates were reduced in isolated perfused hLpL0 hearts. Following abdominal aortic constriction expression levels of genes regulating FA and glucose metabolism were acutely up-regulated in control and hLpL0 mice, yet all hLpL0 mice died within 48 h of abdominal aortic constriction. Older hLpL0 mice developed cardiac dysfunction characterized by decreased fractional shortening and interstitial and perivascular fibrosis. HLpL0 hearts had increased expression of several genes associated with transforming growth factor-beta signaling. Thus, long term reduction of lipoprotein FA uptake is associated with impaired cardiac function despite a compensatory increase in glucose utilization.     

Insulin-induced phosphorylation of ENaC correlates with increased sodium channel function in A6 cells

The purpose of this study was to determine whether there is a correlation between phosphorylation and activity of the epithelial sodium channel (ENaC). The three subunits that form the channel were immunoprecipitated from A6 cells by using specific polyclonal antibodies after labeling cells with ³⁵S or ³²P. When immune complexes were resolved on SDS-PAGE, the -subunit migrated at 85 and 65 kDa, the -subunit at 115 and 100 kDa, and the -subunit at 90 kDa. In the resting state all three subunits were phosphorylated. The -subunit was phosphorylated only in the 65-kDa band, suggesting that the posttranslational modification that gives rise to the rapidly migrating form of is a requirement for phosphorylation. Stimulation with 100 nM insulin for 30 min increased phosphorylation of -, -, and -subunits approximately twofold. Exposure to 1 µM aldosterone for 16 h increased protein abundance and phosphorylation proportionately in the three subunits. When insulin was applied to cells pretreated with aldosterone, phosphorylation was also increased approximately twofold, but the total amount of phosphorylated substrate was larger than in control conditions because of the action of aldosterone. This result might explain the synergistic increase in sodium transport under the same conditions. The protein kinase C inhibitor chelerythrine abolished insulin effects and decreased sodium transport and subunit phosphorylation. Together, our findings suggest that ENaC activity is controlled by subunit phosphorylation in cells that endogenously express the channel and the machinery for hormonal stimulation of sodium transport. epithelial sodium channel; aldosterone; sodium transport     

Requirement for ERK activation in acetone extract identified from Bupleurum scorzonerifolium induced A549 tumor cell apoptosis and keratin 8 phosphorylation

We previously demonstrated that the crude acetone extract of Bupleurum scorzonerifolium (AE-BS) 60 microg/ml has anti-proliferation activity and apoptosis effects to A549 human lung cancer cells. They can also cause tumor cell arrest in G2/M phase. To better understand its target protein in A549 cell, two-dimensional electrophoresis and liquid chromatography-tandem mass spectrometry were applied. The modification of keratin 8 was identified. By immunoblot, the expression of phosphorylated keratin 8 at Ser-73 was increased from 2.0 to 3.0-fold after AE-BS treatment 24 to 48 hr respectively as compared with untreated A549 control cells. Furthermore, the A549 cells were pretreated with 50 microM PD98059, a specific inhibitor of the upstream regulator of ERK1/2, or with the p38 kinase inhibitor 20 microM SB203580 or JNK inhibitor 20 microM SP600125 for 30 min, followed by 24 h of incubation with AE-BS, PD98059 can inhibit K8-Ser-73 hyperphosphorylation and prevented cell apoptosis which was induced by AE-BS significantly. By immunoblot, AE-BS also can induce ERK 1/2 phosphorylation. In conclusion, our data indicate that the AE-BS induced tumor apoptosis in A549 cells was related to ERK 1/2 activation. The molecular mechanism of hyperphosphorylation of K8 on Ser-73 was associated with ERK 1/2 activation rather than JNK and p38 kinase. The apoptosis induced by AE-BS may be related to K8 phosphorylation.     

Loss of parietal cell superoxide dismutase leads to gastric oxidative stress and increased injury susceptibility in mice

Mitochondrial superoxide dismutase (SOD2) prevents accumulation of the superoxide that arises as a consequence of oxidative phosphorylation. However, SOD2 is a target of oxidative/nitrosative inactivation, and reduced SOD2 activity has been demonstrated to contribute to portal hypertensive gastropathy. We investigated the consequences of gastric parietal cell-specific SOD2 deficiency on mitochondrial function and gastric injury susceptibility. Mice expressing Cre recombinase under control of the parietal cell Atpase4b gene promoter were crossed with mice harboring loxP sequences flanking the sod2 gene (SOD2 floxed mice). Cre-positive mice and Cre-negative littermates (controls) were used in studies of SOD2 expression, parietal cell function (ATP synthesis, acid secretion, and mitochondrial enzymatic activity), increased oxidative/nitrosative stress, and gastric susceptibility to acute injury. Parietal cell SOD2 deficiency was accompanied by a 20% (P < 0.05) reduction in total gastric SOD activity and a 93% (P < 0.001) reduction in gastric SOD2 activity. In SOD2-deficient mice, mitochondrial aconitase and ATP synthase activities were impaired by 36% (P < 0.0001) and 44% (P < 0.005), respectively. Gastric tissue ATP content was reduced by 34% (P < 0.002). Basal acid secretion and peak secretagogue (histamine)-induced acid secretion were reduced by 43% (P < 0.0001) and 40% (P < 0.0005), respectively. There was a fourfold (P < 0.02) increase in gastric mucosal apoptosis and 41% (P < 0.001) greater alcohol-induced gastric damage in the parietal cell SOD2-deficient mice. Our findings indicate that loss of parietal cell SOD2 leads to mitochondrial dysfunction, resulting in perturbed energy metabolism, impaired parietal cell function, and increased gastric mucosal oxidative stress. These alterations render the gastric mucosa significantly more susceptible to acute injury.     

Overexpression of TRIM24 correlates with tumor progression in non-small cell lung cancer

The objective of the current study was to investigate the expression pattern and clinicopathological significance of TRIM24 in patients with non-small cell lung cancer (NSCLC). The expression profile of TRIM24 in NSCLC tissues and adjacent noncancerous lung tissues was detected by immunohistochemistry. TRIM24 was found to be overexpressed in 81 of 113 (71.7%) human lung cancer samples and correlated with p-TNM stage (p = 0.0006), poor differentiation (p = 0.004), Ki67 index (p<0.0001), cyclin D1(p = 0.0096) and p-Rb expression (p = 0.0318). In addition, depleting TRIM24 expression by small interfering RNA inhibited growth and invasion in lung cell lines. Moreover, TRIM24 depletion induced cell cycle arrest at the G1/S boundary and induced apoptosis. Western blotting analysis revealed that knockdown of TRIM24 decreased the protein levels of Cyclin A, Cyclin B, Cyclin D1, cyclin E and p-Rb and increased P27 expression. These results indicate that TRIM24 plays an important role in NSCLC progression.     

Mutual regulation between DNA-PKcs and snail1 leads to increased genomic instability and aggressive tumor characteristics

Although the roles of DNA-dependent protein kinase catalytic subunits (DNA-PKcs) in the non-homologous end joining (NHEJ) of DNA repair are well-recognized, the biological mechanisms and regulators by DNA-PKcs besides DNA repair, have not been clearly described. Here, we show that active DNA-PKcs caused by ionizing radiation, phosphorylated Snail1 at serine (Ser) 100, led to increased Snail1 stability. Furthermore, phosphorylated Snail1 at Ser100 reciprocally inhibited the kinase activity of DNA-PKcs, resulting in an inhibition of DNA repair activity. Moreover, Snail1 phosphorylation by DNA-PKcs was involved in genomic instability and aggressive tumor characteristics. Our results describe novel cellular mechanisms that affect genomic instability, sensitivity to DNA-damaging agents, and the migration of tumor cells by reciprocal regulation between DNA-PKcs and Snail1.     

Nutrient-dependent and insulin-stimulated phosphorylation of insulin receptor substrate-1 on serine 302 correlates with increased insulin signaling

Ser/Thr phosphorylation of insulin receptor substrate IRS-1 regulates insulin signaling, but the relevant phosphorylated residues and their potential functions during insulin-stimulated signal transduction are difficult to resolve. We used a sequence-specific polyclonal antibody directed against phosphorylated Ser(302) to study IRS-1-mediated signaling during insulin and insulin-like growth factor IGF-I stimulation. Insulin or IGF-I stimulated phosphorylation of Ser(302) in various cell backgrounds and in murine muscle. Wortmannin or rapamycin inhibited Ser(302) phosphorylation, and amino acids or glucose stimulated Ser(302) phosphorylation, suggesting a role for the mTOR cascade. The Ser(302) kinase associates with IRS-1 during immunoprecipitation, but its identity is unknown. The NH(2)-terminal c-Jun kinase did not phosphorylate Ser(302). Replacing Ser(302) with alanine significantly reduced insulin-stimulated tyrosine phosphorylation of IRS-1 and p85 binding and reduced insulin-stimulated phosphorylation of p70(S6K), ribosomal S6 protein, and 4E-BP1; however, this mutation had no effect on insulin-stimulated Akt or glycogen synthase kinase 3beta phosphorylation. Replacing Ser(302) with alanine reduced insulin/IGF-I-stimulated DNA synthesis. We conclude that Ser(302) phosphorylation integrates nutrient availability with insulin/IGF-I signaling to promote mitogenesis and cell growth.     

Expression of mast cell proteases correlates with mast cell maturation and angiogenesis during tumor progression

Tumor cells are surrounded by infiltrating inflammatory cells, such as lymphocytes, neutrophils, macrophages, and mast cells. A body of evidence indicates that mast cells are associated with various types of tumors. Although role of mast cells can be directly related to their granule content, their function in angiogenesis and tumor progression remains obscure. This study aims to understand the role of mast cells in these processes. Tumors were chemically induced in BALB/c mice and tumor progression was divided into Phases I, II and III. Phase I tumors exhibited a large number of mast cells, which increased in phase II and remained unchanged in phase III. The expression of mouse mast cell protease (mMCP)-4, mMCP-5, mMCP-6, mMCP-7, and carboxypeptidase A were analyzed at the 3 stages. Our results show that with the exception of mMCP-4 expression of these mast cell chymase (mMCP-5), tryptases (mMCP-6 and 7), and carboxypeptidase A (mMC-CPA) increased during tumor progression. Chymase and tryptase activity increased at all stages of tumor progression whereas the number of mast cells remained constant from phase II to III. The number of new blood vessels increased significantly in phase I, while in phases II and III an enlargement of existing blood vessels occurred. In vitro, mMCP-6 and 7 are able to induce vessel formation. The present study suggests that mast cells are involved in induction of angiogenesis in the early stages of tumor development and in modulating blood vessel growth in the later stages of tumor progression.     

Loss of the tumor suppressor Snf5 leads to aberrant activation of the Hedgehog-Gli pathway

Aberrant activation of the Hedgehog (Hh) pathway can drive tumorigenesis. To investigate the mechanism by which glioma-associated oncogene family zinc finger-1 (GLI1), a crucial effector of Hh signaling, regulates Hh pathway activation, we searched for GLI1-interacting proteins. We report that the chromatin remodeling protein SNF5 (encoded by SMARCB1, hereafter called SNF5), which is inactivated in human malignant rhabdoid tumors (MRTs), interacts with GLI1. We show that Snf5 localizes to Gli1-regulated promoters and that loss of Snf5 leads to activation of the Hh-Gli pathway. Conversely, re-expression of SNF5 in MRT cells represses GLI1. Consistent with this, we show the presence of a Hh-Gli-activated gene expression profile in primary MRTs and show that GLI1 drives the growth of SNF5-deficient MRT cells in vitro and in vivo. Therefore, our studies reveal that SNF5 is a key mediator of Hh signaling and that aberrant activation of GLI1 is a previously undescribed targetable mechanism contributing to the growth of MRT cells.     

Loss of circadian clock gene expression is associated with tumor progression in breast cancer

Several studies suggest a link between circadian rhythm disturbances and tumorigenesis. However, the association between circadian clock genes and prognosis in breast cancer has not been systematically studied. Therefore, we examined the expression of 17 clock components in tumors from 766 node-negative breast cancer patients that were untreated in both neoadjuvant and adjuvant settings. In addition, their association with metastasis-free survival (MFS) and correlation to clinicopathological parameters were investigated. Aiming to estimate functionality of the clockwork, we studied clock gene expression relationships by correlation analysis. Higher expression of several clock genes (e.g., CLOCK, PER1, PER2, PER3, CRY2, NPAS2 and RORC) was found to be associated with longer MFS in univariate Cox regression analyses (HR<1 and FDR-adjusted P < 0.05). Stratification according to molecular subtype revealed prognostic relevance for PER1, PER3, CRY2 and NFIL3 in the ER+/HER2- subgroup, CLOCK and NPAS2 in the ER-/HER2- subtype, and ARNTL2 in HER2+ breast cancer. In the multivariate Cox model, only PER3 (HR = 0.66; P = 0.016) and RORC (HR = 0.42; P = 0.003) were found to be associated with survival outcome independent of established clinicopathological parameters. Pairwise correlations between functionally-related clock genes (e.g., PER2-PER3 and CRY2-PER3) were stronger in ER+, HER2- and low-grade carcinomas; whereas, weaker correlation coefficients were observed in ER- and HER2+ tumors, high-grade tumors and tumors that progressed to metastatic disease. In conclusion, loss of clock genes is associated with worse prognosis in breast cancer. Coordinated co-expression of clock genes, indicative of a functional circadian clock, is maintained in ER+, HER2-, low grade and non-metastasizing tumors but is compromised in more aggressive carcinomas.