Targeted Replacement of HSF1 Phosphorylation Sites at S303/S307 with Alanine Residues in Mice Increases Cell Proliferation and Drug Resistance.

Mammalian heat shock factor HSF1 transcriptional activity is controlled by a multitude of phosphorylations that occur under physiological conditions or following exposure of cells to a variety of stresses. One set of HSF1 phosphorylation is on serine 303 and serine 307 (S303/S307). These HSF1 phosphorylation sites are known to repress its transcriptional activity. Here, we describe a knock-in mouse model where these two serine residues were replaced by alanine residues and have determined the impact of these mutations on cellular proliferation and drug resistance. Our previous study using this mouse model indicated the susceptibility of the mutant mice to become obese with age due to an increase in basal levels of heat shock proteins (HSPs) and chronic inflammation. Since HSF1 transcriptional activity is increased in many tumor types, this mouse model may be a useful tool for studies related to cellular transformation and cancer.

Dusp26 phosphatase regulates mitochondrial respiration and oxidative stress and protects neuronal cell death.

The dual specificity protein phosphatases (Dusps) control dephosphorylation of mitogen-activated protein kinases (MAPKs) as well as other substrates. Here, we report that Dusp26, which is highly expressed in neuroblastoma cells and primary neurons is targeted to the mitochondrial outer membrane via its NH2-terminal mitochondrial targeting sequence. Loss of Dusp26 has a significant impact on mitochondrial function that is associated with increased levels of reactive oxygen species (ROS), reduction in ATP generation, reduction in mitochondria motility and release of mitochondrial HtrA2 protease into the cytoplasm. The mitochondrial dysregulation in dusp26-deficient neuroblastoma cells leads to the inhibition of cell proliferation and cell death. In vivo, Dusp26 is highly expressed in neurons in different brain regions, including cortex and midbrain (MB). Ablation of Dusp26 in mouse model leads to dopaminergic (DA) neuronal cell loss in the substantia nigra par compacta (SNpc), inflammatory response in MB and striatum, and phenotypes that are normally associated with Neurodegenerative diseases. Consistent with the data from our mouse model, Dusp26 expressing cells are significantly reduced in the SNpc of Parkinson's Disease patients. The underlying mechanism of DA neuronal death is that loss of Dusp26 in neurons increases mitochondrial ROS and concurrent activation of MAPK/p38 signaling pathway and inflammatory response. Our results suggest that regulation of mitochondrial-associated protein phosphorylation is essential for the maintenance of mitochondrial homeostasis and dysregulation of this process may contribute to the initiation and development of neurodegenerative diseases.

Increased Circulating Cathepsin L in Patients with Coronary Artery Disease.

Cathepsin L (CatL) is a potent collagenase involved in atherosclerotic vascular remodeling and dysfunction in animals and humans. This study investigated the hypothesis that plasma CatL is associated with the prevalence of coronary artery disease (CAD). Between February May 2011 and January 2013, 206 consecutive subjects were enrolled from among patients who underwent coronary angiography and percutaneous coronary intervention treatment. Age-matched subjects (n = 215) served as controls. Plasma CatL and high-sensitive C-reactive protein (hs-CRP) and high-density lipoprotein cholesterol were measured. The patients with CAD had significantly higher plasma CatL levels compared to the controls (1.4 ± 0.4 versus 0.4 ± 0.2 ng/mL, P < 0.001), and the patients with acute coronary syndrome had significantly higher plasma CatL levels compared to those with stable angina pectoris (1.7 ± 0.7 versus 0.8 ± 0.4 ng/mL, P < 0.01). Linear regression analysis showed that overall, the plasma CatL levels were inversely correlated with the high-density lipoprotein levels (r = -0.32, P < 0.01) and positively with hs-CRP levels (r = 0.35, P < 0.01). Multiple logistic regression analyses shows that cathepsin L levels were independent predictors of CAD (add ratio, 1.8; 95% CI, 1.2 to 2.1; P < 0.01). These data demonstrated that increased levels of plasma CatL are closely associated with the presence of CAD and that circulating CatL serves as a useful biomarker for CAD.

HSF1-Mediated Control of Cellular Energy Metabolism and mTORC1 Activation Drive Acute T-Cell Lymphoblastic Leukemia Progression.

Deregulated oncogenic signaling linked to PI3K/AKT and mTORC1 pathway activation is a hallmark of human T-cell acute leukemia (T-ALL) pathogenesis and contributes to leukemic cell resistance and adverse prognosis. Notably, although the multiagent chemotherapy of leukemia leads to a high rate of complete remission, options for salvage therapy for relapsed/refractory disease are limited due to the serious side effects of augmenting cytotoxic chemotherapy. We report that ablation of HSF1, a key transcriptional regulator of the chaperone response and cellular bioenergetics, from mouse T-ALL tumors driven by PTEN loss or human T-ALL cell lines, has significant therapeutic effects in reducing tumor burden and sensitizing malignant cell death. From a mechanistic perspective, the enhanced sensitivity of T-ALLs to HSF1 depletion resides in the reduced MAPK-ERK signaling and metabolic and ATP-producing capacity of malignant cells lacking HSF1 activity. Impaired mitochondrial ATP production and decreased intracellular amino acid content in HSF1-deficient T-ALL cells trigger an energy-saving adaptive response featured by attenuation of the mTORC1 activity, which is coregulated by ATP, and its downstream target proteins (p70S6K and 4E-BP). This leads to protein translation attenuation that diminishes oncogenic signals and malignant cell growth. Collectively, these metabolic alterations in the absence of HSF1 activity reveal cancer cell liabilities and have a profound negative impact on T-ALL progression. IMPLICATIONS: Targeting HSF1 and HSF1-dependent cancer-specific anabolic and protein homeostasis programs has a significant therapeutic potential for T-ALL and may prevent progression of relapsed/refractory disease.

The Molecular Chaperone Heat Shock Protein 70 Controls Liver Cancer Initiation and Progression by Regulating Adaptive DNA Damage and Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Signaling Pathways.

Delineating the mechanisms that drive hepatic injury and hepatocellular carcinoma (HCC) progression is critical for development of novel treatments for recurrent and advanced HCC but also for the development of diagnostic and preventive strategies. Heat shock protein 70 (HSP70) acts in concert with several cochaperones and nucleotide exchange factors and plays an essential role in protein quality control that increases survival by protecting cells against environmental stressors. Specifically, the HSP70-mediated response has been implicated in the pathogenesis of cancer, but the specific mechanisms by which HSP70 may support malignant cell transformation remains to be fully elucidated. Here, we show that genetic ablation of HSP70 markedly impairs HCC initiation and progression by distinct but overlapping pathways. This includes the potentiation of the carcinogen-induced DNA damage response, at the tumor initiation stage, to increase the p53-dependent surveillance response leading to the cell cycle exit or death of genomically damaged differentiated pericentral hepatocytes, and this may also prevent their conversion into more proliferating HCC progenitor cells. Subsequently, activation of a mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) negative feedback pathway diminishes oncogenic signals, thereby attenuating premalignant cell transformation and tumor progression. Modulation of HSP70 function may be a strategy for interfering with oncogenic signals driving liver cell transformation and tumor progression, thus providing an opportunity for human cancer control.

The transcriptional regulator of the chaperone response HSF1 controls hepatic bioenergetics and protein homeostasis.

Metabolic energy reprogramming facilitates adaptations to a variety of stress conditions and cellular dysfunction, but how the energetic demands are monitored and met in response to physiological stimuli remains elusive. Our data support a model demonstrating that heat shock factor 1 (HSF1), a master transcriptional regulator of the chaperone response, has been coopted from its role as a critical protein quality-control regulator to having a central role in systemic energy sensing and for metabolic adaptation to nutrient availability. We found that in the absence of HSF1, levels of NAD+ and ATP are not efficiently sustained in hepatic cells, largely because of transcriptional repression of nicotinamide phosphoribosyltransferase in the NAD+ salvage pathway. Mechanistically, the defect in NAD+ and ATP synthesis linked to a loss of NAD+-dependent deacetylase activity, increased protein acetylation, and impaired mitochondrial integrity. Remarkably, the drop in ATP level caused by HSF1 loss invoked an adaptive response featuring the inhibition of energetically demanding processes, including gluconeogenesis, translation, and lipid synthesis. Our work identifies HSF1 as a central regulator of cellular bioenergetics and protein homeostasis that benefits malignant cell progression and exacerbates development of metabolic diseases.

Point-of-care measurements of platelet inhibition after clopidogrel loading in patients with acute coronary syndrome: comparison of generic and branded clopidogrel bisulfate.

PURPOSE: Platelet-function suppression with antiplatelet therapy is effective in preventing and treating cardiovascular disease. Clopidogrel is a thienopyridine derivative that blocks platelet activation by adenosine diphosphate receptor binding. This study demonstrates the effects of generic clopidogrel bisulfate in comparison to branded clopidogrel bisulfate in patients with acute coronary syndromes. METHODS: This prospective, 2-arm, single-center, open-label trial used 1:1 randomization to assign patients to receive generic or branded clopidogrel bisulfate. Patients with unstable angina or non-ST-segment elevation myocardial infarction and scheduled to undergo coronary angiography were enrolled. Platelet function was measured with a P2Y12 assay and reported in P2Y12 reaction units (PRU) and aspirin reaction units (ARU) after randomization. Platelet function was measured at 2, 4, 8, and 24 hours after 600-mg clopidogrel loading. The clinical outcome was checked at 1 month after coronary angiography. FINDINGS: Ninety-five patients were enrolled and randomized to the generic or branded group. Ninety patients (62 men [69%], 28 women [31%]; mean age, 58 years) completed the study protocol. The clinical characteristics were similar between the 2 groups. The difference in the baseline PRU measurements between the generic and branded groups was not significant (274.8 [59.7] vs 285.4 [62.4], respectively; P = 0.414). There were significant differences in 2-hour PRU (231.1 [71.3] vs 266.9 [67.4]; P = 0.017) and 4-hour PRU (227.3 [80.4] vs 265.7 [71.0]; P = 0.020); however, 24-hour PRU (200.5 [82.1] vs 220.6 [75.8]; P = 0.253) was similar. No death, myocardial infarction, target lesion revascularization, stent thrombosis, or Thrombolysis in Myocardial Infarction-defined major bleeding complications were reported during in-hospital stay or 1-month follow-up. IMPLICATION: In patients with ACS, loading of generic clopidogrel bisulfate was associated with an antiplatelet effect comparable to that of branded clopidogrel bisulfate. ClinicalTrials.gov identifier: NCT02060786.

Inactivation of heat shock factor Hsf4 induces cellular senescence and suppresses tumorigenesis in vivo.

Studies suggest that Hsf4 expression correlates with its role in cell growth and differentiation. However, the role of Hsf4 in tumorigenesis in vivo remains unexplored. In this article, we provide evidence that absence of the Hsf4 gene suppresses evolution of spontaneous tumors arising in p53- or Arf-deficient mice. Furthermore, deletion of hsf4 alters the tumor spectrum by significantly inhibiting development of lymphomas that are normally observed in the majority of mice lacking p53 or Arf tumor suppressor genes. Using mouse embryo fibroblasts deficient in the hsf4 gene, we have found that these cells exhibit reduced proliferation that is associated with induction of senescence and senescence-associated ß-galactosidase (SA-ß-gal). Cellular senescence in hsf4-deficient cells is associated with the increased expression of the cyclin-dependent kinase inhibitors, p21 and p27 proteins. Consistent with the cellular senescence observed in vitro, specific normal tissues of hsf4(-/-) mice and tumors that arose in mice deficient in both hsf4 and p53 genes exhibit increased SA-ß-gal activity and elevated levels of p27 compared with wild-type mice. These results suggest that hsf4 deletion-induced senescence is also present in vivo. Our results therefore indicate that Hsf4 is involved in modulation of cellular senescence, which can be exploited during cancer therapy.

Heat shock transcription factor 1 is a key determinant of HCC development by regulating hepatic steatosis and metabolic syndrome.

Hepatocellular carcinoma (HCC) occurrence and progression are linked tightly to progressive hepatic metabolic syndrome associated with insulin resistance, hepatic steatosis, and chronic inflammation. Heat shock transcription factor 1 (HSF1), a major transactivator of stress proteins, increases survival by protecting cells against environmental stressors. It has been implicated in the pathogenesis of cancer, but specific mechanisms by which HSF1 supports cancer development remain elusive. We propose a pathogenic mechanism whereby HSF1 activation promotes growth of premalignant cells and HCC development by stimulating lipid biosynthesis and perpetuating chronic hepatic metabolic disease induced by carcinogens. Our work shows that inactivation of HSF1 impairs cancer progression, mitigating adverse effects of carcinogens on hepatic metabolism by enhancing insulin sensitivity and sensitizing activation of AMP-activated protein kinase (AMPK), an important regulator of energy homeostasis and inhibitor of lipid synthesis. HSF1 is a potential target for the control of hepatic steatosis, hepatic insulin resistance, and HCC development.

Heat shock factor 1 deficiency via its downstream target gene alphaB-crystallin (Hspb5) impairs p53 degradation.

Heat shock factor Hsf1 regulates the stress-inducibility of heat shock proteins (Hsps) or molecular chaperones. One of the functions attributed to Hsps is their participation in folding and degradation of proteins. We recently showed that hsf1(-/-) cells accumulate ubiquitinated proteins. However, a direct role for Hsf1 in stability of specific proteins such as p53 has not been elucidated. We present evidence that cells deficient in hsf1 accumulate wild-type p53 protein. We further show that hsf1(-/-) cells express lower levels of alphaB-crystallin and cells deficient in alphaB-crystallin also accumulate p53 protein. Reports indicate that alphaB-crystallin binds to Fbx4 ubiquitin ligase, and they target cyclin D1 for degradation through a pathway involving the SCF (Skp1-Cul1-F-box) complex. Towards determining a mechanism for p53 degradation involving alphaB-crystallin and Hsf1, we have found that ectopic expression of Fbx4 in wild-type mouse embryo fibroblasts (MEFs) expressing mutant p53 (p53R175H) leads to increase in its degradation, while MEFs deficient in hsf1 or alphaBcry are defective in degradation of this p53 protein. In addition, immunoprecipitated p53R175H from wild-type MEFs is able to pull-down both alphaB-crystallin and Fbx4. Finally, immunoprecipitated wild-type p53 from doxorubicin treated U2OS cells can pull-down endogenous alphaB-crystallin and Fbx4. These results indicate that hsf1- and alphaBcry-deficient cells accumulate p53 due to reduced levels of alphaB-crystallin in these cells. Elevated levels of p53 in hsf1- and alphaBcry-deficient cells lead to their increased sensitivity to DNA damaging agents. These data reveal a novel mechanism for protein degradation through Hsf1 and alphaB-crystallin.

Relationship between mitochondrial DNA mutations and clinical characteristics in human lung cancer.

Mitochondrial DNA (mtDNA) is known for its high frequencies of polymorphisms and mutations, some of which are related to various diseases, including cancers. However, roles of mutations and polymorphisms in some diseases are among heated debate, especially for cancer. To investigate the possible role of mtDNA mutations in lung cancer, we sequenced complete mtDNA of lung cancer tissues, corresponding normal (i.e., non-cancerous) lung tissues, and peripheral blood samples from 55 lung cancer patients and examined the relationship between mtDNA mutations or polymorphisms and clinical parameters. We identified 56 mutations in 33 (60%) of the 55 patients, including 48 point mutations, four single-nucleotide insertions, and four single-nucleotide deletions. Nineteen of these mutations resulted in amino acid substitution. These missense mtDNA mutations were distributed in 9 of 13 mitochondrial DNA coding genes. Three hundred eighty eight polymorphisms were identified among the 55 patients. Seventy-three polymorphisms resulted in amino acid substitution. There was no association of incidence of specific mtDNA mutation or polymorphism with patients' gender, age at diagnosis, smoking history, tumor type or tumor stage (P>0.05). This study revealed a variety of mtDNA mutations and mtDNA polymorphisms in human lung cancer, some of which might be involved in human lung carcinogenesis.

[Mitochondrial DNA mutations in lung cancer].

BACKGROUND AND OBJECTIVE: Mitochondiral DNA (mtDNA) mutations has been identified in various cancers, but their significance was unknown. This study aimed to detect mtDNA mutations in lung cancer, and to investigate their roles in the carcinogenesis of human lung. METHODS: Total DNA (including nuclear DNA and mtDNA) was extracted from the tumor tissues, corresponding distal non-cancerous lung tissues, and peripheral lymphocytes derived from 58 patients with lung cancer. Fifty-eight overlapping fragments and covering complete sequence of mtDNA were amplified by nested PCR, and the PCR products were sequenced directly with the cycle sequencing methods. The mtDNA mutations in the tumor tissue were determined by comparing with corresponding and peripheral lymphocytes. RESULTS: Sixty-six mutations were identified in 36 cases (62.1%) of lung cancer, including 58 point mutations, 4 insertions, and 4 deletions. These mutations were dispersedly distributed in the full length of mtDNA. The frequency of mutation in D-loop is the highest, in which 18 mutations were detected. No mutation hot spot was found in peptide-coding regions. Among 43 point mutations identified in protein-coding region, 20 were silent mutations. In 8 patients, identical mutations were detected both in the tumor tissues and corresponding distal non-cancerous tissues. CONCLUSION: Most of mtDNA mutations in the lung cancers investigated were occurred randomly and might have no impact on carcinogenesis; whereas the homoplasmic mutations may provide a potential diagnostic marker for lung cancer.