Impaired reciprocal regulation between SIRT6 and TGF-ß signaling in fatty liver.

Dysregulated transforming growth factor-beta (TGF-ß) signaling contributes to fibrotic liver disease and hepatocellular cancer (HCC), both of which are associated with fatty liver disease. SIRT6 limits fibrosis by inhibiting TGF-ß signaling through deacetylating SMAD2 and SMAD3 and limits lipogenesis by inhibiting SREBP1 and SREBP2 activity. Here, we showed that, compared to wild-type mice, high-fat diet-induced fatty liver is worse in TGF-ß signaling-deficient mice (SPTBN1+/- ) and the mutant mice had reduced SIRT6 abundance in the liver. Therefore, we hypothesized that altered reciprocal regulation between TGF-ß signaling and SIRT6 contributes to these liver pathologies. We found that deficiency in SMAD3 or SPTBN1 reduced SIRT6 mRNA and protein abundance and impaired TGF-ß induction of SIRT6 transcripts, and that SMAD3 bound to the SIRT6 promoter, suggesting that an SMAD3-SPTBN1 pathway mediated the induction of SIRT6 in response to TGF-ß. Overexpression of SIRT6 in HCC cells reduced the expression of TGF-ß-induced genes, consistent with the suppressive role of SIRT6 on TGF-ß signaling. Manipulation of SIRT6 abundance in HCC cells altered sterol regulatory element-binding protein (SREBP) activity and overexpression of SIRT6 reduced the amount of acetylated SPTBN1 and the abundance of both SMAD3 and SPTBN1. Furthermore, induction of SREBP target genes in response to SIRT6 overexpression was impaired in SPTBN1 heterozygous cells. Thus, we identified a regulatory loop between SIRT6 and SPTBN1 that represents a potential mechanism for susceptibility to fatty liver in the presence of dysfunctional TGF-ß signaling.

TGF-ß Signaling in Liver, Pancreas, and Gastrointestinal Diseases and Cancer.

Genetic alterations affecting transforming growth factor-ß (TGF-ß) signaling are exceptionally common in diseases and cancers of the gastrointestinal system. As a regulator of tissue renewal, TGF-ß signaling and the downstream SMAD-dependent transcriptional events play complex roles in the transition from a noncancerous disease state to cancer in the gastrointestinal tract, liver, and pancreas. Furthermore, this pathway also regulates the stromal cells and the immune system, which may contribute to evasion of the tumors from immune-mediated elimination. Here, we review the involvement of the TGF-ß pathway mediated by the transcriptional regulators SMADs in disease progression to cancer in the digestive system. The review integrates human genomic studies with animal models that provide clues toward understanding and managing the complexity of the pathway in disease and cancer.

Aldehydes alter TGF-ß signaling and induce obesity and cancer.

Obesity and fatty liver diseases-metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH)-affect over one-third of the global population and are exacerbated in individuals with reduced functional aldehyde dehydrogenase 2 (ALDH2), observed in approximately 560 million people. Current treatment to prevent disease progression to cancer remains inadequate, requiring innovative approaches. We observe that Aldh2-/- and Aldh2-/-Sptbn1+/- mice develop phenotypes of human metabolic syndrome (MetS) and MASH with accumulation of endogenous aldehydes such as 4-hydroxynonenal (4-HNE). Mechanistic studies demonstrate aberrant transforming growth factor ß (TGF-ß) signaling through 4-HNE modification of the SMAD3 adaptor SPTBN1 (ß2-spectrin) to pro-fibrotic and pro-oncogenic phenotypes, which is restored to normal SMAD3 signaling by targeting SPTBN1 with small interfering RNA (siRNA). Significantly, therapeutic inhibition of SPTBN1 blocks MASH and fibrosis in a human model and, additionally, improves glucose handling in Aldh2-/- and Aldh2-/-Sptbn1+/- mice. This study identifies SPTBN1 as a critical regulator of the functional phenotype of toxic aldehyde-induced MASH and a potential therapeutic target.

Mechanistically based blood proteomic markers in the TGF-ß pathway stratify risk of hepatocellular cancer in patients with cirrhosis.

Hepatocellular carcinoma (HCC) is the third leading cause of death from cancer worldwide but is often diagnosed at an advanced incurable stage. Yet, despite the urgent need for blood-based biomarkers for early detection, few studies capture ongoing biology to identify risk-stratifying biomarkers. We address this gap using the TGF-ß pathway because of its biological role in liver disease and cancer, established through rigorous animal models and human studies. Using machine learning methods with blood levels of 108 proteomic markers in the TGF-ß family, we found a pattern that differentiates HCC from non-HCC in a cohort of 216 patients with cirrhosis, which we refer to as TGF-ß based Protein Markers for Early Detection of HCC (TPEARLE) comprising 31 markers. Notably, 20 of the patients with cirrhosis alone presented an HCC-like pattern, suggesting that they may be a group with as yet undetected HCC or at high risk for developing HCC. In addition, we found two other biologically relevant markers, Myostatin and Pyruvate Kinase M2 (PKM2), which were significantly associated with HCC. We tested these for risk stratification of HCC in multivariable models adjusted for demographic and clinical variables, as well as batch and site. These markers reflect ongoing biology in the liver. They potentially indicate the presence of HCC early in its evolution and before it is manifest as a detectable lesion, thereby providing a set of markers that may be able to stratify risk for HCC.

Regulation of endoplasmic reticulum stress in rat cortex by p62/ZIP through the Keap1-Nrf2-ARE signalling pathway after transient focal cerebral ischaemia.

PRIMARY OBJECTIVE: p62/ZIP as the autophagy receptor can transport the misfolded proteins to a macroautophagy-lysosome system for degradation and also create a positive feedback loop between p62/ZIP and Nrf2. However, the role of p62/ZIP on cerebral ischaemia is unclear. The aim of this study was to evaluate the role of p62/ZIP in the regulation of endoplasmic reticulum(ER) stress induced by cerebral ischaemia/reperfusion. RESEARCH DESIGN: Different ischemic periods were designed by transient middle cerebral artery occlusion (tMCAO) using the suture method. METHODS AND PROCEDURES: At 24 hours after reperfusion, the ischaemic brain tissue was studied histologically and biochemically for autophagic, ER stress and Keap1-Nrf2-ARE signalling pathway markers. MAIN OUTCOMES AND RESULTS: Prolongation of ischaemia significantly increased the cortical injury observed in rats and was associated with a gradual increase in the protein expression of ubiquitin-aggregates, Grp78, GADD153/CHOP and p62/ZIP. Autophagy marker Atg12-Atg5 and LC3-PE increased and then decreased. Moreover, p62/ZIP mRNA expression increased and then decreased and was consistent with Nrf2 activation. CONCLUSIONS: p62/ZIP not only plays a key role in scavenging protein aggregates during autophagy, but it may also be involved in preventing oxidative injury and alleviating ER stress through the Keap1-Nrf2-ARE signalling pathway during cerebral ischaemia/reperfusion injury.

Using quantitative immunohistochemistry in patients at high risk for hepatocellular cancer.

Hepatocellular carcinoma (HCC) is the primary form of liver cancer and a major cause of cancer death worldwide. Early detection is key to effective treatment. Yet, early diagnosis is challenging, especially in patients with cirrhosis, who are at high risk of developing HCC. Dysfunction or loss of function of the transforming growth factor ß (TGF-ß) pathway is associated with HCC. Here, using quantitative immunohistochemistry analysis of samples from a multi-institutional repository, we evaluated if differences in TGF-ß receptor abundance were present in tissue from patients with only cirrhosis compared with those with HCC in the context of cirrhosis. We determined that TGFBR2, not TGFBR1, was significantly reduced in HCC tissue compared with cirrhotic tissue. We developed an artificial intelligence (AI)-based process that correctly identified cirrhotic and HCC tissue and confirmed the significant reduction in TGFBR2 in HCC tissue compared with cirrhotic tissue. Thus, we propose that a reduction in TGFBR2 abundance represents a useful biomarker for detecting HCC in the context of cirrhosis and that incorporating this biomarker into an AI-based automated imaging pipeline could reduce variability in diagnosing HCC from biopsy tissue.

Pathogenesis to management of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer whose incidence continues to rise in many parts of the world due to a concomitant rise in many associated risk factors, such as alcohol use and obesity. Although early-stage HCC can be potentially curable through liver resection, liver-directed therapies, or transplantation, patients usually present with intermediate to advanced disease, which continues to be associated with a poor prognosis. This is because HCC is a cancer with significant complexities, including substantial clinical, histopathologic, and genomic heterogeneity. However, the scientific community has made a major effort to better characterize HCC in those aspects via utilizing tissue sampling and histological classification, whole genome sequencing, and developing viable animal models. These efforts ultimately aim to develop clinically relevant biomarkers and discover molecular targets for new therapies. For example, until recently, there was only one approved systemic therapy for advanced or metastatic HCC in the form of sorafenib. Through these efforts, several additional targeted therapies have gained approval in the United States, although much progress remains to be desired. This review will focus on the link between characterizing the pathogenesis of HCC with current and future HCC management.

ß2-spectrin (SPTBN1) as a therapeutic target for diet-induced liver disease and preventing cancer development.

The prevalence of nonalcoholic steatohepatitis (NASH) and liver cancer is increasing. De novo lipogenesis and fibrosis contribute to disease progression and cancerous transformation. Here, we found that ß2-spectrin (SPTBN1) promotes sterol regulatory element (SRE)­binding protein (SREBP)­stimulated lipogenesis and development of liver cancer in mice fed a high-fat diet (HFD) or a western diet (WD). Either hepatocyte-specific knockout of SPTBN1 or siRNA-mediated therapy protected mice from HFD/WD-induced obesity and fibrosis, lipid accumulation, and tissue damage in the liver. Biochemical analysis suggested that HFD/WD induces SPTBN1 and SREBP1 cleavage by CASPASE-3 and that the cleaved products interact to promote expression of genes with sterol response elements. Analysis of human NASH tissue revealed increased SPTBN1 and CASPASE-3 expression. Thus, our data indicate that SPTBN1 represents a potential target for therapeutic intervention in NASH and liver cancer.

Inhibition of autophagic flux by ROS promotes apoptosis during DTT-induced ER/oxidative stress in HeLa cells.

As targets for cancer therapy, endoplasmic reticulum (ER) stress and autophagy are closely linked. However, the signaling pathways responsible for induction of autophagy in response to ER stress and its cellular consequences appear to vary with cell type and stimulus. In the present study, we showed that dithiothreitol (DTT) induced ER stress in HeLa cells in a time- and dose-dependent fashion. With increased ER stress, reactive oxygen species (ROS) production increased and autophagy flux, assessed by intracellular accumulation of LC3B-II and p62, was inhibited. N-acetyl-L-cysteine (NAC), a classic antioxidant, exacerbated cell death induced by 3.2 mM of DTT, but attenuated that induced by 6.4 mM DTT. Low cytotoxic doses of DTT transiently activated c-JNU N-terminal kinase (JNK) and p38, whereas high dose of DTT persistently activated JNK and p38 and simultaneously reduced extracellular signal-regulated kinase (ERK) activity. Combined treatment with DTT and U0126, an inhibitor of ERK upstream activators mitogen-activated protein kinase (MAPK) kinase 1 and 2 (MEK1/2), blocked autophagy flux in HeLa cells. This effect was similar to that caused by a combination of DTT and chloroquine (CQ). These data suggested that insufficient autophagy was accompanied by increased ROS production during DTT-induced ER stress. ROS appeared to regulate MAPK signaling, switching from a pro-survival to a pro-apoptotic signal as ER stress increased. ERK inhibition by ROS during severe ER stress blocked autophagic flux. Impaired autophagic flux, in turn, aggravated ER stress, ultimately leading to cell death. Taken together, our data provide the first reported evidence that ROS may control cell fate through regulating the MAPK pathways and autophagic flux during DTT-induced ER/oxidative stress.

SIRT3 participates in glucose metabolism interruption and apoptosis induced by BH3 mimetic S1 in ovarian cancer cells.

The Bcl-2 antiapoptotic proteins are important cancer therapy targets; however, their role in cancer cell metabolism remains unclear. We found that the BH3-only protein mimetic S1, a novel pan Bcl-2 inhibitor, simultaneously interrupted glucose metabolism and induced apoptosis in human SKOV3 ovarian cancer cells, which was related to the activation of SIRT3, a stress-responsive deacetylase. S1 interrupted the cellular glucose metabolism mainly through causing damage to mitochondrial respiration and inhibiting glycolysis. Moreover, S1 upregulated the gene and protein expression of SIRT3, and induced the translocation of SIRT3 from the nucleus to mitochondria. SIRT3 silencing reversed the effects of S1 on glucose metabolism and apoptosis through increasing the level of HK-II localized to the mitochondria, while a combination of the glycolysis inhibitor 2-DG and S1 intensified the cytotoxicity through further upregulation of SIRT3 expression. This study underscores an essential role of SIRT3 in the antitumor effect of Bcl-2 inhibitors in human ovarian cancer through regulating both metabolism and apoptosis. The manipulation of Bcl-2 inhibitors combined with the use of classic glycolysis inhibitors may be rational strategies to improve ovarian cancer therapy.

Inhibition of JNK3 promotes apoptosis induced by BH3 mimetic S1 in chemoresistant human ovarian cancer cells.

Previous studies have suggested that the novel BH3 mimetic S1 could induce apoptosis in diverse tumor cell lines through endoplasmic reticulum (ER) stress or mitochondrial cell death pathways. The activation of c-Jun N-terminal kinase (JNK) through inositol requiring enzyme-1 (IRE1) is closely connected to ER stress-induced apoptosis. However, the role of JNK is complex, as there are different JNK subtypes and the function of each subtype is still not entirely clear. Here we found that the mRNA expression of JNK3 was continuously high in S1-treated human ovarian cancer SKOV3/DDP cells using a human unfolded protein response (UPR) pathway PCR array. Pharmacological inhibition of JNK3 increased cell sensitivity to apoptosis induced by S1. Furthermore, inhibition of JNK3 induced accumulation of both acidic compartment and p62, and upregulated ROS production. Our results suggest that JNK3 plays a pro-survival role during ER stress through preventing the block of autophagic flux and reducing oxidative stress in SKOV3/DDP cells. Inhibition of JNK3 may be a potential method to enhance the killing effect of the Bcl-2 inhibitor S1.

Sanguinarine-induced apoptosis in lung adenocarcinoma cells is dependent on reactive oxygen species production and endoplasmic reticulum stress.

Sanguinarine (SAN), an alkaloid isolated from plants of the Papaveraceae family, is a compound with multiple biological activities. In the present study, we explored the anticancer properties of SAN in lung cancer using the human lung adenocarcinoma cell line SPC-A1. Our results revealed that SAN inhibited SPC-A1 cell growth and induced apoptosis in a dose-dependent manner. We found that SAN triggered reactive oxygen species (ROS) production, while elimination of ROS by N-acetylcysteine (NAC) reversed the growth inhibition and apoptosis induced by SAN. SAN-induced endoplasmic reticulum (ER) stress resulted in the upregulation of many genes and proteins involved in the unfolded protein response (UPR) pathway, including glucose-regulated protein 78 (GRP78), p-protein kinase R (PKR)-like ER kinase (PERK), p-eukaryotic translation initiation factor 2α (eIF2α), activating transcription factor 4 (ATF4) and CCAAT/enhancer binding protein homologous protein (CHOP). Blocking ER stress with tauroursodeoxycholic acid (TUDCA) markedly reduced SAN-induced inhibition of growth and apoptosis. Furthermore, TUDCA decreased SAN-induced ROS production, and NAC attenuated SAN-induced GRP78 and CHOP expression. Overall, our data indicate that the anticancer effects of SAN in lung cancer cells depend on ROS production and ER stress and that SAN may be a potential agent against lung cancer.

Bcl-2 family proteins are involved in the signal crosstalk between endoplasmic reticulum stress and mitochondrial dysfunction in tumor chemotherapy resistance.

Tumor cells overexpress antiapoptotic proteins of the Bcl-2 (B-cell leukemia/lymphoma-2) family, which can lead to both escape from cell death and resistance to chemotherapeutic drugs. Recent studies suggest that the endoplasmic reticulum (ER) can produce proapoptotic signals, amplifying the apoptotic signaling cascade. The crosstalk between mitochondria and ER plays a decisive role in many cellular events but especially in cell death. Bcl-2 family proteins located in the ER and mitochondria can influence not only the function of the two organelles but also the interaction between them. Therefore, the Bcl-2 family of proteins may also be involved in the mechanism of tumor chemotherapy resistance by influencing crosstalk between the ER and mitochondria. In this review we will briefly discuss evidence to support this concept.

Hyperbaric oxygen enlarges the area of brain damage in MCAO rats by blocking autophagy via ERK1/2 activation.

Hyperbaric oxygen (HBO) is emerging as a therapy for brain ischemia, although its benefits are still debated. The present study aimed to investigate the effect of HBO on brain damage in a rat model of transient focal cerebral ischemia and its underlying mechanism of action. Male Wistar rats, which had suffered 1.5h of transient middle cerebral artery occlusion (tMCAO) and had a Longa's neuron score of 3, were given pure oxygen at 3.0 atm absolute, for 60 min after the third hour of reperfusion. After 24h of reperfusion, rat brains were removed and studied. 2,3,5-triphenyltetrazolium chloride (TTC) and hematoxylin and eosin staining revealed that the infarct ratio in the HBO group increased remarkably when compared with the MCAO group. Up-regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) activation was detected in the HBO group because of reactive oxygen species (ROS) generation. Autophagy appeared to be obstructed in the HBO group. Administration of the ERK1/2 inhibitor U0126 decreased the infarct ratio and improved protein clearance by autophagy in the HBO group. Collectively, these results suggest that HBO enlarges the area of brain damage via reactive oxygen species-induced activation of ERK1/2, which interrupts autophagy flux.

The BH3 mimetic S1 induces endoplasmic reticulum stress-associated apoptosis in cisplatin-resistant human ovarian cancer cells although it activates autophagy.

SKOV3/DDP human ovarian cancer cells have been shown to be resistant to cisplatin. Although the BH3 mimetic S1 induces cell death in several types of tumor cells, it is unclear whether it induces death in drug-resistant cells. Herein, we found that S1 induced endoplasmic reticulum (ER) stress-associated apoptosis in both SKOV3 and SKOV3/DDP cells. S1 activated autophagy at early time points in SKOV3/DDP cells, and inhibition of autophagy increased ER stress-associated apoptosis. Collectively, our data indicate that autophagy plays a protective role, but it cannot protect against S1-induced cell death in cisplatin-resistant SKOV3/DDP cells.

Endoplasmic reticulum is at the crossroads of autophagy, inflammation, and apoptosis signaling pathways and participates in the pathogenesis of diabetes mellitus.

Diabetes mellitus (DM) is a chronic metabolic disease, and its incidence is growing worldwide. The endoplasmic reticulum (ER) is a central component of cellular functions and is involved in protein folding and trafficking, lipid synthesis, and maintenance of calcium homeostasis. The ER is also a sensor of both intra- and extracellular stress and thus participates in monitoring and maintaining cellular homeostasis. Therefore, the ER is one site of interaction between environmental signals and a cell's biological function. The ER is tightly linked to autophagy, inflammation, and apoptosis, and recent evidence suggests that these processes are related to the pathogenesis of DM and its complications. Thus, the ER has been considered an intersection integrating multiple stress responses and playing an important role in metabolism-related diseases including DM. Here, we review the relationship between the ER and autophagy, inflammation, and apoptosis in DM to better understand the molecular mechanisms of this disease.

The BH3 mimetic S1 induces autophagy through ER stress and disruption of Bcl-2/Beclin 1 interaction in human glioma U251 cells.

Previous results showed that a novel BH3 mimetic S1 could induce cell death in a wide range of cancer types in vitro through Bax/Bak-dependent apoptosis. We demonstrated that in addition to mitochondrial pathway apoptosis, endoplasmic reticulum (ER) stress-associated apoptosis was also induced by S1. Moreover, S1 can induce autophagy in U251 cells, which may occur through ER stress and disruption of the association of Bcl-2 and Beclin 1. Inhibition of autophagy by the autophagic inhibitors 3-methyladenine (3-MA) or chloroquine (CQ) increased S1-induced apoptosis. In conclusion, autophagy plays an important role in S1-induced U251 cell death.

p62/SQSTM1 involved in cisplatin resistance in human ovarian cancer cells by clearing ubiquitinated proteins.

Mechanisms of cisplatin resistance in cancer cells are not fully understood. Here, we show a critical role for the ubiquitin-binding protein p62/SQSTM1 in cisplatin resistance in human ovarian cancer cells (HOCCs). Specifically, we found that cisplatin-resistant SKOV3/DDP cells express much higher levels of p62 than do cisplatin-sensitive SKOV3 cells. The protein p62 binds ubiquitinated proteins for transport to autophagic degradation, reducing apoptosis induced by endoplasmic reticulum (ER) stress in SKOV3/DDP cells. Knockdown of p62 or inhibition of autophagy using 3-methyladenine resensitises SKOV3/DDP cells to cisplatin. Collectively, our data indicate that p62 acts as a receptor or adaptor for autophagic degradation of ubiquitinated proteins, and plays an important role in preventing ER stress-induced apoptosis, leading to cisplatin resistance in HOCCs.