Persistent mTORC1 activation due to loss of liver tuberous sclerosis complex 1 promotes liver injury in alcoholic hepatitis.

BACKGROUND AND AIMS: The aim of the study was to investigate the role and mechanisms of tuberous sclerosis complex 1 (TSC1) and mechanistic target of rapamycin complex 1 (mTORC1) in alcohol-associated liver disease. APPROACH AND RESULTS: Liver-specific Tsc1 knockout (L- Tsc1 KO) mice and their matched wild-type mice were subjected to Gao-binge alcohol. Human alcoholic hepatitis (AH) samples were also used for immunohistochemistry staining, western blot, and quantitative real-time PCR (q-PCR) analysis. Human AH and Gao-binge alcohol-fed mice had decreased hepatic TSC1 and increased mTORC1 activation. Gao-binge alcohol markedly increased liver/body weight ratio and serum alanine aminotransferase levels in L- Tsc1 KO mice compared with Gao-binge alcohol-fed wild-type mice. Results from immunohistochemistry staining, western blot, and q-PCR analysis revealed that human AH and Gao-binge alcohol-fed L- Tsc1 KO mouse livers had significantly increased hepatic progenitor cells, macrophages, and neutrophils but decreased HNF4α-positive cells. Gao-binge alcohol-fed L- Tsc1 KO mice also developed severe inflammation and liver fibrosis. Deleting Tsc1 in cholangiocytes but not in hepatocytes promoted cholangiocyte proliferation and aggravated alcohol-induced ductular reactions, fibrosis, inflammation, and liver injury. Pharmacological inhibition of mTORC1 partially reversed hepatomegaly, ductular reaction, fibrosis, inflammatory cell infiltration, and liver injury in alcohol-fed L- Tsc1 KO mice. CONCLUSIONS: Our findings indicate that persistent activation of mTORC1 due to the loss of cholangiocyte TSC1 promotes liver cell repopulation, ductular reaction, inflammation, fibrosis, and liver injury in Gao-binge alcohol-fed L- Tsc1 KO mice, which phenocopy the pathogenesis of human AH.

PERK signaling through C/EBPδ contributes to ER stress-induced expression of immunomodulatory and tumor promoting chemokines by cancer cells.

Cancer cells experience endoplasmic reticulum (ER) stress due to activated oncogenes and conditions of nutrient deprivation and hypoxia. The ensuing unfolded protein response (UPR) is executed by ATF6, IRE1 and PERK pathways. Adaptation to mild ER stress promotes tumor cell survival and aggressiveness. Unmitigated ER stress, however, will result in cell death and is a potential avenue for cancer therapies. Because of this yin-yang nature of ER stress, it is imperative that we fully understand the mechanisms and dynamics of the UPR and its contribution to the complexity of tumor biology. The PERK pathway inhibits global protein synthesis while allowing translation of specific mRNAs, such as the ATF4 transcription factor. Using thapsigargin and tunicamycin to induce acute ER stress, we identified the transcription factor C/EBPδ (CEBPD) as a mediator of PERK signaling to secretion of tumor promoting chemokines. In melanoma and breast cancer cell lines, PERK mediated early induction of C/EBPδ through ATF4-independent pathways that involved at least in part Janus kinases and the STAT3 transcription factor. Transcriptional profiling revealed that C/EBPδ contributed to 20% of thapsigargin response genes including chaperones, components of ER-associated degradation, and apoptosis inhibitors. In addition, C/EBPδ supported the expression of the chemokines CXCL8 (IL-8) and CCL20, which are known for their tumor promoting and immunosuppressive properties. With a paradigm of short-term exposure to thapsigargin, which was sufficient to trigger prolonged activation of the UPR in cancer cells, we found that conditioned media from such cells induced cytokine expression in myeloid cells. In addition, activation of the CXCL8 receptor CXCR1 during thapsigargin exposure supported subsequent sphere formation by cancer cells. Taken together, these investigations elucidated a novel mechanism of ER stress-induced transmissible signals in tumor cells that may be particularly relevant in the context of pharmacological interventions.

The STAT3 inhibitor Stattic acts independently of STAT3 to decrease histone acetylation and modulate gene expression.

Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor involved in many physiological functions including embryonic development and immune responses and is often activated under pathological conditions such as cancer. Strategies to inactivate STAT3 are being pursued as potential anticancer therapies and have led to the identification of Stattic (6-nitrobenzo[b]thiophene-1,1-dioxide) as a "specific" STAT3 inhibitor that is often used to interrogate STAT3-mediated gene expression in vitro and in vivo. Here, we show that Stattic exerts many STAT3-independent effects on cancer cells, calling for reassessment of results previously ascribed to STAT3 functions. Studies of the STAT3-deficient prostate cancer cell line PC-3 (PC3) along with STAT3-proficient breast cancer cell lines (MDA-MB-231, SUM149) revealed that Stattic attenuated histone acetylation and neutralized effects of the histone deacetylase (HDAC) inhibitor romidepsin. In PC3 cells, Stattic alone inhibited gene expression of CCL20 and CCL2, but activated expression of TNFA, CEBPD, SOX2, and MYC. In addition, we found that Stattic promoted autophagy and caused cell death. These data point to profound epigenetic effects of Stattic that are independent of its function as a STAT3 inhibitor. Our results demonstrate that Stattic directly or indirectly reduces histone acetylation and suggest reevaluation of Stattic and related compounds as polypharmacological agents through multipronged cytotoxic effects on cancer cells.

SERPINB3 and B4: From biochemistry to biology.

Human SERPINB3 and SERPINB4 are evolutionary duplicated serine/cysteine protease inhibitors. Genomic analysis indicates that these paralogous genes were encoded from independent loci arising from tandem gene duplication. Although the two molecules share 92% identity of their amino acid sequences, they are distinct in the Reactive Center Loop (RCL) including a hinge region and catalytic sequences which accounts for altered substrate specificity. Elevated expression of the two molecules has been reported to contribute to numerous pathological conditions such as inflammatory diseases and cancer. In this review, we focus on summarizing the biochemical features of SERPINB3/B4 and discussing the mechanistic basis for their biological functions and implications in human diseases.

Vps34 regulates Rab7 and late endocytic trafficking through recruitment of the GTPase-activating protein Armus.

The class III phosphoinositide 3-kinase (PI3K) Vps34 (also known as PIK3C3 in mammals) produces phosphatidylinositol 3-phosphate [PI(3)P] on both early and late endosome membranes to control membrane dynamics. We used Vps34-deficient cells to delineate whether Vps34 has additional roles in endocytic trafficking. In Vps34-/- mouse embryonic fibroblasts (MEFs), transferrin recycling and EEA1 membrane localization were unaffected despite elevated Rab5-GTP levels. Strikingly, a large increase in Rab7-GTP levels, an accumulation of enlarged late endosomes, and decreased EGFR degradation were observed in Vps34-deficient cells. The hyperactivation of Rab7 in Vps34-deficient cells stemmed from the failure to recruit the Rab7 GTPase-activating protein (GAP) Armus (also known as TBC1D2), which binds to PI(3)P, to late endosomes. Protein-lipid overlay and liposome-binding assays reveal that the putative pleckstrin homology (PH) domain in Armus can directly bind to PI(3)P. Elevated Rab7-GTP led to the failure of intraluminal vesicle (ILV) formation and lysosomal maturation. Rab7 silencing and Armus overexpression alleviated the vacuolization seen in Vps34-deficient cells. Taken together, these results demonstrate that Vps34 has a previously unknown role in regulating Rab7 activity and late endosomal trafficking.

SCCA1/SERPINB3 promotes oncogenesis and epithelial-mesenchymal transition via the unfolded protein response and IL6 signaling.

The serine/cysteine protease inhibitor SCCA1 (SERPINB3) is upregulated in many advanced cancers with poor prognosis, but there is limited information about whether it makes functional contributions to malignancy. Here, we show that SCCA1 expression promoted oncogenic transformation and epithelial-mesenchymal transition (EMT) in mammary epithelial cells, and that SCCA1 silencing in breast cancer cells halted their proliferation. SCCA1 overexpression in neu(+) mammary tumors increased the unfolded protein response (UPR), IL6 expression, and inflammatory phenotypes. Mechanistically, SCCA1 induced a prolonged nonlethal increase in the UPR that was sufficient to activate NF-κB and expression of the protumorigenic cytokine IL6. Overall, our findings established that SCCA1 contributes to tumorigenesis by promoting EMT and a UPR-dependent induction of NF-κB and IL6 autocrine signaling that promotes a protumorigenic inflammation.

Oncogenic Ras induces inflammatory cytokine production by upregulating the squamous cell carcinoma antigens SerpinB3/B4.

Mounting evidence indicates that oncogenic Ras can modulate cell autonomous inflammatory cytokine production, although the underlying mechanism remains unclear. Here we show that squamous cell carcinoma antigens 1 and 2 (SCCA1/2), members of the Serpin family of serine/cysteine protease inhibitors, are transcriptionally upregulated by oncogenic Ras via MAPK and the ETS family transcription factor PEA3. Increased SCCA expression leads to inhibition of protein turnover, unfolded protein response, activation of NF-κB and is essential for Ras-mediated cytokine production and tumour growth. Analysis of human colorectal and pancreatic tumour samples reveals a positive correlation between Ras mutation, enhanced SCCA expression and IL-6 expression. These results indicate that SCCA is a Ras-responsive factor that plays an important role in Ras-associated cytokine production and tumorigenesis.

Elevated expression of squamous cell carcinoma antigen (SCCA) is associated with human breast carcinoma.

Squamous cell carcinoma antigen (SCCA) belongs to the serine protease inhibitor (Serpin) family of proteins. Elevated expression of SCCA has been used as a biomarker for aggressive squamous cell carcinoma (SCC) in cancers of the cervix, lung, head and neck, and liver. However, SCCA expression in breast cancer has not been investigated. Immunohistochemical analysis of SCCA expression was performed on tissue microarrays containing breast tumor tissues (n = 1,360) and normal breast epithelium (n = 124). SCCA expression was scored on a tiered scale (0-3) independently by two evaluators blind to the patient's clinical status. SCCA expression was observed in Grade I (0.3%), Grade II (2.5%), and Grade III (9.4%) breast cancers (p<0.0001). Comparing tissues categorized into the three non-metastatic TNM stages, I-III, SCCA positivity was seen in 2.4% of Stage I cancers, 3.1% of Stage II cancers, and 8.6% of Stage III breast cancers (p = 0.0005). No positive staining was observed in normal/non-neoplastic breast tissue (0 out of 124). SCCA expression also correlated to estrogen receptor/progesterone receptor (ER/PR) double-negative tumors (p = 0.0009). Compared to SCCA-negative patients, SCCA-positive patients had both a worse overall survival and recurrence-free survival (p<0.0001 and p<0.0001, respectively). This study shows that SCCA is associated with both advanced stage and high grade human breast carcinoma, and suggests the necessity to further explore the role of SCCA in breast cancer development and treatment.