Regulation of YAP and Wnt signaling by the endosomal protein MAMDC4.

Maintenance of the intestinal epithelium requires constant self-renewal and regeneration. Tight regulation of proliferation and differentiation of intestinal stem cells within the crypt region is critical to maintaining homeostasis. The transcriptional co-factors ß-catenin and YAP are required for proliferation during normal homeostasis as well as intestinal regeneration after injury: aberrant signaling activity results in over proliferation and tumorigenesis. Although both YAP and ß-catenin activity are controlled along canonical pathways, it is becoming increasingly clear that non-canonical regulation of these transcriptional regulators plays a role in fine tuning their activity. We have shown previously that MAMDC4 (Endotubin, AEGP), an integral membrane protein present in endosomes, regulates both YAP and ß-catenin activity in kidney epithelial cells and in the developing intestinal epithelium. Here we show that MAMDC4 interacts with members of the signalosome and mediates cross-talk between YAP and ß-catenin. Interestingly, this cross-talk occurs through a non-canonical pathway involving interactions between AMOT:YAP and AMOT:ß-catenin.

Deleting the mitochondrial respiration negative regulator MCJ enhances the efficacy of CD8+ T cell adoptive therapies in pre-clinical studies.

Mitochondrial respiration is essential for the survival and function of T cells used in adoptive cellular therapies. However, strategies that specifically enhance mitochondrial respiration to promote T cell function remain limited. Here, we investigate methylation-controlled J protein (MCJ), an endogenous negative regulator of mitochondrial complex I expressed in CD8 cells, as a target for improving the efficacy of adoptive T cell therapies. We demonstrate that MCJ inhibits mitochondrial respiration in murine CD8+ CAR-T cells and that deletion of MCJ increases their in vitro and in vivo efficacy against murine B cell leukaemia. Similarly, MCJ deletion in ovalbumin (OVA)-specific CD8+ T cells also increases their efficacy against established OVA-expressing melanoma tumors in vivo. Furthermore, we show for the first time that MCJ is expressed in human CD8 cells and that the level of MCJ expression correlates with the functional activity of CD8+ CAR-T cells. Silencing MCJ expression in human CD8 CAR-T cells increases their mitochondrial metabolism and enhances their anti-tumor activity. Thus, targeting MCJ may represent a potential therapeutic strategy to increase mitochondrial metabolism and improve the efficacy of adoptive T cell therapies.

LPAL2 Suppresses Tumor Growth and Metastasis of Hepatocellular Carcinoma by Modulating MMP9 Expression.

Tumor metastasis is a complex process modulated by both intrinsic and extrinsic factors that ultimately result in poorer patient outcomes, including diminished survival. Pseudogene-derived long non-coding RNAs (lncRNA) play important roles in cancer progression. In the current study, we found that the pseudogene-derived lncRNA LPAL2 is downregulated in hepatocellular carcinoma (HCC) tissues, and further showed that elevated LPAL2 expression is positively correlated with survival outcome. The knockdown of LPAL2 in hepatoma cells induced tumor formation, migration, invasion, sphere formation, and drug resistance. Metalloproteinase 9 (MMP9) was identified as an LPAL2-regulated target gene, consistent with clinical findings that LPAL2 expression is significantly associated with MMP9 expression. Furthermore, patients with a higher expression of LPAL2 and lower expression of MMP9 (LPAL2-high/MMP9-low) had a higher survival rate than those with other combinations. Collectively, our findings establish LPAL2 as a novel tumor suppressor in HCC, and suggest targeting LPAL2 and MMP9 as a therapeutic approach for the treatment of HCC.

Proliferation in the developing intestine is regulated by the endosomal protein Endotubin.

During postnatal intestinal development, the intestinal epithelium is highly proliferative, and this proliferation is regulated by signaling in the intervillous and crypt regions. This signaling is primarily mediated by Wnt, and requires membrane trafficking. However, the mechanisms by which membrane trafficking regulates signaling during this developmental phase are largely unknown. Endotubin (EDTB, MAMDC4) is an endosomal protein that is highly expressed in the apical endocytic complex (AEC) of villus enterocytes during fetal and postnatal development, and knockout of EDTB results in defective formation of the AEC and giant lysosome. Further, knockout of EDTB in cell lines results in decreased proliferation. However, the role of EDTB in proliferation during the development of the intestine is unknown. Using Villin-CreERT2 in EDTBfl/fl mice, we deleted EDTB in the intestine in the early postnatal period, or in enteroids in vitro after isolation of intervillous cells. Loss of EDTB results in decreased proliferation in the developing intestinal epithelium and decreased ability to form enteroids. EDTB is present in cells that contain the stem cell markers LGR5 and OLFM4, indicating that it is expressed in the proliferative compartment. Further, using immunoblot analysis and TCF/LEF-GFP mice as a reporter of Wnt activity, we find that knockout of EDTB results in decreased Wnt signaling. Our results show that EDTB is essential for normal proliferation during the early stages of intestinal development and suggest that this effect is through modulation of Wnt signaling.

LINC01348 suppresses hepatocellular carcinoma metastasis through inhibition of SF3B3-mediated EZH2 pre-mRNA splicing.

Long non-coding RNAs (lncRNA) play crucial roles in hepatocellular carcinoma (HCC) progression. However, the specific functions of lncRNAs in alternative splicing (AS) and the metastatic cascade in liver cancer remain largely unclear. In this study, we identified a novel lncRNA, LINC01348, which was significantly downregulated in HCC and correlated with survival functions in HCC patients. Ectopic expression of LINC01348 induced marked inhibition of cell growth, and metastasis in vitro and in vivo. Conversely, these phenotypes were reversed upon knockdown of LINC01348. Mechanistically, LINC01348 complexed with splicing factor 3b subunit 3 (SF3B3) acted as a modulator of EZH2 pre-mRNA AS, and induced alterations in JNK/c-Jun activity and expression of Snail. Notably, C-terminal truncated HBx (Ct-HBx) negatively regulated LINC01348 through c-Jun signaling. Our data collectively highlight those novel regulatory associations involving LINC01348/SF3B3/EZH2/JNK/c-Jun/Snail are an important determinant of metastasis in HCC cells and support the potential utility of targeting LINC01348 as a therapeutic strategy for HCC.

Mitochondrial ATP fuels ABC transporter-mediated drug efflux in cancer chemoresistance.

Chemotherapy remains the standard of care for most cancers worldwide, however development of chemoresistance due to the presence of the drug-effluxing ATP binding cassette (ABC) transporters remains a significant problem. The development of safe and effective means to overcome chemoresistance is critical for achieving durable remissions in many cancer patients. We have investigated the energetic demands of ABC transporters in the context of the metabolic adaptations of chemoresistant cancer cells. Here we show that ABC transporters use mitochondrial-derived ATP as a source of energy to efflux drugs out of cancer cells. We further demonstrate that the loss of methylation-controlled J protein (MCJ) (also named DnaJC15), an endogenous negative regulator of mitochondrial respiration, in chemoresistant cancer cells boosts their ability to produce ATP from mitochondria and fuel ABC transporters. We have developed MCJ mimetics that can attenuate mitochondrial respiration and safely overcome chemoresistance in vitro and in vivo. Administration of MCJ mimetics in combination with standard chemotherapeutic drugs could therefore become an alternative strategy for treatment of multiple cancers.

Cancer cell-intrinsic expression of MHC II in lung cancer cell lines is actively restricted by MEK/ERK signaling and epigenetic mechanisms.

BACKGROUND: Programmed death 1/programmed death ligand 1 (PD-1/PD-L1) targeted immunotherapy affords clinical benefit in ~20% of unselected patients with lung cancer. The factor(s) that determine whether a tumor responds or fails to respond to immunotherapy remains an active area of investigation. We have previously defined divergent responsiveness of two KRAS-mutant cell lines to PD-1/PD-L1 blockade using an orthotopic, immunocompetent mouse model. Responsiveness to PD-1/PD-L1 checkpoint blockade correlates with an interferon gamma (IFNγ)-inducible gene signature and major histocompatibility complex class II (MHC II) expression by cancer cells. In the current study, we aim to identify therapeutic targets that can be manipulated in order to enhance cancer-cell-specific MHC II expression. METHODS: Responsiveness to IFNγ and induction of MHC II expression was assessed after various treatment conditions in mouse and human non-small cell lung cancer (NSCLC) cell lines using mass cytometric and flow cytometric analysis. RESULTS: Single-cell analysis using mass and flow cytometry demonstrated that IFNγ consistently induced PD-L1 and MHC class I (MHC I) across multiple murine and human NSCLC cell lines. In contrast, MHC II showed highly variable induction following IFNγ treatment both between lines and within lines. In mouse models of NSCLC, MHC II induction was inversely correlated with basal levels of phosphorylated extracellular signal-regulated kinase (ERK) 1/2, suggesting potential mitogen-activated protein (MAP) kinase-dependent antagonism of MHC II expression. To test this, cell lines were subjected to varying levels of stimulation with IFNγ, and assessed for MHC II expression in the presence or absence of mitogen-activated protein kinase kinase (MEK) inhibitors. IFNγ treatment in the presence of MEK inhibitors significantly enhanced MHC II induction across multiple lung cancer lines, with minimal impact on expression of either PD-L1 or MHC I. Inhibition of histone deacetylases (HDACs) also enhanced MHC II expression to a more modest extent. Combined MEK and HDAC inhibition led to greater MHC II expression than either treatment alone. CONCLUSIONS: These studies emphasize the active inhibitory role that epigenetic and ERK signaling cascades have in restricting cancer cell-intrinsic MHC II expression in NSCLC, and suggest that combinatorial blockade of these pathways may engender new responsiveness to checkpoint therapies.

TUG1 Is a Regulator of AFP and Serves as Prognostic Marker in Non-Hepatitis B Non-Hepatitis C Hepatocellular Carcinoma.

Thyroid hormone (T3) and its receptor (TR) are involved in cell metabolism and cancer progression. Hypothyroidism is associated with significantly elevated risk of hepatocellular carcinoma (HCC). Levels of the glycoprotein alpha-fetoprotein (AFP) are increased in the majority of patients with HCC and may be useful in diagnosis and follow-up. However, the relationship between T3/TR and AFP levels in HCC is currently unclear. The expression profiles of long non-coding RNAs (lncRNAs) were compared in microarrays of HepG2-TRα1 cells treated with/without T3 and HCC specimens. The effects of T3 on taurine upregulated gene 1 (TUG1) and AFP expression were validated using qRT-PCR. A correlation between TUG1 and AFP was confirmed via RNAi and clustered regularly interspaced short palindromic repeats (CRISPR) strategies. Finally, overall and recurrence-free survival rates were analyzed using the Kaplan-Meier method and confirmed in online datasets. T3/TR treatment reduced TUG1 expression in vitro, resulting in the downregulation of AFP mRNA. Knockdown of TUG1 suppressed cell cycle progression and soft agar colony formation and induced cellular senescence. Our data support the involvement of TUG1 in the T3/TR-mediated suppression of cell growth. AFP mRNA levels showed strong positive correlations with TUG1 and unfavorable prognosis in patients with non-hepatitis B/non-hepatitis C HCC (NBNC-HCC). T3/TR, TUG1, and AFP may potentially serve as effective prognostic markers for NBNC-HCC.

Uncovering the Role of RNA-Binding Protein hnRNP K in B-Cell Lymphomas.

BACKGROUND: Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an RNA-binding protein that is aberrantly expressed in cancers. We and others have previously shown that reduced hnRNP K expression downmodulates tumor-suppressive programs. However, overexpression of hnRNP K is the more commonly observed clinical phenomenon, yet its functional consequences and clinical significance remain unknown. METHODS: Clinical implications of hnRNP K overexpression were examined through immunohistochemistry on samples from patients with diffuse large B-cell lymphoma who did not harbor MYC alterations (n = 75). A novel transgenic mouse model that overexpresses hnRNP K specifically in B cells was generated to directly examine the role of hnRNP K overexpression in mice (three transgenic lines). Molecular consequences of hnRNP K overexpression were determined through proteomics, formaldehyde-RNA-immunoprecipitation sequencing, and biochemical assays. Therapeutic response to BET-bromodomain inhibition in the context of hnRNP K overexpression was evaluated in vitro and in vivo (n = 3 per group). All statistical tests were two-sided. RESULTS: hnRNP K is overexpressed in diffuse large B-cell lymphoma patients without MYC genomic alterations. This overexpression is associated with dismal overall survival and progression-free survival (P < .001). Overexpression of hnRNP K in transgenic mice resulted in the development of lymphomas and reduced survival (P < .001 for all transgenic lines; Line 171[n = 30]: hazard ratio [HR] = 64.23, 95% confidence interval [CI] = 26.1 to 158.0; Line 173 [n = 31]: HR = 25.27, 95% CI = 10.3 to 62.1; Line 177 [n = 25]: HR = 119.5, 95% CI = 42.7 to 334.2, compared with wild-type mice). Clinical samples, mouse models, global screening assays, and biochemical studies revealed that hnRNP K's oncogenic potential stems from its ability to posttranscriptionally and translationally regulate MYC. Consequently, Hnrnpk overexpression renders cells sensitive to BET-bromodomain-inhibition in both in vitro and transplantation models, which represents a strategy for mitigating hnRNP K-mediated c-Myc activation in patients. CONCLUSION: Our findings indicate that hnRNP K is a bona fide oncogene when overexpressed and represents a novel mechanism for c-Myc activation in the absence of MYC lesions.

Thyroid hormone negatively regulates tumorigenesis through suppression of BC200.

Thyroid hormone (T3) and its receptor (TR) are involved in cancer progression. While deregulation of long non-coding RNA (lncRNA) expression has been detected in many tumor types, the mechanisms underlying specific involvement of lncRNAs in tumorigenicity remain unclear. Experiments from the current study revealed negative regulation of BC200 expression by T3/TR. BC200 was highly expressed in hepatocellular carcinoma (HCC) and effective as an independent prognostic marker. BC200 promoted cell growth and tumor sphere formation, which was mediated via regulation of cell cycle-related genes and stemness markers. Moreover, BC200 protected cyclin E2 mRNA from degradation. Cell growth ability was repressed by T3, but partially enhanced upon BC200 overexpression. Mechanistically, BC200 directly interacted with cyclin E2 and promoted CDK2-cyclin E2 complex formation. Upregulation of cell cycle-related genes in hepatoma samples was positively correlated with BC200 expression. Our collective findings support the utility of a potential therapeutic strategy involving targeting of BC200 for the treatment of HCC.

Long Non-Coding RNAs as Mediators of Tumor Microenvironment and Liver Cancer Cell Communication.

The tumor microenvironment is an important concept that defines cancer development not only through tumor cells themselves but also the surrounding cellular and non-cellular components, including stromal cells, blood vessels, infiltrating inflammatory cells, cancer stem cells (CSC), cytokines, and growth factors, which act in concert to promote tumor cell survival and metastasis. Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies worldwide. Poor prognosis is largely attributable to the high rate of tumor metastasis, highlighting the importance of identifying patients at risk in advance and developing novel therapeutic targets to facilitate effective intervention. Long non-coding RNAs (lncRNA) are a class of non-protein coding transcripts longer than 200 nucleotides frequently dysregulated in various cancer types, which have multiple functions in widespread biological processes, including proliferation, apoptosis, metastasis, and metabolism. lncRNAs are involved in regulation of the tumor microenvironment and reciprocal signaling between cancer cells. Targeting of components of the tumor microenvironment or cancer cells has become a considerable focus of therapeutic research and establishing the effects of different lncRNAs on this network should aid in the development of effective treatment strategies. The current review provides a summary of the essential properties and functional roles of known lncRNAs associated with the tumor microenvironment in HCC.

Taurine up-regulated gene 1 functions as a master regulator to coordinate glycolysis and metastasis in hepatocellular carcinoma.

Cancer cells display altered glucose metabolism characterized by a preference for aerobic glycolysis. The aerobic glycolytic phenotype of hepatocellular carcinoma (HCC) is often correlated with tumor progression and poorer clinical outcomes. However, the issue of whether glycolytic metabolism influences metastasis in HCC remains unclear. In the current study, we showed that knockdown of taurine up-regulated gene 1 (TUG1) induces marked inhibition of cell migration, invasion, and glycolysis through suppression of microRNA (miR)-455-3p. MiR-455-3p, which is transcriptionally repressed by p21, directly targets the 3' untranslated region of adenosine monophosphate-activated protein kinase subunit beta 2 (AMPKß2). The TUG1/miR-455-3p/AMPKß2 axis regulates cell growth, metastasis, and glycolysis through regulation of hexokinase 2 (HK2). TUG1 is clearly associated with HK2 overexpression and unfavorable prognosis in HCC patients. CONCLUSION: Our data collectively highlight that novel regulatory associations among TUG1, miR-455-3p, AMPKß2, and HK2 are an important determinant of glycolytic metabolism and metastasis in HCC cells and support the potential utility of targeting TUG1/HK2 as a therapeutic strategy for HCC. (Hepatology 2018;67:188-203).

Acupuncture lowering blood pressure for secondary prevention of stroke: a study protocol for a multicenter randomized controlled trial.

BACKGROUND: Stroke is the prime cause of morbidity and mortality in the general population, and hypertension will increase the recurrence and mortality of stroke. We report a protocol of a pragmatic randomized controlled trial (RCT) using blood pressure (BP)-lowering acupuncture add-on treatment to treat patients with hypertension and stroke. METHODS: This is a large-scale, multicenter, subject-, assessor- and analyst-blinded, pragmatic RCT. A total of 480 patients with hypertension and ischemic stroke will be randomly assigned to two groups: an experimental group and a control group. The experimental group will receive "HuoXueSanFeng" acupuncture combined with one antihypertensive medication in addition to routine ischemic stroke treatment. The control group will only receive one antihypertensive medication and basic treatments for ischemic stroke. HuoXueSanFeng acupuncture will be given for six sessions weekly for the first 6 weeks and three times weekly for the next 6 weeks. A 9-month follow-up will, thereafter, be conducted. Antihypertensive medication will be adjusted based on BP levels. The primary outcome will be the recurrence of stroke. The secondary outcomes including 24-h ambulatory BP, the TCM syndrome score, the Short Form 36-item Health Survey (SF-36), the National Institute of Health Stroke Scale (NIHSS), as well as the Barthel Index (BI) scale will be assessed at baseline, 6 weeks and 12 weeks post initiating treatments; cardiac ultrasound, carotid artery ultrasound, transcranial Doppler, and lower extremity ultrasound will be evaluated at baseline and 12 weeks after treatment. The safety of acupuncture will also be assessed. DISCUSSION: We aim to determine the clinical effects of controlling BP for secondary prevention of stroke with acupuncture add-on treatment. TRIAL REGISTRATION: ClinicalTrials.gov, ID: NCT02967484 . Registered on 13 February 2017; last updated on 27 June 2017.

A novel splice site mutation of CRYBA3/A1 gene associated with congenital cataract in a Chinese family.

AIM: To identify the disease-causing mutation responsible for the presence of congenital cataract in a Chinese family. METHODS: The study recruited a four-generation Chinese pedigree affected by autosomal dominant congenital cataract (ADCC). Family history and the history of cataract extraction were recorded. Blood samples were collected from individuals for DNA extraction. Direct sequencing of congenital cataract-associated genes was performed. Single-strand conformational polymorphism and bioinformatic analysis were conducted to further study the mutation. RESULTS: Direct sequencing revealed a novel splice site mutation of c.30-2 A>G in the CRYBA3/A1 gene. The mutation co-segregated within all affected individuals in the family and was not found in unaffected members or 100 unrelated normal controls. These results were further confirmed by single-strand conformational polymorphism and bioinformatic analysis using the Human Splicing Finder and MaxEnt online software and Annovar computer software. CONCLUSION: c.30-2 A>G mutation of CRYBA3/A1 gene is a novel mutation and broadens the genetic spectrum of ADCC.

Thyroid hormone-mediated regulation of lipocalin 2 through the Met/FAK pathway in liver cancer.

The thyroid hormone, 3,3',5-triiodo-L-thyronine (T3), regulates cell growth, development and differentiation via interactions with thyroid hormone receptors (TR), but the mechanisms underlying T3-mediated modulation of cancer progression are currently unclear. Lipocalin 2 (LCN2), a tumor-associated protein, is overexpressed in a variety of cancer types. Oligonucleotide microarray, coupled with proteomic analysis, has revealed that LCN2 is positively regulated by T3/TR. However, the physiological role and pathway of T3-mediated regulation of LCN2 in hepatocellular carcinogenesis remain to be characterized. Upregulation of LCN2 after T3 stimulation was observed in a time- and dose-dependent manner. Additionally, TRE on the LCN2 promoter was identified at positions -1444/-1427. Overexpression of LCN2 enhanced tumor cell migration and invasion, and conversely, its knockdown suppressed migration and invasion, both in vitro and in vivo. LCN2-induced migration occurred through activation of the Met/FAK cascade. LCN2 was overexpressed in clinical hepatocellular carcinoma (HCC) patients, compared with normal subjects, and positively correlated with TRα levels. Both TRα and LCN2 showed similar expression patterns in relation to survival rate, tumor grade, tumor stage and vascular invasion. Our findings collectively support a potential role of T3/TR in cancer progression through regulation of LCN2 via the Met/FAK cascade. LCN2 may thus be effectively utilized as a novel marker and therapeutic target in HCC.

Repression of microRNA-130b by thyroid hormone enhances cell motility.

BACKGROUND & AIMS: Thyroid hormone (T3) and its receptor (TR) are involved in cell growth and cancer progression. Although deregulation of microRNA (miRNA) expression has been detected in many tumor types, the mechanisms underlying functional impairment and specific involvement of miRNAs in tumor metastasis remain unclear. In the current study, we aimed to elucidate the involvement of deregulated miRNA-130b (miR-130b) and its target genes mediated by T3/TR in cancer progression. METHODS: Quantitative reverse transcription-PCR, luciferase and chromatin immunoprecipitation assays were performed to identify the miR-130b transcript and the mechanisms implicated in its regulation. The effects of miR-130b on hepatocellular carcinoma (HCC) invasion were further examined in vitro and in vivo. Clinical correlations among miR-130b, TRs and interferon regulatory factor 1 (IRF1) were examined in HCC samples using Spearman correlation analysis. RESULTS: Our experiments disclosed negative regulation of miR-130b expression by T3/TR. Overexpression of miR-130b led to marked inhibition of cell migration and invasion, which was mediated via suppression of IRF1. Cell migration ability was promoted by T3, but partially suppressed upon miR-130b overexpression. Furthermore, miR-130b suppressed expression of epithelial-mesenchymal transition (EMT)-related genes, matrix metalloproteinase-9, phosphorylated mammalian target of rapamycin (mTOR), p-ERK1/2, p-AKT and p-signal transducer and activator of transcription (STAT)-3. Notably, miR-130b was downregulated in hepatoma samples and its expression patterns were inversely correlated with those of TRα1 and IRF1. CONCLUSIONS: Our data collectively highlight a novel pathway interlinking T3/TR, miR-130b, IRF1, the EMT-related genes, p-mTOR, p-STAT3 and the p-AKT cascade, which regulates the motility and invasion of hepatoma cells.