A COMPASS histone H3K4 trimethyltransferase pentamer transactivates drought tolerance and growth/biomass production in Populus trichocarpa.

Histone H3 lysine-4 trimethylation (H3K4me3) activating drought-responsive genes in plants for drought adaptation has long been established, but the underlying regulatory mechanisms are unknown. Here, using yeast two-hybrid, bimolecular fluorescence complementation, biochemical analyses, transient and CRISPR-mediated transgenesis in Populus trichocarpa, we unveiled in this adaptation a regulatory interplay between chromatin regulation and gene transactivation mediated by an epigenetic determinant, a PtrSDG2-1-PtrCOMPASS (complex proteins associated with Set1)-like H3K4me3 complex, PtrSDG2-1-PtrWDR5a-1-PtrRbBP5-1-PtrAsh2-2 (PtrSWRA). Under drought conditions, a transcription factor PtrAREB1-2 interacts with PtrSWRA, forming a PtrSWRA-PtrAREB1-2 pentamer, to recruit PtrSWRA to specific promoter elements of drought-tolerant genes, such as PtrHox2, PtrHox46, and PtrHox52, for depositing H3K4me3 to promote and maintain activated state of such genes for tolerance. CRISPR-edited defects in the pentamer impaired drought tolerance and elevated expression of PtrHox2, PtrHox46, or PtrHox52 improved the tolerance as well as growth in P. trichocarpa. Our findings revealed the identity of the underlying H3K4 trimethyltransferase and its interactive arrangement with the COMPASS for catalysis specificity and efficiency. Furthermore, our study uncovered how the H3K4 trimethyltransferase-COMPASS complex is recruited to the effector genes for elevating H3K4me3 marks for improved drought tolerance and growth/biomass production in plants.

Dimerization of PtrMYB074 and PtrWRKY19 mediates transcriptional activation of PtrbHLH186 for secondary xylem development in Populus trichocarpa.

Wood formation is controlled by transcriptional regulatory networks (TRNs) involving regulatory homeostasis determined by combinations of transcription factor (TF)-DNA and TF-TF interactions. Functions of TF-TF interactions in wood formation are still in the early stages of identification. PtrMYB074 is a woody dicot-specific TF in a TRN for wood formation in Populus trichocarpa. Here, using yeast two-hybrid and bimolecular fluorescence complementation, we conducted a genome-wide screening for PtrMYB074 interactors and identified 54 PtrMYB074-TF pairs. Of these pairs, 53 are novel. We focused on the PtrMYB074-PtrWRKY19 pair, the most highly expressed and xylem-specific interactor, and its direct transregulatory target, PtrbHLH186, the xylem-specific one of the pair's only two direct TF target genes. Using transient and CRISPR-mediated transgenesis in P. trichocarpa coupled with chromatin immunoprecipitation and electrophoretic mobility shift assays, we demonstrated that PtrMYB074 is recruited by PtrWRKY19 and that the PtrMYB074-PtrWRKY19 dimers are required to transactive PtrbHLH186. Overexpressing PtrbHLH186 in P. trichocarpa resulted in retarded plant growth, increased guaiacyl lignin, a higher proportion of smaller stem vessels and strong drought-tolerant phenotypes. Knowledge of the PtrMYB074-PtrWRKY19-PtrbHLH186 regulation may help design genetic controls of optimal growth and wood formation to maximize beneficial wood properties while minimizing negative effects on growth.

The cytochrome P450 enzyme CYP24A1 increases proliferation of mutant KRAS-dependent lung adenocarcinoma independent of its catalytic activity.

We previously reported that overexpression of cytochrome P450 family 24 subfamily A member 1 (CYP24A1) increases lung cancer cell proliferation by activating RAS signaling and that CYP24A1 knockdown inhibits tumor growth. However, the mechanism of CYP24A1-mediated cancer cell proliferation remains unclear. Here, we conducted cell synchronization and biochemical experiments in lung adenocarcinoma cells, revealing a link between CYP24A1 and anaphase-promoting complex (APC), a key cell cycle regulator. We demonstrate that CYP24A1 expression is cell cycle-dependent; it was higher in the G2-M phase and diminished upon G1 entry. CYP24A1 has a functional destruction box (D-box) motif that allows binding with two APC adaptors, CDC20-homologue 1 (CDH1) and cell division cycle 20 (CDC20). Unlike other APC substrates, however, CYP24A1 acted as a pseudo-substrate, inhibiting CDH1 activity and promoting mitotic progression. Conversely, overexpression of a CYP24A1 D-box mutant compromised CDH1 binding, allowing CDH1 hyperactivation, thereby hastening degradation of its substrates cyclin B1 and CDC20, and accumulation of the CDC20 substrate p21, prolonging mitotic exit. These activities also occurred with a CYP24A1 isoform 2 lacking the catalytic cysteine (Cys-462), suggesting that CYP24A1's oncogenic potential is independent of its catalytic activity. CYP24A1 degradation reduced clonogenic survival of mutant KRAS-driven lung cancer cells, and calcitriol treatment increased CYP24A1 levels and tumor burden in Lsl-KRASG12D mice. These results disclose a catalytic activity-independent growth-promoting role of CYP24A1 in mutant KRAS-driven lung cancer. This suggests that CYP24A1 could be therapeutically targeted in lung cancers in which its expression is high.

Isoforms of RNF128 Regulate the Stability of Mutant P53 in Barrett's Esophageal Cells.

BACKGROUND & AIMS: Barrett's esophagus (BE) can progress to dysplasia and esophageal adenocarcinoma (EAC), accompanied by mutations in TP53 that increase the stability of its product, p53. We analyzed BE tissues for messenger RNAs (mRNAs) that associate with BE progression and identified one that affects the stabilization of p53. METHODS: We obtained 54 BE samples collected from patients with high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC), from 1992 through 2015, and performed RNA sequence analyses, including isoform-specific analyses. We performed reverse-transcription polymerase chain reaction analyses of 166 samples and immunohistochemical analyses of tissue microarrays that contained BE tissues from 100 patients with HGD or EAC and normal esophageal squamous mucosa (controls). Proteins were expressed from transfected plasmids or knocked down with small interfering RNAs in BE cells and analyzed by immunoblots and in immunoprecipitation and ubiquitin ligase assays. Athymic nude mice bearing EAC xenograft tumors (grown from OE-33 cells) were given intraperitoneal injections of simvastatin; tumor growth was monitored and tumors were collected and analyzed by immunoblotting for levels of RNF128, p53, and acetylated p53. RESULTS: Progression of BE to HGD or EAC associated with changes in expression of mRNAs that encoded mucins and promoted inflammation and activation of ATM and the DNA damage response. As tissues progressed from BE to HGD to EAC, they increased expression of mRNAs encoding isoform 1 of RNF128 (Iso1) and decreased expression of Iso2 of RNF128. RNF128 is an E3 ubiquitin ligase that targets p53 for degradation. Incubation of BE cells with interferon gamma caused them to increase expression of Iso1 and reduce expression of Iso2. Iso1 was heavily glycosylated with limited ubiquitin ligase activity for p53, resulting in p53 stabilization. Knockdown of Iso1 in BE and EAC cells led to degradation of the mutant form of p53 and reduced clonogenic survival. In contrast, Iso2 was a potent ligase that reduced levels of the mutant form of p53 in BE cells. In BE cells, Iso2 was hypoglycosylated and degraded, via ATM and GSK3ß-mediated phosphorylation and activation of the beta-TrCP1-containing SCF ubiquitin ligase complex. Simvastatin, which degrades the mutant form of p53, also degraded RNF128 Iso1 protein in BE cells and slowed growth of EAC xenograft tumors in mice. CONCLUSIONS: We found that isoform 2 of RNF128 is decreased in BE cells, resulting in increased levels of mutant p53, whereas isoform 1 of RNF128 is increased in BE cells, further promoting the stabilization of mutant p53.

Constitutively Higher Level of GSTT2 in Esophageal Tissues From African Americans Protects Cells Against DNA Damage.

BACKGROUND & AIMS: African American and European American individuals have a similar prevalence of gastroesophageal reflux disease (GERD), yet esophageal adenocarcinoma (EAC) disproportionately affects European American individuals. We investigated whether the esophageal squamous mucosa of African American individuals has features that protect against GERD-induced damage, compared with European American individuals. METHODS: We performed transcriptional profile analysis of esophageal squamous mucosa tissues from 20 African American and 20 European American individuals (24 with no disease and 16 with Barrett's esophagus and/or EAC). We confirmed our findings in a cohort of 56 patients and analyzed DNA samples from patients to identify associated variants. Observations were validated using matched genomic sequence and expression data from lymphoblasts from the 1000 Genomes Project. A panel of esophageal samples from African American and European American subjects was used to confirm allele-related differences in protein levels. The esophageal squamous-derived cell line Het-1A and a rat esophagogastroduodenal anastomosis model for reflux-generated esophageal damage were used to investigate the effects of the DNA-damaging agent cumene-hydroperoxide (cum-OOH) and a chemopreventive cranberry proanthocyanidin (C-PAC) extract, respectively, on levels of protein and messenger RNA (mRNA). RESULTS: We found significantly higher levels of glutathione S-transferase theta 2 (GSTT2) mRNA in squamous mucosa from African American compared with European American individuals and associated these with variants within the GSTT2 locus in African American individuals. We confirmed that 2 previously identified genomic variants at the GSTT2 locus, a 37-kb deletion and a 17-bp promoter duplication, reduce expression of GSTT2 in tissues from European American individuals. The nonduplicated 17-bp promoter was more common in tissue samples from populations of African descendant. GSTT2 protected Het-1A esophageal squamous cells from cum-OOH-induced DNA damage. Addition of C-PAC increased GSTT2 expression in Het-1A cells incubated with cum-OOH and in rats with reflux-induced esophageal damage. C-PAC also reduced levels of DNA damage in reflux-exposed rat esophagi, as observed by reduced levels of phospho-H2A histone family member X. CONCLUSIONS: We found GSTT2 to protect esophageal squamous cells against DNA damage from genotoxic stress and that GSTT2 expression can be induced by C-PAC. Increased levels of GSTT2 in esophageal tissues of African American individuals might protect them from GERD-induced damage and contribute to the low incidence of EAC in this population.

Activation of GATA binding protein 6 (GATA6) sustains oncogenic lineage-survival in esophageal adenocarcinoma.

Gene amplification is a tumor-specific event during malignant transformation. Recent studies have proposed a lineage-dependency (addiction) model of human cancer whereby amplification of certain lineage transcription factors predisposes a survival mechanism in tumor cells. These tumor cells are derived from tissues where the lineage factors play essential developmental and maintenance roles. Here, we show that recurrent amplification at 18q11.2 occurs in 21% of esophageal adenocarcinomas (EAC). Utilization of an integrative genomic strategy reveals a single gene, the embryonic endoderm transcription factor GATA6, as the selected target of the amplification. Overexpression of GATA6 is found in EACs that contain gene amplification. We find that EAC patients whose tumors carry GATA6 amplification have a poorer survival. We show that ectopic expression of GATA6, together with FGFR2 isoform IIIb, increases anchorage-independent growth in immortalized Barrett's esophageal cells. Conversely, siRNA-mediated silencing of GATA6 significantly reduces both cell proliferation and anchorage-independent growth in EAC cells. We further demonstrate that induction of apoptotic/anoikis pathways is triggered upon silencing of GATA6 in EAC cells but not in esophageal squamous cells. We show that activation of p38α signaling and up-regulation of TNF-related apoptosis-inducing ligand are detected in apoptotic EAC cells upon GATA6 deprivation. We conclude that selective gene amplification of GATA6 during EAC development sustains oncogenic lineage-survival of esophageal adenocarcinoma.

Empagliflozin improves cardiac function in rats with chronic heart failure.

The objective of this study is to examine the effect of empagliflozin on cardiac function in rats with chronic heart failure and the possible mechanism. Forty 6-week-old male SD rats were randomly divided into the control group, empagliflozin treatment group, and sham-operated group. SD rats in the control group and empagliflozin treatment group were subjected to ligation of the anterior descending coronary artery to induce an acute myocardial infarction model. SD rats in the sham-operated group were only subjected to threading of the anterior descending branch of the coronary artery without ligation. On the second day after surgery, the control group and sham operation group were given physiological saline by gavage, while the empagliflozin treatment group was given empagliflozin (30 mg/kg/day) by gavage. Sixteen weeks later, cardiac function, intracellular reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP), serum brain natriuretic peptide, hypersensitive C-reactive protein (hs-CRP), iNOS expression levels, and myocardial morphological changes were observed. Compared with that in the control group, heart function in the empagliflozin-treated group was significantly improved, MMP was increased, intracellular ROS levels were decreased, and NT-proBNP and hs-CRP were significantly reduced, and HE staining showed that the cell oedema was less than that in the control group, tissue arrangement was more orderly, and iNOS expression was inhibited. Empagliflozin can improve cardiac function in rats with chronic heart failure, and the mechanism may involve inhibiting inflammation, reducing myocardial oxidative stress, and improving myocardial fibrosis.

ISG15/GRAIL1/CD3 axis influences survival of patients with esophageal adenocarcinoma.

Immunosuppression is a common feature of esophageal adenocarcinoma (EAC) and has been linked to poor overall survival (OS). We hypothesized that upstream factors might negatively influence CD3 levels and T cell activity, thus promoting immunosuppression and worse survival. We used clinical data and patient samples of those who progressed from Barrett's to dysplasia to EAC, investigated gene (RNA-Seq) and protein (tissue microarray) expression, and performed cell biology studies to delineate a pathway impacting CD3 protein stability that might influence EAC outcome. We showed that the loss of both CD3-ε expression and CD3+ T cell number correlated with worse OS in EAC. The gene related to anergy in lymphocytes isoform 1 (GRAIL1), which is the prominent isoform in EACs, degraded (ε, γ, δ) CD3s and inactivated T cells. In contrast, isoform 2 (GRAIL2), which is reduced in EACs, stabilized CD3s. Further, GRAIL1-mediated CD3 degradation was facilitated by interferon-stimulated gene 15 (ISG15), a ubiquitin-like protein. Consequently, the overexpression of a ligase-dead GRAIL1, ISG15 knockdown, or the overexpression of a conjugation-defective ISG15-leucine-arginine-glycine-glycine mutant could increase CD3 levels. Together, we identified an ISG15/GRAIL1/mutant p53 amplification loop negatively influencing CD3 levels and T cell activity, thus promoting immunosuppression in EAC.

Potentiating the radiation-induced type I interferon antitumoral immune response by ATM inhibition in pancreatic cancer.

Radiotherapy induces a type I interferon-mediated (T1IFN-mediated) antitumoral immune response that we hypothesized could be potentiated by a first-in-class ataxia telangiectasia mutated (ATM) inhibitor, leading to enhanced innate immune signaling, T1IFN expression, and sensitization to immunotherapy in pancreatic cancer. We evaluated the effects of AZD1390 or a structurally related compound, AZD0156, on innate immune signaling and found that both inhibitors enhanced radiation-induced T1IFN expression via the POLIII/RIG-I/MAVS pathway. In immunocompetent syngeneic mouse models of pancreatic cancer, ATM inhibitor enhanced radiation-induced antitumoral immune responses and sensitized tumors to anti-PD-L1, producing immunogenic memory and durable tumor control. Therapeutic responses were associated with increased intratumoral CD8+ T cell frequency and effector function. Tumor control was dependent on CD8+ T cells, as therapeutic efficacy was blunted in CD8+ T cell-depleted mice. Adaptive immune responses to combination therapy provided systemic control of contralateral tumors outside of the radiation field. Taken together, we show that a clinical candidate ATM inhibitor enhances radiation-induced T1IFN, leading to both innate and subsequent adaptive antitumoral immune responses and sensitization of otherwise resistant pancreatic cancer to immunotherapy.

GRAIL1 stabilizes misfolded mutant p53 through a ubiquitin ligase-independent, chaperone regulatory function.

Frequent (>70%) TP53 mutations often promote its protein stabilization, driving esophageal adenocarcinoma (EAC) development linked to poor survival and therapy resistance. We previously reported that during Barrett's (BE) progression to EAC, an isoform switch occurs in the E3 ubiquitin ligase RNF128 (aka GRAIL - gene related to anergy in lymphocytes), enriching isoform 1 (hereby GRAIL1) and, stabilizing the mutant p53 protein. Consequently, GRAIL1 knockdown degrades mutant p53. But how GRAIL1 stabilizes the mutant p53 protein remains unclear. In search for a mechanism, here we performed biochemical and cell biology studies to identify that GRAIL has a binding domain (315-PMCKCDILKA-325) for Hsp40/DNAJ. This interaction can influence DNAJ chaperone activity to modulate misfolded mutant p53 stability. As predicted, either the overexpression of a GRAIL fragment (Frag-J) encompassing the DNAJ binding domain, or a cell permeable peptide (Pep-J) encoding the above 10 amino acids, can bind and inhibit DNAJ-Hsp70 co-chaperone activity thus degrading misfolded mutant p53. Consequently, either Frag-J or Pep-J can reduce the survival of mutant p53 containing dysplastic BE and EAC cells and inhibit growth of patient-derived dysplastic BE organoids (PDOs) in 3D cultures. The misfolded mutant p53 targeting and growth inhibitory effects of Pep-J is comparable to simvastatin, a cholesterol lowering drug, that can degrade misfolded mutant p53 also via inhibiting DNAJA1, although by a distinct mechanism. Implications: We identified a novel ubiquitin ligase independent, chaperone regulating domain in GRAIL and further synthesized a first-in-class novel misfolded mutant p53 degrading peptide having future translational potential.

Metabolism drives macrophage heterogeneity in the tumor microenvironment.

Tumor-associated macrophages (TAMs) are a major cellular component in the tumor microenvironment (TME). However, the relationship between the phenotype and metabolic pattern of TAMs remains poorly understood. We performed single-cell transcriptome profiling on hepatic TAMs from mice bearing liver metastatic tumors. We find that TAMs manifest high heterogeneity at the levels of transcription, development, metabolism, and function. Integrative analyses and validation experiments indicate that increased purine metabolism is a feature of TAMs with pro-tumor and terminal differentiation phenotypes. Like mouse TAMs, human TAMs are highly heterogeneous. Human TAMs with increased purine metabolism exhibit a pro-tumor phenotype and correlate with poor therapeutic efficacy to immune checkpoint blockade. Altogether, our work demonstrates that TAMs are developmentally, metabolically, and functionally heterogeneous and purine metabolism may be a key metabolic feature of a pro-tumor macrophage population.

DNA-PK Inhibition and Radiation Promote Antitumoral Immunity through RNA Polymerase III in Pancreatic Cancer.

Targeting the DNA damage response in combination with radiation enhances type I interferon (T1IFN)-driven innate immune signaling. It is not understood, however, whether DNA-dependent protein kinase (DNA-PK), the kinase critical for repairing the majority of radiation-induced DNA double-strand breaks in cancer cells, is immunomodulatory. We show that combining radiation with DNA-PK inhibition increases cytosolic double-stranded DNA and tumoral T1IFN signaling in a cyclic GMP-AMP synthase (cGAS)- and stimulator of interferon genes (STING)-independent, but an RNA polymerase III (POL III), retinoic acid-inducible gene I (RIG-I), and antiviral-signaling protein (MAVS)-dependent manner. Although DNA-PK inhibition and radiation also promote programmed death-ligand 1 (PD-L1) expression, the use of anti-PD-L1 in combination with radiation and DNA-PK inhibitor potentiates antitumor immunity in pancreatic cancer models. Our findings demonstrate a novel mechanism for the antitumoral immune effects of DNA-PK inhibitor and radiation that leads to increased sensitivity to anti-PD-L1 in poorly immunogenic pancreatic cancers. IMPLICATIONS: Our work nominates a novel therapeutic strategy as well as its cellular mechanisms pertinent for future clinical trials combining M3814, radiation, and anti-PD-L1 antibody in patients with pancreatic cancer.

UBCH5 Family Members Differentially Impact Stabilization of Mutant p53 via RNF128 Iso1 During Barrett's Progression to Esophageal Adenocarcinoma.

BACKGROUND & AIMS: TP53 mutations underlie Barrett's esophagus (BE) progression to dysplasia and cancer. During BE progression, the ubiquitin ligase (E3) RNF128/GRAIL switches expression from isoform 2 (Iso2) to Iso1, stabilizing mutant p53. However, the ubiquitin-conjugating enzyme (E2) that partners with Iso1 to stabilize mutant p53 is unknown. METHODS: Single-cell RNA sequencing of paired normal esophagus and BE tissues identified candidate E2s, further investigated in expression data from BE to esophageal adenocarcinoma (EAC) progression samples. Biochemical and cellular studies helped clarify the role of RNF128-E2 on mutant p53 stability. RESULTS: The UBE2D family member 2D3 (UBCH5C) is the most abundant E2 in normal esophagus. However, during BE to EAC progression, loss of UBE2D3 copy number and reduced expression of RNF128 Iso2 were noted, 2 known p53 degraders. In contrast, expression of UBE2D1 (UBCH5A) and RNF128 Iso1 in dysplastic BE and EAC forms an inactive E2-E3 complex, stabilizing mutant p53. To destabilize mutant p53, we targeted RNF128 Iso1 either by mutating asparagine (N48, 59, and 101) residues to block glycosylation to facilitate ß-TrCP1-mediated degradation or by mutating proline (P54 and 105) residues to restore p53 polyubiquitinating ability. In addition, either loss of UBCH5A catalytic activity, or disruption of the Iso1-UBCH5A interaction promoted Iso1 loss. Consequently, overexpression of either catalytically dead or Iso1-binding-deficient UBCH5A mutants destabilized Iso1 to degrade mutant p53, thus compromising the clonogenic survival of mutant p53-dependent BE cells. CONCLUSIONS: Loss of RNF128 Iso2-UBCH5C and persistence of the Iso1-UBCH5A complex favors mutant p53 stability to promote BE cell survival. Therefore, targeting of Iso1-UBCH5A may provide a novel therapeutic strategy to prevent BE progression.

Circular RNA circHIPK3 modulates autophagy via MIR124-3p-STAT3-PRKAA/AMPKα signaling in STK11 mutant lung cancer.

The role of circular RNA in cancer is emerging. A newly reported circular RNA HIPK3 (circHIPK3) is critical in cell proliferation of various cancer types, although its role in non-small cell lung cancer (NSCLC), has yet to be elucidated. Our results provided evidence that silencing of circHIPK3 significantly impaired cell proliferation, migration, invasion and induced macroautophagy/autophagy. Mechanistically, we uncovered that autophagy was induced upon loss of circHIPK3 via the MIR124-3p-STAT3-PRKAA/AMPKa axis in STK11 mutant lung cancer cell lines (A549 and H838). STAT3 abrogation as well as transfection with a MIR124-3p mimic, recapitulated the induction of autophagy. We also demonstrated antagonistic regulation on autophagy between circHIPK3 and linear HIPK3 (linHIPK3). We therefore propose that the ratio between circHIPK3 and linHIPK3 (C:L ratio) may reflect autophagy levels in cancer cells. We observed that a high C:L ratio (>0.49) was an indicator of poor survival, especially in advanced-stage NSCLC patients. These results support that circHIPK3 is a key autophagy regulator in a subset of lung cancer and has potential clinical use as a prognostic factor. The circular RNA HIPK3 (circHIPK3) functions as an oncogene and autophagy regulator may potential use as a prognostic marker and therapeutic target in lung cancer.Abbreviations 3-MA: 3-methyladenine; AMPK: AMP-activated protein kinase; ATG7: autophagy related 7; Baf-A: bafilomycin A1; BECN1: beclin 1; circHIPK3: circular HIPK3; CQ: chloroquine; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; HIPK3: homeodomain interacting protein kinase 3; IL6R: interleukin 6 receptor; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; NSCLC: non-small cell lung cancer; RFP: red fluorescent protein; RPS6KB1/S6K: ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; STAT3: signal transducer and activator of transcription 3; STK11: serine/threonine kinase 11.

Autoantibody profiles reveal ubiquilin 1 as a humoral immune response target in lung adenocarcinoma.

There is considerable evidence that the presence of cancer can elicit a humoral immune response to specific proteins in the host, and these resulting autoantibodies may have potential as noninvasive biomarkers. To characterize the autoantibody repertoire present in the sera of patients with lung adenocarcinoma, we developed a high-density peptide microarray derived from biopanning a lung cancer phage display library. Using a 2,304-element microarray, we interrogated a total of 250 sera from Michigan lung cancer patients and noncancer controls to develop an "autoantibody profile" of lung adenocarcinoma. A set of 22 discriminating peptides derived from a training set of 125 serum samples from lung adenocarcinoma patients and control subjects was found to predict cancer status with 85% sensitivity and 86% specificity in an independent test set of 125 sera. Sequencing of the immunoreactive phage-peptide clones identified candidate humoral immune response targets in lung adenocarcinoma, including ubiquilin 1, a protein that regulates the degradation of several ubiquitin-dependent proteasome substrates. An independent validation set of 122 serum samples from Pittsburgh was examined using two overlapping clones of ubiquilin 1 that showed 0.79 and 0.74 of the area under the receiver operating characteristics curve, respectively. Significantly increased levels of both ubiquilin 1 mRNA and protein, as well as reduced levels of the phosphorylated form of this protein, were detected in lung tumors. Immunofluorescence using anti-ubiquilin 1 antibodies confirmed intracellular expression within tumors cells. These studies indicate that autoantibody profiles, as well as individual candidates, may be useful for the noninvasive detection of lung adenocarcinoma.

Serum microRNA Signature Is Capable of Early Diagnosis for Non-Small Cell Lung Cancer.

Despite decades of efforts, non-small-cell lung cancer (NSCLC) remains the leading cause of cancer mortality globally primarily due to the challenge in early detection of the cancer. Being an important player in cancer development, the dysregulated miRNAs have been shown promising values as non-invasive diagnostic and prognostic biomarkers for NSCLC. The aim of our study is to access the efficacy and reliability of a potential circulating miRNA panel in early diagnosis of NSCLC. We first selected eight candidate miRNAs, miR-146b, miR-205, miR-29c, miR-31, miR-30b, miR-337, miR-411, and miR-708, which have been shown frequently aberrant in primary NSCLC patients based on our previous studies and other reports. The serum level of each of these miRNAs was evaluated by quantitative real-time PCR (qRT-PCR) in training and testing sets. We found that 5 out of 8 miRNAs (miR-146b, miR-205, miR-29c, miR-30b, and miR-337) were significantly up-regulated in NSCLCs patients compared to healthy or cancer-free controls in both training and testing sets. Based on the logistic regression model, a 4-miRNAs set (miR-146b, miR-205, miR-29c and miR-30b) was picked out of the 5 miRNAs owing to its excellent diagnostic power for NSCLC patients in the training set (AUC=0.99, accuracy=95.00%), the testing set (AUC=0.93, accuracy=89.69%), and the training-testing combined set ( AUC=0.96, accuracy=92.00%). When pathological subtypes of NSCLC are compared, this 4-miRNA panel carried a relatively higher prediction power and higher sensitivity for adenocarcinoma (AC) (AUC=0.98, sensitivity=99.10%) than for squamous cell carcinoma (SCC) (AUC=0.93, sensitivity=90.32%). Additionally, this panel demonstrated a comparable diagnostic capacity for stage I (AUC=0.96) and stage II-III (AUC=0.95) of NSCLC, suggesting its role in reflecting the tumor load. Importantly, the high levels of miR-146b and miR-29c in serum were significantly associated with poor 5-year overall survival (OS) (both p=0.04). Further survival analysis showed that high level of miR-146b in serum is specifically correlated with poor survival rate in SCC patients (p=0.0035) but not in AC patients (p=0.83), consistent with our previous finding that the high tissue expression of miR-146b in lung cancer specimen is indicative of a poor prognosis for SCC patients. Altogether, our study demonstrated that the 4-miRNA panel is a novel, sensitive and non-invasive serum marker for the early diagnosis of NSCLC.

LncRNA MIR22HG abrogation inhibits proliferation and induces apoptosis in esophageal adenocarcinoma cells via activation of the STAT3/c-Myc/FAK signaling.

Long non-coding RNAs (lncRNAs) have involved in human malignancies and played an important role in gene regulations. The dysregulation of lncRNA MIR22HG has been reported in several cancers. However, the role of MIR22HG in esophageal adenocarcinoma (EAC) is poorly understood. Loss of function approaches were used to investigate the biological role of MIR22HG in EAC cells. The effects of MIR22HG on cell proliferation were evaluated by WST-1 and colony formation assays. The effects of MIR22HG on cell migration and invasion were examined using transwell assays. QRT-PCR and Western blot were used to evaluate the mRNA and protein expression of related genes. In this study, abrogation of MIR22HG inhibited cell proliferation, colony formation, invasion and migration in EAC 3 cell lines (OE33, OE19 and FLO-1). Mechanistically, MIR22HG silencing decreased the expression of STAT3/c-Myc/p-FAK proteins and induced apoptosis in EAC cell lines. These results delineate a novel mechanism of MIR22HG in EAC, and may provide potential targets by developing lncRNA-based therapies for EAC.

LINC00857 knockdown inhibits cell proliferation and induces apoptosis via involving STAT3 and MET oncogenic proteins in esophageal adenocarcinoma.

Esophageal adenocarcinoma (EAC) is one of the leading causes of cancer-related death worldwide, and the molecular biology of this cancer remains poorly understood. Recent evidence indicates that long non-coding RNAs are dysregulated in a variety of cancers including EAC. In this study, siRNA mediated gene knockdown, Western blot, RT-PCR, as well as oncogenic function assay were performed. We found that the cell proliferation, colony formation, invasion and migration were decreased after LINC00857 knockdown in EAC cell lines. We also found that knockdown LINC00857 could induce apoptosis. Mechanistically, we found that the MET, STAT3, c-Myc and p-CREB proteins were decreased after LINC00857 knockdown. Our study suggests that LINC00857 may play an important oncogenic role in EAC via STAT3 and MET signaling.

Validation of a serum 4-microRNA signature for the detection of lung cancer.

BACKGROUND: Our previous studies have identified a serum-based 4-microRNA (4-miRNA) signature that may help distinguish patients with lung cancer (LC) from non-cancer controls (NCs). Here, we used an extended independent cohort of 398 subjects to further validate the diagnostic ability of this 4-miRNA signature. METHODS: Using quantitative reverse transcription polymerase chain reaction (qRT-PCR), expression of the 4-miRNAs was assessed in a total of 398 sera that included 213 LC patients and 185 NCs. A logistic regression model using training-test sets, receiver operating characteristic (ROC) curve analysis and t-test were used to test the impact of varying expression of these miRNAs on its diagnostic accuracy for LC. The cell proliferation and colony formation affected by these miRNAs, as well as gene ontology (GO) analysis of miRNA target genes were performed. RESULTS: The levels of the 4-miRNAs were significantly higher in the serum of patients with LCs as compared to NCs. Using a logistic regression prediction model based on training and test sets analysis, we obtained the area under the curve (AUC) of 0.921 [95% confidence interval (CI), 0.876-0.966] on the test set with specificity 90.6%, sensitivity 77.9%, accuracy 84.1%, positive predictive value (PPV) 89.8% and negative predictive value (NPV) 79.5%. CONCLUSIONS: We have verified that this serum 4-miRNA signature could provide a promising noninvasive biomarker for the prediction of LC, particularly in patients with indeterminate lung nodules on screening CT scans.

Silencing of Long Noncoding RNA MIR22HG Triggers Cell Survival/Death Signaling via Oncogenes YBX1, MET, and p21 in Lung Cancer.

The long noncoding RNA (lncRNA) MIR22HG has previously been identified as a prognostic marker in hepatocellular carcinoma. Here, we performed a comprehensive analysis of lncRNA expression profiles from RNA-Seq data and report that MIR22HG plays a similar role in lung cancer. Analysis of 918 lung cancer and normal lung tissues and lung cancer cell lines revealed that MIR22HG was significantly downregulated in lung cancer; this decreased expression was associated with poor patient survival. MIR22HG bound and stabilized the YBX1 protein. Silencing of MIR22HG triggered both cell survival and cell death signaling through dysregulation of the oncogenes YBX1, MET, and p21. In this MIR22HG network, p21 played an oncogenic role by promoting cell proliferation and antiapoptosis in lung cancers. MIR22HG played a tumor-suppressive role as indicated by inhibition of multiple cell cycle-related genes in human primary lung tumors. These data show that MIR22HG has potential as a new diagnostic and prognostic marker and as a therapeutic target for lung cancer.Significance: The lncRNA MIR22HG functions as a tumor suppressor, with potential use a diagnostic/prognostic marker and therapeutic target in lung cancer. Cancer Res; 78(12); 3207-19. ©2018 AACR.