High Tissue Expression Research Articles

Clinical Limitations of Tissue Annexin A2 Level as a Predictor of Postoperative Overall Survival in Patients with Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the second common cause of cancer-related death in Taiwan. Tumor recurrence is frequently observed in HCC patients receiving surgical resection, resulting in unsatisfactory overall survival (OS). Therefore, it is pivotal to identify effective prognostic makers, so that intensive surveillance or adjuvant treatments can be applied to predictively unfavorable patients. Previous studies indicated that Annexin A2 (ANXA2) was an effective prognostic marker in several cancers, including HCC. However, the prognostic value of ANXA2 in Taiwanese HCC patients remains unclear, where a great proportion of patients had chronic hepatitis B with liver cirrhosis. Here, ANXA2 was highly expressed in HCC tissues compared with para-neoplastic noncancerous tissues. Furthermore, high ANXA2 expression in HCC tissues independently predicted shorter OS. In subgroup analysis, however, ANXA2 expression could not effectively predict OS in the following subgroups: female, age > 65 years old, Child–Pugh classification B, hepatitis B virus surface antigen negative or anti-hepatitis C antibody positive, alcoholism, tumor number >1, presence of micro- or macrovascular invasion, absence of capsule, non-cirrhosis and high alpha-fetoprotein. In conclusion, ANXA2 expression in HCC tissues could predict postoperative OS. However, the predictive value was limited in patients with specific clinical conditions.

METTL3 promotes the initiation and metastasis of ovarian cancer by inhibiting CCNG2 expression via promoting the maturation of pri-microRNA-1246

Ovarian cancer is a common gynecological malignant tumor with a high mortality rate and poor prognosis. There is inadequate knowledge of the molecular mechanisms underlying ovarian cancer. We examined the expression of methyltransferase-like 3 (METTL3) in tumor specimens using RT-qPCR, immunohistochemistry, and Western blot analysis, and tested the methylation of METTL3 by MSP. Levels of METTL3, miR-1246, pri-miR-1246 and CCNG2 were then analyzed and their effects on cell biological processes were also investigated, using in vivo assay to validate the in vitro findings. METTL3 showed hypomethylation and high expression in ovarian cancer tissues and cells. Hypomethylation of METTL3 was pronounced in ovarian cancer samples, which was negatively associated with patient survival. Decreased METTL3 inhibited the proliferation and migration of ovarian cancer cells and promoted apoptosis, while METTL3 overexpression exerted opposite effects. Mechanistically, METTL3 aggravated ovarian cancer by targeting miR-1246, while miR-1246 targeted and inhibited CCNG2 expression. High expression of METTL3 downregulated CCNG2, promoted the metabolism and growth of transplanted tumors in nude mice, and inhibited apoptosis. The current study highlights the promoting role of METTL3 in the development of ovarian cancer, and presents new targets for its treatment.

SSRP1 affects the growth and apoptosis of gastric cancer cells through AKT pathway.

BackgroundWe aimed to determine the SSRP1's potential influence on the apoptosis and proliferation of gastric cancer (GC) cells and its regulatory mechanism.MethodsSSRP1 expression in GC cells and tissues was detected via quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The interrelation between clinicopathological characteristics of GC patients and SSRP1 expression was analysed via x2 test, and the correlation between SSRP1 expression and overall survival rate was analysed using Kaplan-Meier survival analysis. After the knockdown of SSRP1 in AGS cells, the SSRP1 expression, colony formation ability, cell viability, cell cycle changes, apoptosis rate, and migration and invasion ability were detected through qRT-PCR, colony formation assay, CCK8 assay, flow cytometry and transwell test, respectively. Finally, the effects of down-regulation of SSRP1 on the expressions of phosphorylated-protein kinase B (p-AKT), B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) were explored using Western blotting.ResultsSSRP1 displayed a high expression in GC cells and tissues. SSRP1 expression was closely interrelated to the TNM stage, lymph node metastasis and tumour size. The survival rate of patients was markedly shorter in the high expression group than in the lower expression group. After the knockdown of SSRP1 in cells, the viability and colony formation ability of AGS cells were inhibited. In addition, the cell ratio in the G1 phase was increased, while that in the S phase declined, and the cell invasion and migration were obviously weakened. It was found from Western blotting that the knockdown of SSRP1 could evidently suppress the protein levels of Bcl-2 and p-AKT but promote the protein expression of Bax, indicating that silencing SSRP1 can inhibit the proliferative capacity and increase the number of GC cells through inactivating the AKT signalling pathway.ConclusionsSSRP1 rose up in GC tissues and cells. Reduction of SSRP1 can inhibit the proliferative capacity and increase the number of GC cells through inactivating the AKT signalling pathway.

Discovery and construction of prognostic model for clear cell renal cell carcinoma based on single-cell and bulk transcriptome analysis.

BackgroundClear cell renal cell carcinoma (ccRCC) is the most common malignant kidney tumor in adults. Single-cell transcriptome sequencing can provide accurate gene expression data of individual cells. Integrated single-cell and bulk transcriptome data from ccRCC samples provide comprehensive information, which allows the discovery of new understandings of ccRCC and the construction of a novel prognostic model for ccRCC patients.MethodsSingle-cell transcriptome sequencing data was preprocessed by using the Seurat package in R software. Principal component analysis (PCA) and the t-distributed stochastic neighbor embedding (t-SNE) algorithm were used to perform cluster classification. Two subtypes of cancer cells were identified, pseudotime trajectory analysis and gene ontology (GO) analysis were conducted with the monocle and clusterProfiler packages. Two novel cancer cell biomarkers were identified according to the single-cell sequencing and were confirmed by The Cancer Genome Atlas (TCGA) data. T cell-related marker genes according to single-cell sequencing were screened by a combination of Kaplan-Meier (KM) analysis, univariate Cox analysis, least absolute shrinkage and selection operator (Lasso) regression and multivariate Cox analysis of TCGA data. Four survival predicting genes were screened out to develop a risk score model. A nomogram consisting of the risk score and clinical information was constructed to predict the prognosis for ccRCC patients.ResultsA total of 5,933 cells were included in the study after quality control. Fifteen cell clusters were classified by PCA and t-SNE algorithm. Two clusters of cancer cells with distinct differentiation status were identified. Besides, GO analysis revealed that biological processes were different between the two subgroups. Egl-9 family hypoxia-inducible factor 3 (EGLN3) and nucleolar protein 3 (NOL3) were specifically expressed in cancer cell clusters, bulk RNA sequencing data from TCGA confirmed their high expression in ccRCC tissues. GTSE1, CENPF, SMC2 and H2AFV were screened out and applied to the construction of risk score model. A nomogram was generated to predict prognosis of ccRCC by combing the risk score and clinical parameters.ConclusionsWe integrated single-cell and bulk transcriptome data from ccRCC in this study. Two subtypes of ccRCC cells with different biological characteristics and two potential biomarkers of ccRCC were discovered. A novel prognostic model was constructed for clinical application.

Serum Anti-PDLIM1 Autoantibody as Diagnostic Marker in Ovarian Cancer.

BackgroundSerum autoantibodies (AAbs) against tumor-associated antigens (TAAs) could be useful biomarkers for cancer detection. This study aims to evaluate the diagnostic value of autoantibody against PDLIM1 for improving the detection of ovarian cancer (OC).MethodsImmunohistochemistry (IHC) test in tissue array containing 280 OC tissues, 20 adjacent tissues, and 8 normal ovarian tissues was performed to analyze the expression of PDLIM1 in tissues. Enzyme-linked immunosorbent assay (ELISA) was employed to measure the autoantibody to PDLIM1 in 545 sera samples from 182 patients with OC, 181 patients with ovarian benign diseases, and 182 healthy controls.ResultsThe results of IHC indicated that 84.3% (236/280) OC tissues were positively stained with PDLIM1, while no positive staining was found in adjacent or normal ovarian tissues. The frequency of anti-PDLIM1 autoantibody was significantly higher in OC patients than that in healthy and ovarian benign controls in both training (n=122) and validation (n=423) sets. The area under the curves (AUCs) of anti-PDLIM1 autoantibody for discriminating OC from healthy controls were 0.765 in training set and 0.740 in validation set, and the AUC of anti-PDLIM1 autoantibody for discriminating OC from ovarian benign controls was 0.757 in validation set. Overall, it was able to distinguish 35.7% of OC, 40.6% of patients with early-stage, and 39.5% of patients with late-stage. When combined with CA125, the AUC increased to 0.846, and 79.2% of OC were detected, which is statistically higher than CA125 (61.7%) or anti-PDLIM1(35.7%) alone (p<0.001). Also, anti-PDLIM1 autoantibody could identify 15% (18/120) of patients that were negative with CA125 (CA125 <35 U/ml).ConclusionsThe anti-PDLIM1 autoantibody response in OC patients was positively correlated with PDLIM1 high expression in OC tissues, suggesting that the autoantibody against PDLIM1 might have the potential to be a novel serological biomarker of OC, serving as a complementary measure of CA125, which could improve the power of OC detection.

Effects of EFNA1 on cell phenotype and prognosis of esophageal carcinoma

BackgroundTo investigate the expression and clinical significance of EFNA1 in broad-spectrum tumors, and to evaluate its relationship with prognosis and biological functions of esophageal carcinoma (ESCA).MethodsEFNA1 expression in various cancers was analyzed according to the data in the TCGA database. The clinical data were integrated, to analyze the relationship with ESCA clinical parameters and prognosis, and EFNA1 expression in ESCA tissue samples was detected by immunohistochemistry (IHC). Based on bioinformatics, the functional background of EFNA1 overexpression was analyzed. EFNA1 knockout cell model was established by EFNA1-shRNA transfecting ESCA cells, and the effect of knocking down EFNA1 on the proliferation of ESCA cells was detected by MTT.ResultsAmong 7563 samples from TCGA, the EFNA1 gene highly expressed in 15 samples with common cancers and endangered the prognosis of patients with tumors. Its overexpression in ESCA and its influence on the prognosis were most significant. EFNA1 expression in 80 samples with ESCA and their paired samples was tested by IHC to verify its high expression (paired t test, P < 0.001) in ESCA tissues. It was found that EFNA1 expression was related to clinical factors (TNM staging, P = 0.031; lymph node metastasis, P = 0.043; infiltration, P = 0.016). Meanwhile, EFNA1 was found to be an independent risk factor based on the COX multi-factor analysis. And to further explore the importance of EFNA1 in tumors, EC-9706 and ECA109 cells were screened from 8 ESCA-related cell lines to build EFNA1 knockdown cell models. The results showed that EFNA1 knockdown significantly inhibited the proliferation of tumor cells (P < 0.05). In terms of molecular mechanism, EFNA1 related genes were significantly enriched in the proliferative pathway according to the pathway enrichment analysis. It was found that knocking down EFNA1 did inhibit cell proliferation based on cell experiments.ConclusionsEFNA1 overexpression in ESCA tissue is related to the prognosis of patients. Knocking down EFNA1 can significantly inhibit the proliferation of ESCA cells.

CLEC4s as Potential Therapeutic Targets in Hepatocellular Carcinoma Microenvironment.

Immunosuppressive tumor microenvironment in hepatocellular carcinoma (HCC) is critical in tumor development. C-type (Ca2+ -dependent) lectin (CLEC) receptors, essential in innate pattern recognition, have potential regulatory effects on immune cell trafficking and modulatory effects on cancer cell activity. However, information on the expression and prognostic value of CLECs in HCC is scanty. Herein, we explored the potential role of CLECs in HCC based on TCGA, ONCOMINE, GEPIA, UALCAN, cBioPortal, Metascape, TRRUST, and TIMER databases. Results demonstrated a significantly higher mRNA level of CLEC4A and CLEC4L in HCC tissues than normal liver tissues. Contrarily, we found significantly low CLEC4G/H1/H2/M expression in HCC tissues. The IHC analysis revealed the following: Absence of CLEC4A/J/K/M in normal and liver cancer tissues; high CLEC4C expression in HCC tissues; low expression and zero detection of CLEC4D/E/H1/H2/L in HCC tissues and normal tissues, respectively. And the HepG2 and LX-2 were used to verify the expression level of CLEC4s via qRT-PCR in vitro. Furthermore, the expression of CLEC4H1 (ASGR1) and CLEC4H2 (ASGR2) exhibited a significant relation to clinical stages. However, the expression of CLEC4A, CLEC4D, CLEC4E, CLEC4J (FCER2), CLEC4K (CD207), CLEC4G, CLEC4H1, CLEC4M, and CLEC4H2 decreased with tumor progression. Patients expressing higher CLEC4H1/H2 levels had longer overall survival than patients exhibiting lower expression. Moreover, CLEC4A/D/E/J/K/G/H1/M/H2 had significant down-regulated levels of promoter methylation. The expression level of CLEC4s was correlated with the infiltration of B cells, CD8 + T cells, CD4 + T cells, macrophage cells, neutrophil cells, and dendritic cells. Functional analysis revealed the potential role of CLECL4s in virus infection, including COVID-19. Also, hsa-miR-4278 and hsa-miR-324-5p, two potential miRNA targets of CLEC4s, were uncovered. This article demonstrates that CLEC4 is crucial for the development of HCC and is associated with infiltration of various immune cells, providing evidence for new immunotherapy targets in HCC.

Translation initiation factor eIF2Bε promotes Wnt-mediated clonogenicity and global translation in intestinal epithelial cells

Modulation of global mRNA translation, which is essential for intestinal stem cell function, is controlled by Wnt signaling. Loss of tumor supressor APC in stem cells drives adenoma formation through hyperactivion of Wnt signaling and dysregulated translational control. It is unclear whether factors that coordinate global translation in the intestinal epithelium are needed for APC-driven malignant transformation. Here we identified nucleotide exchange factor eIF2Bε as a translation initiation factor involved in Wnt-mediated intestinal epithelial stemness. Using eIF2BεArg191His mice with a homozygous point mutation that leads to dysfunction in the enzymatic activity, we demonstrate that eIF2Bε is involved in small intestinal crypt formation, stemness marker expression, and secreted Paneth cell-derived granule formation. Wnt hyperactivation in ex vivo eIF2BεArg191His organoids, using a GSK3β inhibitor to mimic Apc driven transformation, shows that eIF2Bε is essential for Wnt-mediated clonogenicity and associated increase of the global translational capacity. Finally, we observe high eIF2Bε expression in human colonic adenoma tissues, exposing eIF2Bε as a potential target of CRC stem cells with aberrant Wnt signaling.

MiR-1 Targeted Downregulation of Bcl-2 Increases Chemosensitivity of Lung Cancer Cells.

Objective: To investigate the expression of B cell lymphoma-2 (Bcl-2) in lung cancer cells and the effect of the miR-1/Bcl-2 axis on the chemosensitivity of lung cancer. Materials and Methods: Real-time quantitative PCR and western blotting were used to detect the expression of Bcl-2 in human embryonic lung fibroblasts and lung cancer cells. The effects of siRNA directed against Bcl-2, in lung cancer tissue samples was detected by immunohistochemistry; these results were used to develop prognostic models. Bioinformatic analyses, dual luciferase reporter gene technology, and western blotting technology were used to explore the targeted regulation of miR-1 on bcl-2. The effect of miR-1 on the chemosensitivity of lung cancer cells was measured using the MTT assay. Results: Compared with human embryonic lung fibroblasts, Bcl-2 was highly expressed in the lung cancer cells, especially in H460 cells. After silencing Bcl-2 with siRNA, the sensitivity of the cells to cisplatin (CDDP) increased. Immunohistochemical results and prognostic analysis revealed that high Bcl-2 expression in lung cancer tissues was negatively correlated with prognosis of lung cancer patients; A dual luciferase reporter assay combined with western blotting confirmed that miR-1 can bind to the Bcl-23' UTR region and regulate its expression. Overexpression of miR-1 in lung cancer cells (H460 and A549) increased the sensitivity of these cells to CDDP. Conclusion: Bcl-2 is upregulated in lung cancer cells, which is negatively correlated with the patient prognosis. miR-1 affects the chemosensitivity of lung cancer cells by targeting Bcl-2. These data should provide a theoretical basis for refining the molecular mechanisms of chemoresistance in lung cancer.

Rapid Genomic Evolution Drives the Diversification of Male Reproductive Genes in Dung Beetles.

The molecular basis for the evolution of novel phenotypes is a central question in evolutionary biology. In recent years, dung beetles have emerged as models for novel trait evolution as they possess distinct precopulatory traits such as sexually dimorphic horns on their head and thorax. Here, we use functional and evolutionary genomics to investigate the origins and the evolution of postcopulatory reproductive traits in male dung beetles. Male ejaculates that underlie postcopulatory sexual selection are excellent candidates to study novel trait evolution as they are complex, fast evolving, and often highly divergent in insects. We assemble de novo transcriptomes of male accessory glands and testes of a widespread dung beetle, Catharsius molossus, and we perform an evolutionary analysis of closely and distantly related insect genomes. Our results show there is rapid innovation at the genomic level even among closely related dung beetles. Genomic expansion and contraction drive the divergence of male reproductive traits and their functions. The birth of scores of completely novel reproductive genes is reinforced by the recruitment of these genes for high expression in male reproductive tissues, especially in the accessory glands. We find that male accessory glands of C. molossus are specialized for secretory function and express female, egg, and embryo-related genes as well as serine protease inhibitors, whilst the testes are specialized for spermatogenesis and sperm function. Finally, we touch upon putative functions of these evolutionary novelties using structure-function analysis as these proteins bear no homology to any other known proteins.

MiR-186-5p suppresses cell migration, invasion, and epithelial mesenchymal transition in bladder cancer by targeting RAB27A/B.

Bladder cancer (BCa) is a common malignancy in the urinary system. Ras-related protein Rab-27A (RAB27A) and Ras-related protein Rab-27B (RAB27B) have been verified to be closely related to the development of many tumors. Since the role of both RAB27A and RAB27B in BCa have not been reported, we intended to explore the function and mechanism of RAB27A and RAB27B in BCa development. Reverse transcription quantitative polymerase chain reaction revealed that RAB27A/B showed high expression in BCa tissues and cells. Cell counting kit-8, wound healing and Transwell assays as well as western blot analyses revealed that silencing RAB27A/B suppressed BCa cell proliferation, migration and invasion as well as the epithelial-mesenchymal transition (EMT) process. Based on bioinformatics analysis and our experiments, microRNA-186-5p (miR-186-5p) was found to be the upstream miRNA of RAB27A/B in BCa. MiR-186-5p expression was significantly downregulated in BCa cells and tissues. MiR-186-5p directly targeted the 3'-untranslated region (3'-UTR) of RAB27A/B and downregulated both mRNA and protein levels of RAB27A/B in BCa cells. MiR-186-5p overexpression suppressed BCa cell proliferation, migration and invasion as well as the EMT process in vitro and inhibited tumor growth in vivo. Overexpressing RAB27A/B rescued the inhibitory effect of miR-186-5p on malignant phenotypes of BCa cells. Furthermore, miR-186-5p inactivated the phosphatidylinositol 3-Kinase (PI3K)/mitogen-activated protein kinase (MAPK) signaling pathway by downregulating the expression of RAB27A/B, as shown in western blot analysis. Overall, miR-186-5p suppressed BCa cell proliferation, migration, invasion and EMT process and inhibited xenograft tumor growth by targeting RAB27A/B to inactivate the PI3K/MAPK signaling.

Poly(A)-Specific Ribonuclease (PARN) Promotes the Proliferation, Migration and Metastasis of Esophageal Cancer

Background: Esophageal cancer leads to the majority of cancer deaths in the world. Surgery, radiotherapy and chemotherapy are the main treatment methods, but due to its high recurrence and metastasis rate, the overall prognosis is poor. Poly(A)-specific ribonuclease (PARN) is a multifunctional enzyme molecule, which is involved in the occurrence and development of tumors. This study explores the relationship between PARN and the proliferation, metastasis and prognosis of esophageal cancer. Methods: The esophageal cancer tissues and adjacent normal tissues were collected from 91 patients with esophageal cancer after surgery. The expression level of PARN was detected by immunohistochemistry (IHC) using tissue microarray (TMA). PARN expression and its relationships with clinicopathological characteristics and prognosis was detected. In addition, stable PARN knockdown esophageal cancer cell lines TE-1 and Eca-109 were used to study its effect on the phenotype and behavior of tumor cells. Meanwhile, the effect of PARN silencing on tumor growth in vivo was verified by xenotransplantation model in nude mice. Finally, we examined possible downstream signaling mechanisms of PARN in esophageal cancer. Findings: PARN showed high expression in esophageal carcinoma tissues and its expression was significantly correlated with lymphatic metastasis and prognosis of patients. In the culture of PARN knockdown esophageal cancer cell lines, the growth and proliferation were significantly inhibited, and the invasion and migration ability of tumor cells were also significantly decreased. In addition, inhibition of PARN suppressed tumor growth and increased survival time in xenograft murine models. In the PARN knockdown group, BIM, IGFBP-5 and p21 were significantly increased, while the expression levels of anti-apoptotic proteins Survivin and STNF-R1 were significantly down-regulated. In addition, the expression levels of Akt, p-Akt, PIK3CA and CCND1 downstream of the signaling pathway regulating esophageal cancer progression were significantly down-regulated. Interpretation: These results suggest a previously undetected role of PARN as a suppressor of tumor development and its associations of prognosis in esophageal cancer through anti-apoptotic effect. Funding Information: None. Declaration of Interests: The authors declare no conflicts of interests. Ethics Approval Statement: Ethical approval for the study was approved by the Research Ethics Committee of the Second Affiliated Hospital of Guangxi Medical University and Zhujiang Hospital Affiliated to Southern Medical University, and written patient consent was obtained from all patients. Nude mice xenograft model - This study was approved by the Guangxi Medical University Ethics Committee.

LncRNA FEZF1-AS1 regulates biological behaviors of cervical cancer by targeting miRNA-1254.

AimThe purpose of this research was to evaluate lncRNA FEZF1‐AS1 in cervical cancer development and clinical significance.Materials and methodsCollecting cervical cancer tissues, measuring FEZF1‐AS1 expression, and analysis correlation between FEZF1‐AS1 and prognosis. In cell vitro study, using MTT assay to measure cell proliferation, evaluating cell apoptosis by flow cytometry, measuring cell invasion and migration by Transwell and wound healing assay; lncRNA FEZF1‐AS1 and miR‐1254 gene expressions were evaluated by RT‐qPCR assay; relative protein (Smurf1, E‐cadherin, Vimentin, N‐cadherin, AKT, p‐AKT, c‐Myc, and ZEB1) expressions were measured by Western blot assay. The correlation among FEZF1‐AS1, miR‐1254, and Smurf1 were analysis by dual luciferase reporter gene assay.ResultsBy clinical analysis, lncRNA FEZF1‐AS1 was high expression in cervical cancer tissues and high expression was closely correlated with poor prognosis in cervical cancer patients. In vitro study, the SiHa and HeLa cell biologically including cell proliferation, migration, and invasion of si‐FEZF1‐AS1 group which knockdown lncRNA FEZF1‐AS1 were significantly depressed (p < .001, respectively). However, with miR‐1254 expression inhibiting, the cell biological activities were significantly increased in si‐FEZF1‐AS1+miRNA inhibitor groups (p < .001, respectively).ConclusionlncRNA FEZF1‐AS1 might be an oncological role in cervical cancer; lncRNA FEZF1‐AS1 knockdown had antitumor effects with miR‐1254 activating in cervical cancer by in vitro study.

The Lrat-/- Rat: CRISPR/Cas9 Construction and Phenotyping of a New Animal Model for Retinitis Pigmentosa.

Purpose: We developed and phenotyped a pigmented knockout rat model for lecithin retinol acyltransferase (LRAT) using CRISPR/Cas9. The introduced mutation (c.12delA) is based on a patient group harboring a homologous homozygous frameshift mutation in the LRAT gene (c.12delC), causing a dysfunctional visual (retinoid) cycle. Methods: The introduced mutation was confirmed by DNA and RNA sequencing. The expression of Lrat was determined on both the RNA and protein level in wildtype and knockout animals using RT-PCR and immunohistochemistry. The retinal structure and function, as well as the visual behavior of the Lrat−/− and control rats, were characterized using scanning laser ophthalmoscopy (SLO), optical coherence tomography (OCT), electroretinography (ERG) and vision-based behavioral assays. Results: Wildtype animals had high Lrat mRNA expression in multiple tissues, including the eye and liver. In contrast, hardly any expression was detected in Lrat−/− animals. LRAT protein was abundantly present in wildtype animals and absent in Lrat−/− animals. Lrat−/− animals showed progressively reduced ERG potentials compared to wildtype controls from two weeks of age onwards. Vison-based behavioral assays confirmed reduced vision. Structural abnormalities, such as overall retinal thinning, were observed in Lrat−/− animals. The retinal thickness in knockout rats was decreased to roughly 80% by four months of age. No functional or structural differences were observed between wildtype and heterozygote animals. Conclusions: Our Lrat−/− rat is a new animal model for retinal dystrophy, especially for the LRAT-subtype of early-onset retinal dystrophies. This model has advantages over the existing mouse models and the RCS rat strain and can be used for translational studies of retinal dystrophies.

High-Expression of Cytoplasmic Poly (A) Binding Protein 1 (PABPC1) as a Prognostic Biomarker for Early-Stage Esophageal Squamous Cell Carcinoma

Background and ObjectivePoly (A) binding protein cytoplasmic 1 (PABPC1) plays a crucial role in the regulation of RNA polyadenylation, translation initiation, and mRNA stability and may be involved in tumorigenesis. Herein, we set out to identify the prognostic value of PABPC1 expression in esophageal squamous cell carcinoma (ESCC).MethodsUsing quantitative real-time PCR (qRT-PCR) and immunohistochemical analysis, the present study investigated mRNA and protein expressions of PABPC1 in 231 ESCCs and their paired adjacent normal epithelial tissues.ResultsWe observed a reduction in the average mRNA expression of PABPC1 in ESCC tissue specimen, but the mRNA expression of PABPC1 was significantly higher (P<0.001) in ESCC tissues with high PABPC1 expression and lower (P=0.033) in tissues with low PABPC1 expression. In immunohistochemical analysis, positive expression of the PABPC1 protein was identified in 179 ESCC tissue specimens (179/231, 77.5%), while the percentage of ESCC tissue specimens with high expression of PABPC1 was found to be 41.1% (95/231). PABPC1 expression was found to be significantly correlated with lymph node metastasis (LNM) (P=0.011), pathological stage (P=0.021), tumor recurrence (P<0.001), and the outcome (P<0.001) of patients with ESCC. High expression of PABPC1 was associated with poor overall survival (OS) of ESCC patients (P<0.001) among all pathological stages, particularly in the early stages (pStage-I and -II), and identified to be an independent prognostic factor for OS of patients with ESCC in multivariate analysis (HR=2.622; 95% CI, 1.68–4.129). Comparatively, the expression of Ki-67, p53, and nm23 was not associated with OS.ConclusionIn this study, we discovered that PABPC1 is a prognostic biomarker and a therapeutic target for ESCC, particularly early-stage ESCC.

Abstract 1860: Bispecific claudin-6 x CD3 antibodies in a 2 + 1 format demonstrate selectivity and activity on human ovarian cancer cells

Abstract There is a large unmet need for targeted therapies to treat ovarian cancer and other solid tumors. A promising strategy is the use of T cell engaging bispecific antibodies that recruit T cells to kill cancer cells by simultaneously binding to tumor-associated antigens (TAA) on cancer cells and CD3 on T cells. Effective and safe use of these therapeutics depends on selective targeting of cancer cells over normal tissue, feasible when a TAA is more highly expressed on cancer cells versus normal tissues. Selectivity can also be improved by using a mixed valency “2 + 1” bispecific format, coupled with target affinity tuning, to achieve avid binding to the TAA. Claudin-6 (CLDN6) is a tetraspan membrane protein, a member of the claudin family of tight junction proteins, and has been identified as a promising TAA for ovarian cancer due to its high expression on ovarian and other cancer tissues and low expression on normal tissue. However, a complicating factor is that many proteins in the claudin family have high sequence identity. Most similar to CLDN6 is CLDN9, and the two proteins differ at only 3/76 residues in their extracellular loops, creating a challenge for the development of highly selective CLDN6 antibodies. We analyzed CLDN6 and CLDN9 expression using a combination of normal tissue gene expression data (GTEx), cancer cell genomics data (TCGA), and immunohistochemistry (IHC) on a panel of cancer and normal tissues. Data confirmed the tumor-specific expression pattern of CLDN6 but indicated high CLDN9 expression in normal tissues, suggesting that strong antibody selectivity for CLDN6 versus CLDN9 is critical. We created CLDN6-selective T cell engaging bispecific antibodies by starting with a highly selective monoclonal antibody humanized from a mouse hybridoma. Selectivity was further improved by engineering the CDR regions. Variant antibodies were screened for binding to transiently transfected HEK293E cells that highly expressed either CLDN6, CLDN9, or other homologous claudin family members. Selective variants were converted into the XmAb® 2 + 1 bispecific antibody format and tested in T cell-dependent cellular cytotoxicity (TDCC) assays using cancer cell lines expressing CLDN6, or HEK293E cells transfected with claudin homologs. A set of lead CLDN6 x CD3 bispecifics displaying a range of potencies on CLDN6+ cancer cell lines and high selectivity for CLDN6 over CLDN9 and other homologous claudins was identified. Tolerability and pharmacokinetics of these molecules are currently being assessed in non-human primates, and activity will be evaluated in humanized mouse models of ovarian cancer. Citation Format: Matthew S. Faber, Sung-Hyung Lee, Yoon Kyung Kim, Jing Qi, Kendra N. Avery, Duc-Hanh T. Nguyen, Rumana Rashid, Araz Eivazi, Seung Y. Chu, Juan E. Diaz, Connie Ardila, Ruschelle Love, Alex Nisthal, Norman J. Barlow, Christine Bonzon, Umesh S. Muchhal, Matthew J. Bernett, John R. Desjarlais. Bispecific claudin-6 x CD3 antibodies in a 2 + 1 format demonstrate selectivity and activity on human ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1860.

High membrane expression of CMTM6 in hepatocellular carcinoma is associated with tumor recurrence

CKLF‐like MARVEL transmembrane domain–containing protein 6 (CMTM6) maintains membrane PD‐L1 expression by controlling its endosomal recycling. However, in patients with hepatocellular carcinoma (HCC), the correlation among CMTM6, B7 family ligands, and CD8‐positive cytotoxic T lymphocytes (CTLs), and the molecular function of CMTM6 in HCC have not been established. We performed immunohistochemistry to evaluate the relationships among CMTM6 expression, clinicopathological factors, B7 family ligands expression, and CTL infiltration in HCC samples. Moreover, we established CMTM6‐knockout human HCC cell lines to evaluate the function of human CMTM6 in immune regulation and tumor viability. CMTM6 expression was positively associated with membrane B7 family ligands expression and CTL infiltration in HCC samples. High CMTM6 expression in HCC tissues was associated with the expression of the proliferation marker Ki‐67 and shorter recurrence‐free survival. In vitro analysis showed the downregulation of membrane B7 family ligands and proliferation potency in the CMTM6‐knockout human HCC cell line. High membrane CMTM6 expression was associated with tumor recurrence and proliferation via the regulation of membranous B7 family ligands expression. Thus, CMTM6 might be a biomarker to predict the risk of HCC recurrence and a therapeutic target to suppress tumor growth and increase CTL activity.

Circ_0030586 inhibits cell proliferation and stemness in bladder cancer by inactivating the ERK signaling via miR-665/NR4A3 axis

Increasing evidence reveals that circular RNAs (circRNAs) serve as oncogenes or tumor suppressors in the development of various tumors including bladder cancer (BCa). In this study, we explored the function and mechanism of circ_0030586 (also named circABCC4, ATP binding cassette subfamily C member 4) in BCa. The expression of circ_0030586 was significantly decreased in BCa tissues and cells, as suggested by RT-qPCR. The circular characteristics of circ_0030586 were verified by agarose gel electrophoresis and RNase R treatment. Colony formation, 5-Ethynyl-2′-deoxyuridine and sphere formation assays revealed that overexpression of circ_0030586 suppressed BCa cell proliferation and stemness in vitro. According to xenograft experiment, circ_0030586 overexpression suppressed tumor growth in vivo. Mechanistically, RNA pulldown and luciferase reporter assays were carried out to explore the interaction between genes. Circ_0030586 served as a competing endogenous RNA (ceRNA) for hsa-miR-665 to upregulate the expression of nuclear receptor subfamily 4 group A member 3 (NR4A3) which is a downstream target gene of miR-665 in BCa. MiR-665 exhibited high expression in BCa tissues and cells while NR4A3 expression was downregulated in BCa. MiR-665 overexpression or NR4A3 silencing reversed the suppressive effect of circ_0030586 overexpression on BCa cell proliferation and stemness. Moreover, western blot analysis revealed that circ_0030586 inactivated the extracellular signal-regulated kinase (ERK) pathway by upregulating NR4A3 expression. In conclusion, circ_0030586 inhibits BCa cell proliferation and stemness by serving as a ceRNA for miR-665 to upregulate NR4A3 expression and thus inactivate the ERK signaling.

MiR-875 Serves as A Candidate Biomarker for Detection and Prognosis and Is Correlated with PHH3 Index Levels in Breast Cancer Patients

BackgroundmiRNAs play crucial roles in cancers. This study investigated the potential value of miR-875 to be a detective or/and prognostic marker and evaluate the correlation between its expression and PHH3 index levels in breast cancer. MethodsmiR-875 expression was determined in breast cancer serum and tissues by a quantitative real-time reverse transcription-polymerase chain reaction. The detective value of serum miR-875 expression for breast cancer was assessed by receiver operating characteristic analysis. Then the associations of miR-875 expression with clinical characteristics of patients and overall survival were evaluated by χ2 test and Kaplan-Meier curve methods, respectively. ResultsThe expression of miR-875 was increased in both breast cancer serum and tissues compared with respective controls. The high miR-875 expression in serum and tissues was associated with positive lymph node metastasis and advanced TNM stages. Besides, miR-875 expression in tissues was positively associated with the PHH3 index. And serum miR-875 could screen breast cancer patients from healthy individuals. Moreover, breast cancer patients with both high expression of miR-875 and PHH3 index had shorter overall survival. ConclusionThis study suggested that miR-875 expression may be suitable as a potential breast cancer detection and prognosis biomarker. And the miR-875 expression in tissues was positively associated with the PHH3 index, suggesting that miR-875 expression may be involved in tumor proliferation of breast cancer.

High Tissue TLR5 Expression Predicts Better Outcomes in Colorectal Cancer Patients

Background: Colorectal cancer (CRC), the third most common cancer globally, caused 881,000 cancer deaths in 2018. Toll-like receptors (TLRs), the primary sensors of pathogen-associated molecular patterns and damage-associated molecular patterns, activate innate and adaptive immune systems and participate in the development of an inflammatory tumor microenvironment. We aimed to explore the prognostic value of TLR3, TLR5, TLR7, and TLR9 tissue expressions in CRC patients. Methods: Using immunohistochemistry, we analyzed tissue microarray samples from 825 CRC patients who underwent surgery between 1982 and 2002 at the Department of Surgery, Helsinki University Hospital, Finland. After analyzing a pilot series of 205 tissue samples, we included only TLR5 and TLR7 in the remainder of the patient series. We evaluated the associations between TLR5 and TLR7 tissue expressions, clinicopathologic variables, and survival. Using the Kaplan-Meier method, we generated survival curves, determining significance using the log-rank test. Univariate and multivariate survival analyses relied on the Cox proportional hazards model. Results: The 5-year disease-specific survival was 55.9% among TLR5-negative (95% confidence interval [CI] 50.6–61.2%) and 61.9% (95% CI 56.6–67.2%; p = 0.011, log-rank test) among TLR5-positive patients. In the Cox multivariate survival analysis adjusted for age, sex, stage, location, and grade, positive TLR5 immunoexpression (hazard ratio [HR] 0.74; 95% CI 0.59–0.92; p = 0.007) served as an independent positive prognostic factor. TLR7 immunoexpression exhibited no prognostic value in the survival analysis across the entire cohort (HR 0.97; 95% CI 0.78–1.20; p = 0.754) nor in subgroup analyses. Conclusions: We show for the first time that a high TLR5 tumor tissue expression associates with a better prognosis in CRC patients.