The SMARCA4 gene encodes a key ATPase subunit of the SWI/SNF (BAF) chromatin-remodeling complex, which plays an essential role in regulating transcription and cellular differentiation. Loss-of-function alterations of SMARCA4 are common in various human cancers, including sarcomas; however, rare SMARCA4 fusion events, presumably resulting in gain of function, have also been reported. We present two pediatric soft-tissue sarcomas harboring a novel, recurrent in-frame fusion between SMARCA4 and VEZF1 (Vascular Endothelial Zinc Finger 1), a transcription factor important for vascular development and angiogenesis. The predicted fusion protein contains the N-terminal QLQ protein interaction domain of SMARCA4 and preserves most of VEZF1's C2H2 zinc-finger DNA-binding domains. Interestingly, another component of the BAF complex, SS18, has also been reported to be fused to VEZF1 in uterine sarcoma. We propose that fusion of BAF complex components to VEZF1 leads to aberrant recruitment of chromatin-remodeling activity to VEZF1 target loci, resulting in altered chromatin architecture, dysregulated VEZF1-dependent transcription, and tumorigenesis.

Recent studies have highlighted the pivotal role of the cGAS-STING pathway in cancer immunotherapy. However, clinical trials with cGAS-STING pathway agonists have faced setbacks thanks to their short biological half-life, lack of tumor specificity, and potential to promote tumor immune evasion. To address these challenges, a novel exosome-based drug delivery platform, termed cmExoaCD11b is developed, designed to precisely target and reprogram the tumor microenvironment (TME) in situ for pancreatic cancer immunotherapy. cmExoaCD11b is engineered to encapsulate high copy numbers of IL-12 mRNA and 2'3'-cGAMP (cGAMP) and is functionalized with CD11b antibodies for targeted delivery to macrophages. Notably, cmExoaCD11b facilitated the repolarization of M2 macrophages to M1 phenotype, thereby reprogramming the TME and enhancing the secretion of pro-inflammatory cytokines. This immunomodulatory effect reversed the immunosuppressive milieu of the TME and significantly inhibited tumor progression. More importantly, cmExoaCD11b exhibited robust therapeutic efficacy in both murine pancreatic cancer and patient-derived xenograft models. These results suggest that cmExoaCD11b represents a promising approach for overcoming immunosuppression in pancreatic cancer, paving the way for its potential application in cancer immunotherapy.

Somatic mutations in DNA methyltransferase 3 A (DNMT3A) are frequently observed in patients with hematological malignancies. Hematopoietic stem/progenitor cells (HSPCs) with mutated DNMT3A demonstrate increased self-renewal activity and skewed lineage differentiation. However, the molecular mechanisms underlying these changes remain largely unexplored. In this study, we show that Dnmt3a loss leads to the upregulation of endogenous retroviruses (ERVs) in HSPCs, subsequently activating the cGAS-STING pathway and triggering inflammatory responses in these cells. Both genetic and pharmacological inhibition of STING effectively corrects the increased self-renewal activity and differentiation skewing induced by Dnmt3a deficiency in mice. Notably, targeting STING showed inhibited acute myeloid leukemia (AML) development in a Dnmt3a-KO; Flt3-ITD AML model, comparable to AC220, an FDA-approved FLT3-ITD inhibitor. A patient-derived xenograft (PDX) model further demonstrated that targeting STING effectively alleviates the leukemic burden of DNMT3A-mutant AML. Collectively, our findings highlight a critical role for STING in hematopoietic disorders induced by DNMT3A mutations and propose STING as a potential therapeutic target for preventing the progression of DNMT3A mutation-associated leukemia.

Lung adenocarcinoma (LUAD) remains the leading cause of cancer deaths worldwide. Apurinic/apyrimidinic endonuclease 1 (APE1), an enzyme integral to DNA repair and redox signaling, is notably upregulated in LUAD. Here we reveal that APE1 amplification, primarily via allele duplication, strongly correlates with poor prognosis in LUAD patients. Using human LUAD cell lines and a KRAS-driven mouse model, we showed that APE1 deletion hampered cell proliferation and tumor growth, highlighting its role in tumorigenesis. Mechanistically, APE1 promoted the transcription of urea cycle genes CPS1 and ARG2 by modulating the presence of G-quadruplex (G4) structures in their promoter regions. APE1 loss disrupted the urea cycle and pyrimidine metabolism, inducing metabolic reprogramming and growth arrest, which could be rescued by CPS1 or pyrimidine restoration. These findings uncover APE1's role in transcriptional regulation of urea cycle metabolic reprogramming via G4 structure, providing a potential therapeutic target LUAD patients with elevated APE1 expression.

Abstract Background: The epigenetic modification of LncRNA N6-methyladenosine (m6A) methylation plays a wide role in the invasion and metastasis of various malignant tumors. However, the role of LncRNA-ATB m6A methylation modification in colorectal cancer (CRC) has not been systematically elucidated. Methods: Detect the regulatory effect of ALKBH5 on LncRNA-ATB by Luciferin reporter gene. LncRNA-ATB expression was detected by IHC and qPCR in CRC tissues and cells. Transwell and scratch tests were used to detect the effect of ALKBH5 overexpression after LncRNA-ATB knockdown and replacement on the invasion and migration ability of CRC cells. Western blotting was used to detect the expression changes of MMPs proteins. Results: ALKBH5 could regulate the methylation level of the LncRNA-ATB 3’ UTR region (p<0.05). ALKBH5 overexpression in CRC cells led to a significant decrease in the expression level of LncRNA-ATB (p<0.05). LncRNA-ATB exhibited elevated expression in CRC cells and demonstrated significantly higher levels in CRC tissues compared to paracancerous tissues (p<0.05), indicating the potential tumor-promoting effect of LncRNA-ATB in CRC, with the mechanism possibly regulated by ALKBH5-mediated m6A demethylation. ALKBH5 overexpression inhibited the proliferation and migration of CRC cells after LncRNA-ATB knockdown. However, this inhibitory effect was attenuated upon LncRNA-ATB rescue, accompanied by an increase in MMP9 and a decrease in TIMP1. Conclusion: LncRNA-ATB plays a crucial role in ALKBH5-mediated m6A demethylation, regulating the invasion and metastasis of CRC. The ALKBH5-LncRNA-ATB m6A demethylation-MMPs signaling axis has been identified as a key regulatory pathway promoting CRC migration. m6A modification of lncRNA-ATB modulated by ALKBH5 provides a theoretical basis for novel therapeutic strategies for patients with metastatic CRC. FUNDING: This study was supported by the National Natural Science Foundation of China (Grant No. 81702414), Natural Science Foundation of Fujian Province of China (Grant No. 2020J05306) and Xiamen Science and Technology Planning Project (Grant No. 3502Z20194003). Citation Format: Yu Lang#, Jia-Lin Lin#, Jia-Peng Kang, Lu Yang, Dan Zhou, Can-Bin Fang*, Feng Ye*, Wei-wei Tang*. The role and mechanism of m6A methylation modification of LncRNA-ATB in regulating the invasion and metastasis of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3169.

Even though acute myeloid leukemia (AML) patients with a RUNX1::RUNX1T1 (AE) fusion have a relatively favorable prognosis, approximately 50% relapse within 2.5 years and develop resistance to subsequent chemotherapy [1]. It is therefore imperative to identify novel therapeutic targets for AE leukemia to improve outcomes. In this study, we unveil that targeting STING effectively suppresses the growth of AE leukemia cells. Both genetic and pharmacological inhibition of STING lead to the diminish of AE leukemia cells. Importantly, in a mouse primary AE leukemia model, STING deletion significantly attenuates leukemogenesis and prolongs the animals’ lifespan. Blocking the downstream inflammatory pathway of STING yields similar effects to STING inhibition in AE leukemia cells, highlighting the pivotal role of STING-dependent inflammatory responses in sustaining the survival of AE leukemia cells. Moreover, through a genome-wide CRISPR screen, we identified fatty acid desaturase 2 (FADS2) as a non-canonical factor downstream of STING inhibition that mediates cell death. Inhibition of STING releases FADS2 activity, consequently inducing the synthesis of polyunsaturated fatty acids (PUFAs) and triggering lipid peroxidation-associated cell death [2]. Taken together, these findings reveal a critical function of STING in the survival of AE-positive AML cells and suggest STING to be a potential therapeutic target for clinical intervention in these patients.

Oxidized methylcytidines 5-hydroxymethyl-20deoxycytidine (5hmdC) and 5-formy-20deoxycytidine (5fdC) are deaminated by cytidine deaminase (CDA) into genome-toxic variants of uridine, triggering DNA damage and cell death. These compounds are promising chemotherapeutic agents for cancer cells that are resistant to pyrimidine derivative drugs, such as decitabine and cytarabine, which are inactivated by CDA. In our study, we found that cancer cells infected with mycoplasma exhibited a markedly increased sensitivity to 5hmdC and 5fdC, which was independent of CDA expression of cancer cells. In vitro biochemical assay showed that the homologous CDA protein from mycoplasma was capable of deaminating 5hmdC and 5fdC into their uridine form. Moreover, mycoplasma infection increased the sensitivity of cancer cells to 5hmdC and 5fdC, whereas administration of Tetrahydrouridine (THU) attenuated this effect, suggesting that mycoplasma CDA confers a similar effect as human CDA. As mycoplasma infection occurs in many primary tumors, our findings suggest that intratumoral microbes could enhance the tumor-killing effect and expand the utility of oxidized methylcytidines in cancer treatment.& COPY; 2023 Elsevier Inc. All rights reserved.

Somatic loss-of-function mutations of the dioxygenase Ten-eleven translocation-2 (TET2) occur frequently in individuals with clonal hematopoiesis (CH) and acute myeloid leukemia (AML). These common hematopoietic disorders can be recapitulated in mouse models. However, the underlying mechanisms by which the deficiency in TET2 promotes these disorders remain unclear. Here we show that the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) pathway is activated to mediate the effect of TET2 deficiency in dysregulated hematopoiesis in mouse models. DNA damage arising in Tet2-deficient hematopoietic stem/progenitor cells (HSPCs) leads to activation of the cGAS-STING pathway which in turn promotes the enhanced self-renewal and development of CH. Notably, both pharmacological inhibition and genetic deletion of STING suppresses Tet2 mutation-induced aberrant hematopoiesis. In patient-derived xenograft (PDX) models, STING inhibition specifically attenuates the proliferation of leukemia cells from TET2-mutated individuals. These observations suggest that the development of CH associated with TET2 mutations is powered through chronic inflammation dependent on the activated cGAS-STING pathway and that STING may represent a potential target for intervention of relevant hematopoietic diseases.

Background: Histone methylation (HM) is an abundant form of histone modification and recent studies have highlighted it strongly related to tumorigenicity and progression. However, histone methylation modification patterns and potential roles in gastric cancer have not been systematically elucidated. Method: In this study, we curated 53 histone methyltransferase regulators and 9 histone demethylase regulators. Identified histone methylation(HM)modification clusters based on the expression of 62 HM regulators. Principal component analysis (PCA) served to construct histone methylation score (HMScore) base on differential genes among three HM clusters. Evaluating the relationship between HMScore and clinical prognosis, tumor mutation burden (TMB), microsatellite instability (MSI), immune cell infiltration and immunotherapy. Results: We identified distinct three HM modification clusters in patients with gastric cancer based on the expression of 62 HM regulators, which were association with different clinical prognosis, biological pathways and tumor microenvironment characterization. Based on HMScore to quantitatively evaluate the histone methylation modification patterns in gastric cancer patients. We founded that patients with a low-HMScore group(score<2.276) was association with poor prognosis. In addition, high-HMScore group(score≥2.276) displayed tumor mutation burden-high (TMB-H) and microsatellite instability-high (MSI-H), and correlation analysis indicated that HMScore was positively correlated with tumor mutation burden. Immune infiltration correlation analysis showed that HMScore strongly negative related to MDSC cell, mast cell and regulatory T cell. Survival analysis indicated that patients with TMB-H and high-HMScore were related to long clinical prognosis in gastric cancer. Moreover, patients with high-HMScore group had high abundance of PD-L1, and high-HMScore was confirmed sensitive response to immunotherapy in gastric cancer. Conclusion: This study provided a new model to evaluate individual gastric cancer histone methylation modification patterns. HMScore was benefit to evaluate clinical prognosis value and guide strategies to improve treatment in gastric cancer. Citation Format: Jiapeng Kang, Lu Yang, Jialin Lin, Dan Zhou, Canbin Fang, Feng Ye, Weiwei Tang. Quantification of histone methylation regulator-associated modification patterns was a potential biomarker for prognosis and associated with immunotherapy in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5750.

Somatic loss-of-function mutations of the dioxygenase Ten-eleven translocation-2 (TET2) occur frequently in individuals with clonal hematopoiesis (CH) and acute myeloid leukemia (AML). These common hematopoietic disorders can be recapitulated in mouse models. However, the underlying mechanisms by which the deficiency in TET2 promotes these disorders remain largely unknown. Here we show that the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) pathway is activated to mediate the effect of TET2 deficiency in leukemogenesis in mouse models. DNA damage arising in Tet2 -deficient hematopoietic stem/progenitor cells (HSPCs) leads to activation of the cGAS-STING pathway which in turn induces the development of CH and myeloid transformation. Notably, both pharmacological inhibition and genetic deletion of STING suppresses Tet2 mutation-induced aberrant myelopoiesis. In patient-derived xenograft (PDX) models, STING inhibition specifically attenuates the proliferation of leukemia cells from TET2-mutated individuals. These observations suggest that the hematopoietic transformation associated with TET2 mutations is powered through sterile inflammation dependent on the activated cGAS-STING pathway, and that STING may represent a potential target for intervention of relevant hematopoietic malignancies. Key points Tet2 deficiency leads to DNA damage which in turn activates the cGAS-STING pathway to induce an inflammatory response Blocking STING in TET2-mutated hematopoietic stem/progenitor cells suppresses clonal hematopoiesis in mice and leukemogenesis in patient-derived xenograft models

N6‐methyladenosine (m6A) has emerged as the most prevalent post‐transcriptional modification on mRNA that contributes prominently to tumorigenesis. However, the specific function of m6A methyltransferase methyltransferase‐like 3 (METTL3) in colorectal cancer (CRC) remains elusive. Herein, we explored the biological function of METTL3 in CRC progression. Clinically, METTL3 was frequently upregulated in CRC tissues, cell lines, and plasma samples and its high expression predicted poor prognosis of CRC patients. Functionally, knockdown of METTL3 significantly repressed CRC cell proliferation and migration in vitro, while its overexpression accelerated CRC tumor formation and metastasis both in vitro and in vivo. Mechanistically, METTL3 epigenetically repressed YPEL5 in an m6A‐YTHDF2‐dependent manner by targeting the m6A site in the coding sequence region of the YPEL5 transcript. Moreover, overexpression of YPEL5 significantly reduced CCNB1 and PCNA expression. Collectively, we identified the pivotal role of METTL3‐catalyzed m6A modification in CRC tumorigenesis, wherein it facilitates CRC tumor growth and metastasis through suppressing YPEL5 expression in an m6A‐YTHDF2‐dependent manner, suggesting a promising strategy for the diagnosis and therapy of CRC.

Abstract Tumor specific alterations in miRNA provide ideal biomarkers for early diagnosis of colorectal cancer(CRC). Methylation changes play an important role in the miRNA regulation mechanism involved in tumorigenesis. Therefore, it is important to find miRNA methylation markers related to tumor proliferation and metastasis, and to explore its key regulatory mechanisms. Previous integrated bioinformatics analysis suggested that reduced expression and hypermethation of miR-146a have detected in majority of CRC. However, its biological behavior in CRC remains unclear. This article aims to explore its epigenetic regulation and mechanism in the development of CRC. We collected peripheral blood and tissues from 60 patients with advanced primary CRC, analyzed their clinicopathological characteristics. The mRNA and methylation difference was detected by qPCR and MSP respectively, and the significance in the diagnosis of CRC was analyzed. The research was further confirmed in SW480, SW620, HCT116 and HT29 CRC cell lines, as well as CRC animal models treated with demethylated drugs–5-aza-miR2-oxyeytidineine(5-AZA) and various gene transfection, in order to explore the key downstream regulatory network of miR-146a methylation changes and the effect of methylation inhibitors in the development of CRC. We found that the percentage of down-regulated and methylation of miR-146a in both tissues and plasmas of CRC was significantly higher than that of normal samples, and the expression of miR-146a could be recovered after demethylation treatment in cancer, which was associated with clinical stage. miR-146a methylation was significantly correlated with grading, but not gender, tumor location, histological type or TNM, it may be used as a predictor of poor prognosis. Diagnostic evaluation of miR-146a methylation in tissue and plasma was evaluated using ROC curve, area under regional curve, which indicated that miR-146a methylation could be used as a biomarker for the diagnosis of CRC. Fluorescein report system showed that miR-146a targeted VASN protein, and while miR-146a was overexpressed after transfection into CRC cells, the expression levels of VASN, TGF-β, VEGF-C, MMP9, and Vimentin were significantly reduced, while the expression of TIMP1 and Ecadherin were up-regulated. Additionally, the miR-146a mRNA in CRC tissues was also negatively correlated with the expression of VASN. VASN Silencing down-regulated TGF-β expression, accompanied by a decrease of MMP9. At the same time, after 5-aza demethylation or siVASN, proliferation activity of CRC cells decreased, accompanied by varying degrees of decline in membrane penetrating and migration ability, suggesting that miR-146a may regulate the proliferation and metastasis by regulating VASN/TGF-β relatived pathway. In vivo animal models, it was further confirmed that overexpressed miR-146a was consistent with the effect of 5-aza intervention or siVASN, which could significantly inhibit the occurrence of liver metastasis of CRC. Demethylated drugs treatment or VASN knockdown also significantly inhibited tumor growth in nude mice with CRC. miR-146a was a potential epigenetic silencing tumor suppressor in CRC, and the methylation of miR-146a may play an important role in the diagnosis and prediction of tumor progression in CRC as a new tumor marker. Citation Format: Weiwei Tang, Dan Zhou, Hanxiang An, Jiapeng Kang, Mingquan Cai, Ru Zeng, Jing Song, Bin Hu, Jiabian Lian, Qin Lin, Lilin Chen, Feng Ye. miR-146a methylation regulates proliferation and metastasis by targeted activation of VASN/TGF-beta signaling pathway in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2443.

BackgroundGastric cancer (GC) is the third leading cause of cancer-related death worldwide, partially due to the lack of effective screening strategies. Thus, there is a stringent need for non-invasive biomarkers to improve patient diagnostic efficiency in GC.MethodsThis study initially filtered messenger RNAs (mRNAs) as prospective biomarkers through bioinformatics analysis. Clinical validation was conducted using circulating mRNA in plasma from patients with GC. Relationships between expression levels of target genes and clinicopathological characteristics were calculated. Then, associations of these selected biomarkers with overall survival (OS) were analyzed using the Kaplan-Meier plotter online tool.ResultsBased on a comprehensive analysis of transcriptional expression profiles across 5 microarrays, top 3 over- and underexpressed mRNAs in GC were generated. Compared with normal controls, expression levels ofcollagen type VI alpha 3 chain(COL6A3),serpin family H member 1 (SERPINH1)andpleckstrin homology and RhoGEF domain containing G1 (PLEKHG1)were significantly upregulated in GC plasmas. Receiver-operating characteristic (ROC) curves on the diagnostic efficacy of plasmaCOL6A3,SERPINH1andPLEKHG1mRNAs in GC showed that the area under the ROC (AUC) was 0.720, 0.698 and 0.833, respectively. Combined, these three biomarkers showed an elevated AUC of 0.907. Interestingly, the higherCOL6A3level was significantly correlated with lymph node metastasis and poor prognosis in GC patients. High level ofSERPINH1mRNA expression was correlated with advanced age, poor differentiation, lower OS, andPLEKHG1was also associated with poor OS in GC patients.ConclusionOur results suggested that circulatingCOL6A3,SERPINH1andPLEKHG1mRNAs could be putative noninvasive biomarkers for GC diagnosis and prognosis.

PURPOSE:JAM3, an adhesion and transmigration regulatory element, is abundantly expressed in intestinal epithelial cells. However, its expression and function in colorectal cancer (CRC) remain unknown. In this study, we explored its epigenetic mechanism and biological role in CRC. PATIENTS AND METHODS:Bioinformatics analysis was used to analyze the expression and methylation level of JAM3 in CRC. Methylation and expression status of JAM3 were then validated by quantitative methylation-specific PCR (qMSP) and quantitative PCR in tissues, plasma samples, and cell lines. Flow cytometry, Western blot, transwell, siRNA, colony formation, and transfection were used to evaluate the biological function of JAM3. RESULTS:We initially found that JAM3 was frequently methylated and downregulated in CRC based on bioinformatics tools. qMSP validation showed that the methylation levels of JAM3 were increased in 75% (18/24) of CRC tissues, 61% (11/18) plasma samples, and all four CRC cell lines and were significantly associated with tumor stage in CRC tissues. Moreover, JAM3 was downregulated in primary CRC tissues, plasma samples, and CRC cell lines as compared with that in nonmalignant controls, although its expression could be recovered after demethylation treatment. Restoration of JAM3 repressed CRC cell viability, colony formation, and migration. In addition, siRNA-mediated depletion of JAM3 in NCM460 cells improved the clonogenicity and migration capability, whereas it suppressed cell apoptosis and cell-cycle arrest. These functional effects were accompanied with alterations of several epithelial cell markers, including E-cadherin, vimentin, phosphor-β-catenin (ser552), and TJP1, which were responsible for epithelial-mesenchymal transition. CONCLUSION:The findings indicated that JAM3 may be a novel tumor suppressor gene with epigenetic reduction in CRC and can be used as a potential noninvasive biomarker for CRC diagnosis.

Background: Compared with the classical tumor markers, ctDNA has higher accuracy and specificity while being noninvasive. Based on the prophase work, we explore the role of ctDNA methylation in the detection of PCDH18 as a new tumor marker compared with CEA in the clinical diagnosis and progressive evaluation of colorectal cancer (CRC). Methods: We collected peripheral blood from 60 patients with advanced primary CRC before and after two courses of treatment, as well as from 60 healthy individuals, and the clinicopathologic characteristics were analyzed. The changes of CEA levels before and after treatment were dynamically monitored by electrochemiluminescence (ECL). The methylation status of PCDH18 was detected by qMSP, the correlation between them was also statistically analyzed, and the value of different indexes in the diagnosis and monitoring of tumor progression of CRC were compared. Results: We found that the percentage of methylation of PCDH18 in plasma of CRC before treatment was significantly higher than that of normal plasma samples. The difference (p < 0.01) suggests that PCDH18 methylation may be involved in the carcinogenesis of CRC. PCDH18 methylation was not significantly correlated with sex, tumor location, histological type, tumor differentiation, TNM or CEA(P > 0.05). To further explore the relationship between the methylation of PCDH18 and the risk of CRC, the results showed that with the increase of methylation level of PCDH18, the risk of CRC and tumor progression increased significantly, while in CEA group, the OR > 1 only in the group with CEA elevation as the dividing point, but the P value was not statistically significant. The sensitivity and specificity of PCDH18 ctDNA methylation combined with CEA in the diagnosis of progression of CRC were 90.0% and 67.6%, respectively. The area under the curve (AUC) reached 0.861. Further analysis showed that PCDH18 ctDNA methylation was 67.86% while CEA was negative in CRC progression patients, indicating that PCDH18 ctDNA methylation could significantly increase the detection of progression in comparison with CEA. Conclusions: The methylation of PCDH18 ctDNA may play an important role in the diagnosis and prediction of tumor progression in CRC as a new tumor marker and can significantly increase the detection of tumor progression in CEA-negative patients. W-W. Tang, H-X. An and D. Zhou contributed equally to this work. Legal entity responsible for the study: Clinical Research Ethics Committee of the First Affiliated Hospital of Xiamen University. Funding: This study was supported by the National Natural Science Foundation of China (No. 81702414), the Fujian Provincial Health and Family Planning Commission Foundation of Youth scientific research project (No. 2015-2-43) and Xiamen Science and Technology Bureau Foundation of science and technology project for the benefit of the people (No. 3502Z20164010). Disclosure: All authors have declared no conflicts of interest.

Lung cancer (LC) remains associated with significant mortality worldwide. The lack of reliable noninvasive biomarkers and targeted therapies contributes to poor survival rate. Herein, we initially took advantage of the public microarray data from Oncomine database to filter messenger RNAs (mRNAs) as potential biomarkers. Subsequently, clinical validation was applied to identify candidate noninvasive biomarkers in plasma from patients with LC. Through comprehensive analysis of transcriptional expression profiles across 12 studies, top 6 over- and underexpressed mRNAs were generated. Then, a pair of matched plasma samples from LC patient and normal control was detected by RT-PCR, and three genes with positive bands were selected for further validation. Finally, qPCR was conducted to further assess values of the three identified genes. We displayed with high confidence that two cell-free mRNAs (HJURP and ADAMTS8) were expressed at significantly levels compared to normal controls. Receiver-operating characteristic (ROC) curves on the diagnostic efficacy of plasma HJURP and ADAMTS8 mRNAs in LC diagnosis showed that the area under the ROC (AUC) was 0.6960 and 0.6877; sensitivity was 66.0% and 83.7%; specificity was 78.6% and 71.4%, respectively. Combined ROC analyses using these two biomarkers revealed an elevated AUC of 0.75. Furthermore, the higher HJURP level could be associated with early-stage LC while lower ADAMTS8 level could be correlated with non-small cell lung cancer. Collectively, circulating HJURP and ADAMTS8 mRNAs are promising noninvasive biomarkers for LC diagnosis. Our integrative strategy provides new insights into novel noninvasive biomarker identification for other types of cancer.

Protocadherin18 (PCDH18) was found to be preferentially methylated and inactivated in colorectal cancer (CRC) using bioinformatics tools. However, its biologic role in tumorgenesis remains unclear. Herein, we aimed to elucidate its epigenetic regulation and biological functions in CRC. The methylation status of PCDH18 was significant higher in CRC tissues than in adjacent non-tumor tissues (median, 15.17% vs. median, 0.4438%). Expression level of PCDH18 was significantly lower in primary CRCs than in nonmalignant tissues. Importantly, methylation status of PCDH18 in cell-free DNA of CRC patients was also significantly higher than in healthy subjects. PCDH18 was readily expressed in NCM460 cells, but downregulated in 100% (4/4) of CRC cell lines by promoter methylation, despite its expression could be restored through demethylation treatment. Overexpression of PCDH18 suppressed CRC cell viability, colony formation and migration. Meanwhile, the depletion of PCDH18 by siRNA in NCM460 cells enhanced the colonogenicity and migration ability and promoted β-catenin nuclear accumulation, whereas it inhibited cell cycle arrest. These effects were associated with upregulation of phospho-GSK-3β and cyclin D1, and downregulation of caspase3 and p21. Our results suggested that PCDH18 was a putative tumor suppressor with epigenetic silencing in CRC and a potential biomarker for CRC diagnosis.

Breast cancer deaths are mainly attributed to metastasis, while epithelial to mesenchymal transition (EMT) plays a pivotal role in promoting cancer cell metastasis. EMT is activated by various stimuli, resulting in different therapeutic responses. The TGFβ signaling pathway is a canonical driver of EMT, and many molecular drugs are developed to target this pathway. However, the alternations of this pathway in BC remain elusive. The aim of this study was to investigate the relationship between the canonical TGFβ-Smad signaling pathway and breast cancer pathogenesis. Herein, we investigated the mutational, transcriptional, epigenetic and post-transcriptional alternations of 14 core members in this pathway through bioinformatics approach by analyses of 56,496 BC patients from various public databases, including COSMIC, Oncomine, DiseaseMeth and Starbase. A protein-protein interaction network was derived from Cytoscape software. Associations of these selected members with relapse free survival (RFS), overall survival (OS), distant metastases free survival (DMFS) and post progression survival (PPS) were performed using the Kaplan-Meier plotter online tool. Our integrated analyses revealed that Smad4 was the most frequently mutated member of the TGFβ-Smad pathway in BC with a mutation frequency of 0.85% (30/3537). The expression levels of TGFβ1, TGFβ3, Smad1, Smad2 and Smad7 were significantly higher, whereas TGFβ2, ALK1, ALK2, ALK5, TGFβR2, Smad3, Smad4, Smad5 and Smad6 were downregulated in BC compared with normal subjects (P<0.001). Evaluation of epigenetic alteration identified that the promoters of ALK1 (P=0.000e+00), ALK2 (P=2.22e-16) and ALK5 (P=1.625e-12) were hypermethylated in 1,274 BC samples. In contrast, Smad2 (P=6.56e-04) was significantly hypomethylated in BC. Although all 14 members had potential diagnostic significances with different prediction power (AUC ranging from 0.788 to 1.00) in various subtypes of BC, only TGFβ3 could predict RFS (HR=0.69, 95% CI [0.62-0.77]), OS (HR=0.69, 95% CI [0.56-0.86]), DMFS (HR=0.8, 95% CI [0.66-0.97]) and PPS (HR=0.76, 95% CI [0.6-0.97]) for patients with BC in an independent manner. Additionally, 11 out of 14 members were associated with RFS in luminal A BC. Our results indicated that TGFβ-Smad pathway core members could serve as novel diagnostic and prognostic biomarkers in BC.

PURPOSE. To investigate the exact mechanism by which keratocytes promote corneal neovascularization.METHODS. The expression of matrix metalloproteinase 13 (MMP13), cluster of differentiation 146 (CD146), VEGFa, VEGFc, VEGF receptor (r)2, and VEGFr3 by normal and alkali-burned rat corneas was determined via quantitative (q)RT-PCR and/or Western blot analysis or in situ hybridization. Corneal neovascularization was observed under a slit lamp microscope and evaluated via immunohistochemistry. The cells that expressed MMP13 in the corneas were determined via sequential immunohistochemistry and in situ hybridization. The degradation of type I collagen was evaluated via the detection of hydroxyproline content and Western blot analysis. The effects of VEGFa and VEGFc on MMP13 expression were determined via luciferase reporter assay for the MMP13 promoter and primary keratocyte culture.RESULTS. Matrix metalloproteinase 13 was predominantly expressed by epithelial cells in normal rat corneas, but it was expressed by cells in corneal stromas after alkali burns. The formation of new blood vessels was consistent with MMP13 expression and attenuated by a selective MMP13 inhibitor in alkali-burned corneas. Keratocytes were the major cells expressing MMP13 in corneal stromas after alkali burns. Through MMP13 expression, keratocytes directly degraded collagen type I to create stromal spaces, which were convenient for newly formed blood vessels to grow into. Expression of MMP13 and collagen type I degradation via keratocytes were induced by VEGFc through VEGFr3 and inhibited by antibodies for VEGFc and VEGFr3.CONCLUSIONS. Keratocytes could directly degrade type I collagen and create stromal spaces, promoting corneal neovascularization through VEGFc/VEGFr3-induced MMP13 expression.

PURPOSEThe purpose is to investigate the mechanism of subconjunctival fibrosis caused by benzalkonium chloride (BAC), which is the most common preservative in ophthalmic preparations.METHODSThe left eyes of male Sprague-Dawley rats were topically treated with 0.01% BAC or PBS twice daily for 1 month. Primary conjunctival fibroblasts (CFs) were exposed for 24 hours to 0.00005% BAC, 0.000075% BAC, 0.000075% BAC + LY2157299 (a selective transforming growth factor β receptor type I inhibitor); 0.000075% BAC + NS-398 (a selective cyclooxygenase-2 inhibitor) and PBS, respectively. The pathological changes of the bulbar conjunctival tissue of rats were examined using hematoxylin-eosin (HE), Van Gieson's (vG), periodic acid-Schiff (PAS) stains, or immunohistochemisty (IHC). The expression of the extracellular matrix (ECM), the transforming growth factor β (TGF-β) signaling pathway-related molecules, and cyclooxygenase-2 (COX-2) in bulbar conjunctival tissues and CFs were detected using Western blot (WB) and quantitative real-time RT-PCR (qRT-PCR).RESULTSRats treated with 0.01% BAC exhibited a slight increase of the fibroblast density and a more compact collagen deposition in the bulbar subepithelial connective tissues in comparison with rats treated with PBS. Western blot and qRT-PCR analyses showed that the expression of ECM, TGF-β signaling pathway-related molecules, and COX-2 were markedly increased in the bulbar conjunctival tissues of rats exposed to 0.01% BAC and in CFs exposed to 0.00005% and 0.000075% BAC. In conjunctival fibroblasts, BAC-induced ECM expression was clearly decreased by LY2157299, while the BAC-induced activation of the TGF-β1/Smad3 signaling pathway was greatly attenuated by NS-398.CONCLUSIONSSubconjunctival fibrosis BAC-induced is a consequence of excessive ECM production of CFs through the COX-2-modulated activation of a TGF-β1/Smad3 signaling pathway.