Machine learning-based establishment and validation of age-related patterns for predicting prognosis in non-small cell lung cancer within the context of the tumor microenvironment.

Lung cancer (LC) is a leading cause of cancer-related mortality worldwide, with non-small cell lung cancer (NSCLC) accounting for over 80% of cases. The impact of aging on clinical outcomes in NSCLC remains poorly understood, particularly with respect to the immune response. In this study, we explored the effects of aging on NSCLC using 307 genes associated with human aging from the Human Ageing Genomic Resources. We identified 53 aging-associated genes that significantly correlate with overall survival of NSCLC patients, including the clinically validated gene BUB1B. Furthermore, we developed an aging-associated enrichment score to categorize patients based on their aging subtypes and evaluated their prognostic and therapeutic response values in LC. Our analyses yielded two aging-associated subtypes with unique profiles in the tumor microenvironment, demonstrating varying responses to immunotherapy. Consensus clustering based on transcriptome profiles provided insights into the effects of aging on NSCLC and highlighted the potential of personalized therapeutic approaches tailored to aging subtypes. Our findings provide a new target and theoretical support for personalized therapeutic approaches in patients with NSCLC, offering insights into the potential impact of aging on cancer outcomes.

Long noncoding RNA LINC01088 inhibits esophageal squamous cell carcinoma progression by targeting the NPM1-HDM2-p53 axis.

Esophageal squamous cell carcinoma (ESCC) is characterized by extensive metastasis and poor prognosis. Long noncoding RNAs (lncRNAs) have been shown to play important roles in ESCC. However, the specific roles of lncRNAs in ESCC tumorigenesis and metastasis remain largely unknown. Here, we investigate LINC01088 in ESCC. Differentially expressed LINC01088 levels are screened from the GEO database. We find that LINC01088 is expressed at low level in collected clinical samples and is correlated with vascular tumor emboli and poor overall survival time of patients after surgery. LINC01088 inhibits not only ESCC cell migration and invasion in vitro, but also tumorigenesis and metastasis in vivo. Mechanistically, LINC01088 directly interacts with nucleophosmin (NPM1) and increases the expression of NPM1 in the nucleoplasm compared to that in the nucleolar region. LINC01088 decreases mutant p53 (mut-p53) expression and rescues the transcriptional activity of p53 by targeting the NPM1-HDM2-p53 axis. LINC01088 may also interfere with the DNA repair function of NPM1 by affecting its translocation. Our results highlight the potential of LINC01088 as a prognostic biomarker and therapeutic target of ESCC.

Inhibiting CBX4 efficiently protects hepatocellular carcinoma cells against sorafenib resistance.

BACKGROUND: This study aimed to investigate the possible role of inhibiting chromobox protein homologue 4 (CBX4) to deregulate of cancer stem cells (CSCs) and to evaluate the contribution of these molecules to sorafenib resistance in advanced hepatocellular carcinoma (HCC). METHODS: HCC cell lines and a xenograft mouse model with resistance to sorafenib were employed to analyse the effects of miR424 on CSC characteristics. RNA expression was analysed by RT-PCR and next-generation sequencing in a cohort of HCC cancer patients and sorafenib-resistant (SR) cell lines, respectively, to validate the key microRNAs and targets in the network. RESULTS: MicroRNA and mRNA profiles of SR cell lines identified miR424 and its direct target CBX4 as significantly associated with stem-cell-like properties, poor survival, and clinical characteristics. Functional experiments demonstrated that miR424 suppressed CBX4 and CBX4 induced nuclear translocation of YAP1 protein but was not associated with protein production. When YAP1 and CBX4 were modulated with CA3 and UNC3866, tumorigenicity and stem-like properties were extremely inhibited, thus indicating that these compounds exerted a strong anti-tumour effect in vivo against SR HCC cells. CONCLUSIONS: Our results revealed that blocking CBX4 expression is critical in response to sorafenib resistance with advanced HCC.

Let-7d inhibits intratumoral macrophage M2 polarization and subsequent tumor angiogenesis by targeting IL-13 and IL-10.

The microRNA let-7d has been reported to be a tumor suppressor in renal cell carcinoma (RCC). Tumor-associated macrophages (TAM) are M2-polarized macrophages that can enhance tumor growth and angiogenesis in many human cancers. However, the role of let-7d in TAM-associated RCC progression remains elusive. First, we observed a strongly inverse correlation between let-7d expression and microvessel density in RCC tissues. Furthermore, the proliferation, migration, and tube formation of HUVECs were significantly inhibited by conditioned medium from a coculture system of the phorbol myristate acetate pretreated human THP-1 macrophages and let-7d-overexpressing RCC cells. Moreover, the proportion of M2 macrophages was significantly lower in the group that was cocultured with let-7d-overexpressing RCC cells. Subcutaneous xenografts formed by the injection of let-7d-overexpressing RCC cells together with THP-1 cells resulted in a significant decrease in the M2 macrophage ratio and microvessel density compared with those formed by the injection of control RCC cells with THP-1 cells. In silico and experimental analysis revealed interleukin-10 (IL-10) and IL-13 as let-7d target genes. Importantly, the addition of IL-10 and IL-13 counteracted the inhibitory effects of the conditioned medium from the coculture system with let-7d-overexpressing RCC cells in vitro. Additionally, overexpression of IL-10 and IL-13 reversed the effects of let-7d on macrophage M2 polarization and tumor angiogenesis in vivo. Finally, the expression of IL-10 and IL-13 were inversely correlated with the expression of let-7d in RCC clinical specimens. These results suggest that let-7d may inhibit intratumoral macrophage M2 polarization and subsequent tumor angiogenesis by targeting IL-10 and IL-13.

IncRNA ZNF667-AS1 Suppresses Epithelial Mesenchymal Transformation by Targeting TGF-ß1 in Oral Squamous Cell Carcinoma.

BACKGROUND: IncRNAs perform complex functions and play an essential role in all stages of tumor progression. However, there are few studies that discuss the function of lncRNA ZNF667-AS1in oral squamous cell carcinoma (OSCC). This study aimed at analyzing the expression and biological behavior of lncRNA ZNF667-AS1 in OSCC. METHODS: IncRNA ZNF667-AS1 expression level in OSCC tissues and cell lines was explored by real-time PCR. The role of lncRNA ZNF667-AS1 on prognosis was elucidated. Cell proliferation assay, plate colony formation assay, wound-healing assay, and transwell migration assay were used to detect cell proliferation ability, cell clone formation ability, migration ability, and invasion ability, respectively. The effect of lncRNA ZNF667-AS1 on epithelial mesenchymal transformation (EMT) process was evaluated by western blot and real-time PCR. RESULTS: The expression levels of lncRNA ZNF667-AS1 were decreased in malignant tumor tissues. The OSCC patients with high expression of lncRNA ZNF667-AS1 had a longer survival time. IncRNA ZNF667-AS1 inhibited cell proliferation, cell clone formation ability, invasion and migration. Furthermore, lncRNA ZNF667-AS1 could inhibit the EMT process by suppressing transforming growth factor-ß-1 (TGF-ß1) expression, and TGF-ß1 treatment could partially restore the inhibitory effect. CONCLUSIONS: IncRNA ZNF667-AS1 may act as an antioncogene inhibiting the ability of proliferation, cell clone formation, invasion and migration, and suppress the process of EMT by targeting TGF-ß1. IncRNA ZNF667-AS1 could be a potential therapeutic target and a new predictive biological marker of OSCC.

All-trans retinoic acid (ATRA) inhibits insufficient radiofrequency ablation (IRFA)-induced enrichment of tumor-initiating cells in hepatocellular carcinoma.

OBJECTIVE: Local recurrence of hepatocellular carcinoma (HCC) after radiofrequency ablation (RFA) treatment remains a serious problem. Tumor-initiating cells (TICs) are thought to be responsible for tumor relapse. Here, we investigated the effect of the TIC differentiation inducer, all-trans retinoic acid (ATRA), on RFA and explored the potential molecular mechanisms. METHODS: The proportions of CD133+ and epithelial cell adhesion molecule (EpCAM)+ TICs in recurrent HCC after RFA and primary HCC were first determined in clinic. Then, the effect of heat intervention or insufficient RFA (IRFA) on the malignant potential of HCC cells, including cell migration, sphere formation ability, tumor growth, the proportion of CD133+ and EpCAM+ TICs and expression of stem cell-related genes, was evaluated in vitro andin vivo. Finally, the effect of ATRA on the tumor growth and the proportion of TICs was evaluated. RESULTS: In clinical data, a higher proportion of CD133+ and EpCAM+ TICs was found in recurrent tumors than in primary tumors. In vitro heat intervention promoted the cell migration and sphere formation ability. Additionally, it increased the proportion of CD133+ and EpCAM+ TICs and the expression of stem cell-related genes. In addition, after IRFA the residual tumors in xenografts grew faster and had more TICs than untreated tumors. ATRA remarkably inhibited residual tumor growth after IRFA by elimination of TICs though the PI3K/AKT pathway. Combination treatment with ATRA resulted in longer survival outcomes in mouse xenografts than RFA alone. CONCLUSIONS: ATRA, as a TIC differentiation inducer, could help to improve the effect of RFA treatment, which was partially attributed to its effect against TICs. The data indicated its potential as an alternative drug in the development of better therapeutic strategies for use in combination with RFA.

MiR-21 promotes calcium oxalate-induced renal tubular cell injury by targeting PPARA.

Kidney stone disease is a crystal concretion formed in the kidneys that has been associated with an increased risk of chronic kidney disease. MicroRNAs are functionally involved in kidney injury. Data mining using a microRNA array database suggested that miR-21 may be associated with calcium oxalate monohydrate (COM)-induced renal tubular cell injury. Here, we confirmed that COM exposure significantly upregulated miR-21 expression, inhibited proliferation, promoted apoptosis, and caused lipid accumulation in an immortalized renal tubular cell line (HK-2). Moreover, inhibition of miR-21 enhanced proliferation and decreased apoptosis and lipid accumulation in HK-2 cells upon COM exposure. In a glyoxylate-induced mouse model of renal calcium oxalate deposition, increased miR-21 expression, lipid accumulation, and kidney injury were also observed. In silico analysis and subsequent experimental validation confirmed the peroxisome proliferator-activated receptor (PPAR)-α gene (PPARA) a key gene in fatty acid oxidation, as a direct miR-21 target. Suppression of miR-21 by miRNA antagomiR or activation of PPAR-α by its selective agonist fenofibrate significantly reduced renal lipid accumulation and protected against renal injury in vivo. In addition, miR-21 was significantly increased in urine samples from patients with calcium oxalate renal stones compared with healthy volunteers. In situ hybridization of biopsy samples from patients with nephrocalcinosis revealed that miR-21 was also significantly upregulated compared with normal kidney tissues from patients with renal cell carcinoma who underwent radical nephrectomy. These results suggested that miR-21 promoted calcium oxalate-induced renal tubular cell injury by targeting PPARA, indicating that miR-21 could be a potential therapeutic target and biomarker for nephrolithiasis.

High levels of immunoglobulin expression predict shorter overall survival in patients with acute myeloid leukemia.

OBJECTIVES: It has been believed that immunoglobulins can only be produced by B lymphocytes and plasma cells. We have previously reported that IgG can be expressed in myeloblasts from patients with acute myeloid leukemia (AML) and plays a role in the proliferation and apoptosis of leukemic cells. However, its clinical impact has not been assessed. METHODS: We assessed the expression of different classes of immunoglobulin in peripheral blood and bone marrow samples from 132 AML patients and correlated the levels of expression with clinicopathologic and molecular genetic features, as well as clinical outcome. RESULTS: We found that, in addition to IgG, all classes of immunoglobulin are expressed in myeloblasts, including IgG, IgM, IgA, IgD, IgE, Igκ, and Igλ. The levels of IgG expression (coupled with Igκ or Igλ) are higher than those of IgM, IgA, IgD, and IgE. Using receiver operating characteristic (ROC) curve analysis, we identified two distinct groups of AML patients with differential expression of immunoglobulin and different clinical outcomes. CONCLUSIONS: High levels of immunoglobulin expression are associated with monocytic differentiation, multilineage dysplasia, TET2 and KRAS mutations, and poor overall survival. Assessment of immunoglobulin may serve as a useful marker for prognostic stratification and target therapy.

MicroRNA-130a-3p suppresses cell migration and invasion by inhibition of TBL1XR1-mediated EMT in human gastric carcinoma.

MiR-130a-3p was found to play tumor suppressor role in most human cancers, except for gastric cancer. However, in this study, we demonstrated that miR-130a-3p was significantly down-regulated in gastric carcinoma (GC) tissues compared with adjacent non-neoplastic tissues, and decreased miR-130a-3p expression was associated with shorter overall survival (OS) and was an independent prognostic factor for OS in GC patients. Over-expression of miR-130a-3p remarkably inhibited not only GC cell migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro, but also tumorigenesis and lung metastasis in the chick embryo chorioallantoic membrane (CAM) assay in vivo. Conversely, inhibition of miR-130a-3p resulted in opposite phenotype changes in GC cells. Furthermore, TBL1XR1 was identified as a direct target of miR-130a-3p, and reintroduction of TBL1XR1 into miR-130a-3p-transfected MGC-803 cells reversed the inhibitory effects of miR-130a-3p on GC cell migration, invasion and EMT. Taken together, our data suggested that miR-130a-3p suppressed aggressive phenotype of GC cells partially by direct targeting and decreasing TBL1XR1 and subsequent EMT process.

AZGP1 inhibits soft tissue sarcoma cells invasion and migration.

BACKGROUND: One of the major challenges in soft tissue sarcomas is to identify factors that predict metastasis. AZGP1 is a potential biomarker of cancer progression, but its value in soft tissue sarcomas remains unknown. The aim of this study is to determine the expression level of AZGP1 in soft tissue sarcomas, and to analyze its influence on tumor progression. METHODS: AZGP1 immunohistochemistry (IHC) and RT-PCR were performed in 86 patients with soft tissue sarcomas. The relationships between AZGP1 levels and clinicopathologic features were analyzed. In vitro experiments were performed using fibrosarcoma (HT1080), rhabdomyosarcoma (RD) and synovial sarcoma (SW982) cell lines to corroborate our findings. We used lentiviral over-expression and knockdown assays to examine how changes of AZGP1 expressions might affect cellular migration and invasion. RESULTS: The quantitative RT-PCR results showed that AZGP1 expression was negatively correlated with metastasis and overall survival in soft tissue sarcomas (p < 0.05). Immunohistochemical staining showed lower expression of AZGP1 in patients with metastasis than in those without. Kaplan-Meier survival analysis showed that patients with low expression of AZGP1 had shorter overall (p = 0.056) and metastasis-free survivals (p = 0.038). These findings were corroborated by our in vitro experiments. Over-expression of AZGP1 significantly decreased RD cellular migration and invasion by 64% and 78%, respectively. HT1080 cells migration was inhibited by 2-fold, whereas their invasion was repressed by 7-fold after AZGP1 knockdown. CONCLUSIONS: Our study reveals that reduced AZGP1 expression correlates with in vitro cellular migration and invasion. In vivo, it is associated with higher metastatic risk and shorter survival in patients with soft tissue sarcomas.

Inhibition of growth and metastasis of triple-negative breast cancer targeted by Traditional Chinese Medicine Tubeimu in orthotopic mice models.

OBJECTIVE: Triple-negative breast cancer (TNBC) is highly invasive and metastatic, which is in urgent need of transformative therapeutics. Tubeimu (TBM), the rhizome of Bolbostemma paniculatum (Maxim.) Franquet, is one of the Chinese medicinal herbs used for breast diseases since the ancient times. The present study evaluated the efficacy, especially the anti-metastatic effects of the dichloromethane extract of Tubeimu (ETBM) on TNBC orthotopic mouse models and cell lines. METHODS: We applied real-time imaging on florescent orthotopic TNBC mice model and tested cell migration and invasion abilities with MDA-MB-231 cell line. Digital gene expression sequencing was performed and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis applied to explore the pathways influenced by ETBM. Moreover, quantitative real-time polymerase chain reactions (qRT-PCR) and Western blot were delivered to confirm the gene expression changes. RESULTS: ETBM exhibited noticeable control on tumor metastasis and growth of TNBC tumors with no obvious toxicity. In compliance with this, it also showed inhibition of cell migration and invasion in vitro. Its impact on the changed biological behavior in TNBC may be a result of decreased expression of integrin ß1 (ITGß1), integrin ß8 (ITGß8) and Rho GTPase activating protein 5 (ARHGAP5), which disabled the focal adhesion pathway and caused change in cell morphology. CONCLUSIONS: This study reveals that ETBM has anti-metastatic effects on MDA-MB-231-GFP tumor and may lead to a new therapeutic agent for the integrative treatment of highly invasive TNBC.

Tumor-penetrating Peptide-integrated Thermally Sensitive Liposomal Doxorubicin Enhances Efficacy of Radiofrequency Ablation in Liver Tumors.

Purpose To investigate the role of a tumor-penetrating peptide (internalizing CRGDRGPDC [iRGD])-integrated thermally sensitive liposomal (TSL) doxorubicin (DOX) in combination with radiofrequency (RF) ablation of liver tumors in an animal model. Materials and Methods Approval from the institutional animal care and use committee was obtained. Characterization of iRGD-TSL-DOX was performed in vitro. Next, H22 liver adenocarcinomas were implanted in 138 mice in vivo. The DOX accumulation and cell apoptosis of iRGD-TSL-DOX and TSL-DOX with or without RF were evaluated (n = 5) at different time points after treatment with quantitative analysis or pathologic staining. Mice bearing tumors were randomized into the following six groups (each group, eight mice): no treatment, iRGD-TSL-DOX, TSL-DOX, RF alone, RF ablation followed by TSL-DOX at 30 minutes (TSL-DOX combined with RF), and RF ablation followed by iRGD-TSL-DOX (iRGD-TSL-DOX combined with RF). Kaplan-Meier method was used to estimate the survival curves and log-rank test was used for comparison with statistical software. Results DOX encapsulation efficiency in iRGD-TSL-DOX was 97.5% ± 1.3 (standard deviation) with temperature-dependent drug release capability confirmed in vitro. In vivo, the iRGD-TSL-DOX group had overall higher DOX concentration in the tumor and had maximal difference at 24 hours compared with TSL-DOX group (2.7-fold). RF caused more intense cell apoptosis at 24 hours (median, 65% vs 21%, respectively; P < .001). For end-point survival, the iRGD-TSL-DOX combined with RF group had better survival (median, 32 days) than TSL-DOX combined with RF (median, 27 days; P = .035) or RF alone (median, 21 days; P < .001). Conclusion Conjugation to iRGD helped to improve intratumoral DOX accumulation and further enhanced the activity of TSL-DOX in RF ablation of liver tumors. © RSNA, 2017 Online supplemental material is available for this article.

Does Thermosensitive Liposomal Vinorelbine Improve End-Point Survival after Percutaneous Radiofrequency Ablation of Liver Tumors in a Mouse Model?

Purpose To investigate the role of thermosensitive liposome-encapsulated vinorelbine (Thermo-Vin) in combined radiofrequency (RF) ablation of liver tumors. Materials and Methods Approval from the institutional animal care and use committee was obtained before this study. First, the anticancer efficacy of Thermo-Vin was assessed in vitro (H22 cells) for 72 hours at 37°C or 42°C. Next, 203 H22 liver adenocarcinomas were implanted in 191 mice for in vivo study. Tumors were randomized into seven groups: (a) no treatment, (b) treatment with RF ablation alone, (c) treatment with RF ablation followed by free vinorelbine (Free-Vin) at 30 minutes, (d) treatment with RF ablation followed by empty liposomes (Empty-Lip+RF), (e) treatment with RF ablation followed by Thermo-Vin (5 mg/kg), (f) treatment with RF ablation followed by Thermo-Vin (10 mg/kg), and (g) treatment with RF ablation followed by Thermo-Vin (20 mg/kg). Tumor destruction areas and pathologic changes were compared for different groups at 24 and 72 hours after treatment. Kaplan-Meier analysis was used to compare end-point survival (tumor < 30 mm in diameter). Additionally, the effect of initial tumor size on long-term outcome was analyzed. Results In vitro, both Free-Vin and Thermo-Vin dramatically inhibited H22 cell viability at 24 hours. Likewise, in vivo, 10 mg/kg Thermo-Vin+RF ablation increased tumor destruction compared with RF ablation (P = .001). Intratumoral vinorelbine accumulation with Thermo-Vin+RF increased 15-fold compared with Free-Vin alone. Thermo-Vin substantially increased apoptosis at the coagulation margin and suppressed cellular proliferation in the residual tumor (P < .001). The Thermo-Vin+RF study arm also had better survival than the arm treated with RF ablation alone (mean, 37.6 days ± 20.1 vs 23.4 days ± 5.0; P = .001), the arm treated with Free-Vin+RF (23.3 days ± 1.2, P = .002), or the arm treated with Empty-Lip+RF (20.8 days ± 0.4, P < .001) in animals with medium-sized (10-12-mm) tumors. No significant difference in end-point survival was noted in the treatment arms with large or small tumors. Conclusion Thermo-Vin can effectively increase tumor destruction and improve animal survival. End-point survival is most affected in animals with medium-sized tumors, suggesting that combination therapy should be tailored to tumor size and the expected volume of ablation of the device used. (©) RSNA, 2016 Online supplemental material is available for this article.

Copy number amplification-induced overexpression of lncRNA LOC101927668 facilitates colorectal cancer progression by recruiting hnRNPD to disrupt RBM47/p53/p21 signaling.

BACKGROUND: Somatic copy number alterations (SCNAs) are pivotal in cancer progression and patient prognosis. Dysregulated long non-coding RNAs (lncRNAs), modulated by SCNAs, significantly impact tumorigenesis, including colorectal cancer (CRC). Nonetheless, the functional significance of lncRNAs induced by SCNAs in CRC remains largely unexplored. METHODS: The dysregulated lncRNA LOC101927668, induced by copy number amplification, was identified through comprehensive bioinformatic analyses utilizing multidimensional data. Subsequent in situ hybridization was employed to ascertain the subcellular localization of LOC101927668, and gain- and loss-of-function experiments were conducted to elucidate its role in CRC progression. The downstream targets and signaling pathway influenced by LOC101927668 were identified and validated through a comprehensive approach, encompassing RNA sequencing, RT-qPCR, Western blot analysis, dual-luciferase reporter assay, evaluation of mRNA and protein degradation, and rescue experiments. Analysis of AU-rich elements (AREs) within the mRNA 3' untranslated region (UTR) of the downstream target, along with exploration of putative ARE-binding proteins, was conducted. RNA pull-down, mass spectrometry, RNA immunoprecipitation, and dual-luciferase reporter assays were employed to elucidate potential interacting proteins of LOC101927668 and further delineate the regulatory mechanism between LOC101927668 and its downstream target. Moreover, subcutaneous xenograft and orthotopic liver xenograft tumor models were utilized to evaluate the in vivo impact of LOC101927668 on CRC cells and investigate its correlation with downstream targets. RESULTS: Significantly overexpressed LOC101927668, driven by chr7p22.3-p14.3 amplification, was markedly correlated with unfavorable clinical outcomes in our CRC patient cohort, as well as in TCGA and GEO datasets. Moreover, we demonstrated that enforced expression of LOC101927668 significantly enhanced cell proliferation, migration, and invasion, while its depletion impeded these processes in a p53-dependent manner. Mechanistically, nucleus-localized LOC101927668 recruited hnRNPD and translocated to the cytoplasm, accelerating the destabilization of RBM47 mRNA, a transcription factor of p53. As a nucleocytoplasmic shuttling protein, hnRNPD mediated RBM47 destabilization by binding to the ARE motif within RBM47 3'UTR, thereby suppressing the p53 signaling pathway and facilitating CRC progression. CONCLUSIONS: The overexpression of LOC101927668, driven by SCNAs, facilitates CRC proliferation and metastasis by recruiting hnRNPD, thus perturbing the RBM47/p53/p21 signaling pathway. These findings underscore the pivotal roles of LOC101927668 and highlight its therapeutic potential in anti-CRC interventions.

Let-7c functions as a metastasis suppressor by targeting MMP11 and PBX3 in colorectal cancer.

Accumulating evidence shows that microRNAs, functioning as either oncogenes or tumour suppressors by negatively regulating downstream target genes that are actively involved in tumour initiation and progression, may be promising biomarkers and therapy targets. Data mining through a microRNA chip database indicated that let-7c may be associated with tumour metastasis. Here, we confirmed that down-regulation of let-7c in primary cancer tissues was significantly associated with metastases, advanced TNM stages and poor survival of colorectal cancer patients. Moreover, ectopic expression of let-7c in a highly metastatic Lovo cell line remarkably suppressed cell migration and invasion in vitro by the down-regulation of K-RAS, MMP11 and PBX3, as well as tumour growth and metastases in vivo, whereas inhibition of let-7c in low-metastatic HT29 cells increased cell motility and invasion by the enhanced gene expression of K-RAS, MMP11 and PBX3. Interestingly, the luciferase reporters' activities with the 3'-UTRs of K-RAS, MMP11 and PBX3 were inhibited significantly by let-7c. Importantly, rescue experiments involving the over-expression of these genes without their 3'-UTRs completely reversed the effects of let-7c on tumour metastasis, both in vitro and in vivo. Finally, the levels of let-7c were inversely correlated with those of MMP11 and PBX3, but not with those of K-RAS. Taken together, these results demonstrate that let-7c, apart from its tumour growth suppression role, also functions as a tumour metastasis suppressor in colorectal cancer by directly destabilizing the mRNAs of MMP11 and PBX3 at least.

Targeting SALL4 by Entinostat Inhibits the Malignant Phenotype of Gastric Cancer Cells by Reducing EMT Signaling.

BACKGROUND/AIM: Spalt-like transcription factor 4 (SALL4), as a proto-oncogene, is expressed in various tumors and correlates with poor prognosis of patients. Entinostat, a histone deacetylase (HDAC) inhibitor, has emerged as a potentially promising anti-cancer drug. This study aims to explore the biological role and underlying mechanism of SALL4 targeting by entinostat in gastric cancer. MATERIALS AND METHODS: Online databases were used to exam the link between SALL4 and prognosis. We tested the biological roles of SALL4 in gastric cancer cells. Cell viability and growth were analyzed using the Cell Counting Kit-8 (CCK-8) assay and clone formation assay. Cell migration was assessed using the wound healing assay. The effects of entinostat on gastric cancer cell lines were measured by the CCK-8 assay, clone formation assay, wound healing assay and transwell assay. Epithelial-mesenchymal transition (EMT) signaling pathways were detected by western blot. RESULTS: SALL4 expression was upregulated in gastric cancer tissues and positively correlated with tumor stage and prognosis of patients by TCGA dataset analysis. Knockdown of SALL4 by siRNA inhibited the proliferation and migration of gastric cancer cells. In contrast, SALL4 overexpression by stably transfecting a SALL4-expressing plasmid promoted the proliferation and invasiveness of gastric cancer cells in vitro through alteration of EMT-related genes. In addition, entinostat, a HDAC inhibitor targeting SALL4, could suppress the proliferation, migration, and invasion of gastric cancer cells via regulating expression of EMT-associated proteins. CONCLUSION: SALL4 may be a new therapeutic target for the treatment of gastric cancer, and entinostat is a potential novel agent for the treatment of gastric cancer partially by targeting SALL4.

Long Non-Coding RNA LOC339059 Attenuates IL-6/STAT3-Signaling-Mediated PDL1 Expression and Macrophage M2 Polarization by Interacting with c-Myc in Gastric Cancer.

Background: Long non-coding RNA (lncRNA) was identified as a novel diagnostic biomarker in gastric cancer (GC). However, the functions of lncRNAs in immuno-microenvironments have not been comprehensively explored. In this study, we explored a critical lncRNA, LOC339059, that can predict the clinical prognosis in GC related to the modulation of PD-L1 and determined its influence upon macrophage polarization via the IL-6/STAT3 pathway. Methods: To date, accumulating evidence has demonstrated that the dysregulation of LOC339059 plays an important role in the pathological processes of GC. It acts as a tumor suppressor, regulating GC cell proliferation, migration, invasion, tumorigenesis, and metastasis. A flow cytometry assay showed that the loss of LOC339059 enhanced PDL1 expression and M2 macrophage polarization. RNA sequencing, RNA pull-down, RNA immunoprecipitation, Chip-PCR, and a luciferase reporter assay revealed the pivotal role of signaling alternation between LOC339059 and c-Myc. Results: A lower level of LOC339059 RNA was found in primary GC tissues compared to adjacent tissues, and such a lower level is associated with a poorer survival period (2.5 years) after surgery in patient cohorts. Moreover, we determined important immunological molecular biomarkers. We found that LOC339059 expression was correlated with PD-L1, CTLA4, CD206, and CD204, but not with TIM3, FOXP3, CD3, C33, CD64, or CD80, in a total of 146 GC RNA samples. The gain of LOC339059 in SGC7901 and AGS inhibited biological characteristics of malignancy, such as proliferation, migration, invasion, tumorigenesis, and metastasis. Furthermore, our data gathered following the co-culture of THP-1 and U937 with genomic GC cells indicate that LOC339059 led to a reduction in the macrophage cell ratio, in terms of CD68+/CD206+, to 1/6, whereas the selective knockdown of LOC339059 promoted the abovementioned malignant cell phenotypes, suggesting that it has a tumor-suppressing role in GC. RNA-Seq analyses showed that the gain of LOC339059 repressed the expression of the interleukin family, especially IL-6/STAT3 signaling. The rescue of IL-6 in LOC339059-overexpressing cells reverted the inhibitory effects of the gain of LOC339059 on malignant cell phenotypes. Our experiments verified that the interaction between LOC339059 and c-Myc resulted in less c-Myc binding to the IL-6 promoter, leading to the inactivation of IL-6 transcription. Conclusions: Our results establish that LOC339059 acts as a tumor suppressor in GC by competitively inhibiting c-Myc, resulting in diminished IL-6/STAT3-signaling-mediated PDL1 expression and macrophage M2 polarization.

HDGF promotes gefitinib resistance by activating the PI3K/AKT and MEK/ERK signaling pathways in non-small cell lung cancer.

Hepatoma-derived growth factor (HDGF) expression is associated with poor prognosis in non-small cell lung cancer (NSCLC); however, whether HDGF affects gefitinib resistance in NSCLC remains unknown. This study aimed to explore the role of HDGF in gefitinib resistance in NSCLC and to discover the underlying mechanisms. Stable HDGF knockout or overexpression cell lines were generated to perform experiments in vitro and in vivo. HDGF concentrations were determined using an ELISA kit. HDGF overexpression exacerbated the malignant phenotype of NSCLC cells, while HDGF knockdown exerted the opposite effects. Furthermore, PC-9 cells, which were initially gefitinib-sensitive, became resistant to gefitinib treatment after HDGF overexpression, whereas HDGF knockdown enhanced gefitinib sensitivity in H1975 cells, which were initially gefitinib-resistant. Higher levels of HDGF in plasma or tumor tissue also indicated gefitinib resistance. The effects of HDGF on promoting the gefitinib resistance were largely attenuated by MK2206 (Akt inhibitor) or U0126 (ERK inhibitor). Mechanistically, gefitinib treatment provoked HDGF expression and activated the Akt and ERK pathways, which were independent of EGFR phosphorylation. In summary, HDGF contributes to gefitinib resistance by activating the Akt and ERK signaling pathways. The higher HDGF levels may predict poor efficacy for TKI treatment, thus it has the potential to serve as a new target for overcoming tyrosine kinase inhibitor resistance in combating NSCLC.

A Novel Germline Deletion of p.C630 in RET Causes MTC and Promotes Cell Proliferation and Sensitivity to Pralsetinib.

CONTEXT: Medullary thyroid cancer (MTC) is usually caused by gain-of-function mutations in the proto-oncogene RET. OBJECTIVE: This study aimed to determine the underlying mechanism in a male patient diagnosed with MTC at age 51 years. METHODS: Genomic DNA extracted from leukocytes or tumor tissues of patients was used for next-generation sequencing (NGS)-panel sequencing and Sanger sequencing. Wild-type (WT) and p.C630 deletion RET were expressed in HEK 293T cells. Activation of phosphorylation of the crucial tyrosine-905 of RET and MAPK/ERK was analyzed by Western blotting. The effect of RET mutants on cell viability and colony formation ability was determined by CCK8 assay and a colony forming assay. RESULTS: NGS-Panel sequencing revealed a 3-nucleotide/1-amino acid C630 in-frame deletion in exon 11 of RET (c.1887_1889delGTG p.C630del). In vitro expression showed that phosphorylation of the crucial tyrosine 905 was much stronger in the p.C630del RET mutant than in WT RET, indicating ligand-independent activation of the Ret protein tyrosine kinase. Furthermore, p.C630del RET mutant induced strong activation of the MAPK/ERK pathway. In addition, p.C630del RET mutant cells exhibited increased HEK 293T cell viability and colony formation compared with WT RET cells. Pralsetinib (BLU-667), a highly selective RET inhibitor, inhibited the viability of WT RET and p.C630del RET mutant-transfected HEK 293T cells (IC50s: 18.54 and 16.49 µM after treatment for 24 hours), followed by inhibition of the RET-induced MAPK/ERK pathway. CONCLUSION: The finding in our patient with MTC was a 3-base-pair deletion in exon 11 of RET, a p.C630 deletion not previously reported. The p.C630del RET stimulates cell proliferation by increasing ligand-independent phosphorylation and activation of MAPK/ERK pathway, demonstrating the pathogenic nature of the mutation. We therefore recommend screening panel sequence of RET in MTC patients with indications of a genetic cause.