RNA Editing Enzyme ADAR1 Regulates METTL3 in an Editing Dependent Manner to Promote Breast Cancer Progression via METTL3/ARHGAP5/YTHDF1 Axis.

A-to-I RNA editing and m6A modification are two of the most prevalent types of RNA modifications controlling gene expression in mammals and play very important roles in tumorigenesis and tumor progression. However, the functional roles and correlations of these two RNA modifications remain to be further investigated in cancer. Herein, we show that ADAR1, an A-to-I RNA-editing enzyme, interacts with METTL3 and increases its protein level to promote the proliferation, migration and invasion of breast cancer cells through a mechanism connecting ADAR1, METTL3 and YTHDF1. We show that both ADAR1 and METTL3 are upregulated in breast cancer samples, and ADAR1 positively correlates with METTL3; ADAR1 edits METTL3 mRNA and changes its binding site to miR532-5p, leading to increased METTL3 protein, which further targets ARHGAP5, recognized by YTHDF1. Additionally, we show that loss of ADAR1 significantly inhibits breast cancer growth in vivo. Collectively, our findings identify the ADAR1-METTL3 axis as a novel, important pathway that connects A-to-I editing and m6A RNA modifications during breast cancer progression.

MsrB1 Promotes Proliferation and Invasion of Colorectal Cancer Cells via GSK-3ß/ß-catenin Signaling Axis.

Methionine sulfoxide reductase B1 (MsrB1) can catalyze both free and protein-bound R-methionine sulfoxides (R-MetO) to methionine (Met). It has been reported that MsrB1 plays an important role in the development of HCC and human bone osteosarcoma. However, little is known about the functions of MsrB1 in human colorectal cancer (CRC). Herein, we detected MsrB1 expression level in CRC tissue and cell lines, and investigated the effect of MsrB1 knockdown on CRC phenotypes and possible mechanisms involved in. The results showed that MsrB1 was highly expressed in both CRC tissues and cell lines, and that cell proliferation, migration and invasion were significantly inhibited, but apoptosis was increased after MsrB1 knockdown in colorectal cancer HCT116 and RKO cell lines, compared to control siRNA group. In addition, E-cadherin protein level was increased, vimentin and Snail protein were greatly decreased after knockdown of MsrB1 in cells. Furthermore, pGSK-3ß (Ser9) and ß-catenin protein levels were reduced, the promoter activity of TCF/LEF construction was inhibited after MsrB1 knockdown in cells, suggesting that GSK-3ß/ß-catenin signaling axis was involved in the tumorigenesis of CRC. In conclusion, the oncogenic role and related mechanisms of MsrB1 in CRC discovered in our work determined the potential role of MsrB1 as a biomarker and may provide a new target for clinical therapy of CRC.

Krüppel-like factor 8 regulates VEGFA expression and angiogenesis in hepatocellular carcinoma.

Tumor angiogenesis plays a critical role in hepatocellular carcinoma (HCC) development and progression, but its mechanism is unclear. Krüppel-like factor 8 (KLF8) is a transcription factor that plays an important role in HCC progression. Here, we investigated the role of KLF8 in angiogenesis in HCC and its possible mechanism. Immunohistochemistry, quantitative RT-PCR, western blotting, promoter reporter assays, chromatin immunoprecipitation (ChIP), and chicken chorioallantoic membrane (CAM) and nude mouse tumor models were used to show that the mRNA and protein expression levels of KLF8 and VEGFA are highly correlated in HCC tissue samples. The up-regulation of KLF8 increased VEGFA protein levels and induced VEGFA promoter activity by binding to the CACCC region of the VEGFA promoter. In addition, KLF8 regulated HIF-1α and Focal adhesion kinase (FAK) expression. The PI3K/AKT inhibitor LY294002 inhibited KLF8-induced VEGFA expression, whereas PI3K/AKT signaling pathway proteins, such as P-PDK1(Ser241) and P-AKT(Thr308), were decreased significantly. KLF8-overexpressing HCC cells had a higher potential for inducing angiogenesis. Thus, our results indicate that KLF8 may induce angiogenesis in HCC by binding to the CACCC region of the VEGFA promoter to induce VEGFA promoter activity and through FAK to activate PI3K/AKT signaling to regulate HIF-1α expression levels.

MSX1 induces G0/G1 arrest and apoptosis by suppressing Notch signaling and is frequently methylated in cervical cancer.

PURPOSE: The objectives of this study were to investigate the expression of MSX1 in cervical cells and tissues, the methylation status of the MSX1 promoter, the influence of overexpression of gene MSX1 on the proliferation, migration, and invasion of HeLa and SiHa cells, and finally the possible molecular mechanisms responsible for the suppressive effects of MSX1 upon cervical cancer cells. PATIENTS AND METHODS: Semi-quantitative and quantitative reverse transcription-polymerase chain reactions were used to investigate the expression levels of MSX1, and methylation-specific polymerase chain reaction (MSP) was performed to investigate promoter methylation status in cervical cancer cell lines, primary cervical tissues, and normal cervical tissues. Clone formation, Cell Counting Kit-8 (CCK-8), cell wound scratch, and transwell assays were performed to verify whether MSX1 could inhibit the proliferation and migration of cervical cancer cells. Western blot was used to analyze the effect of MSX1 upon Notch1, Jagged1, c-Myc, cleaved PARP, cleaved caspse-3, and cyclin D1 (CCND1). RESULTS: MSX1 was frequently downregulated or silenced in 60.0% (3/5) of cervical cancer cell lines. The promoter methylation of MSX1 was detected in 42.0% (42/100) of primary tumor tissues, while no methylation was observed in normal cervical tissues. Pharmacological demethylation reduced MSX1 promoter methylation levels and restored the expression of MSX1. The overexpression of MSX1 in cervical cancer cells thus inhibited the proliferation and migration of cervical cancer cells. The overexpression of MSX1 in cervical cancer cells downregulated the expression levels of Notch1, Jagged1, and c-Myc but upregulated the expression levels of CCND1, cleaved PARP, and cleaved caspase-3. CONCLUSION: MSX1 appears to be a functional tumor suppressor that regulates tumorigenesis in cervical cancer by antagonizing Notch signaling.

KLF8 promotes tumorigenesis, invasion and metastasis of colorectal cancer cells by transcriptional activation of FHL2.

The transcription factor Krüppel-like factor (KLF)8 plays an important role in the formation of several human tumors, including colorectal cancer. We recently identified four-and-a-half LIM protein 2 (FHL2) as a critical inducer of the epithelial-to-mesenchymal transition (EMT) and invasion. However, the molecular mechanism by which KLF8 affects FHL2-mediated tumor proliferation, EMT and metastasis remains unknown. Here, we showed that KLF8 overexpression promoted EMT and metastatic phenotypes. KLF8 expression was stimulated by transforming growth factor (TGF)-ß1. Moreover, KLF8 acted as a potential EMT inducer by stimulating vimentin expression and inducing a loss of E-cadherin in stable KLF8-transfected cells. KLF8 overexpression induced a strong increase in FHL2 expression, and a positive correlation between the expression patterns of KLF8 and FHL2 was observed in CRC cells. Promoter reporter and chromatin immunoprecipitation (ChIP) assays demonstrated that KLF8 directly bound to and activated the human FHL2 gene promoter. However, siRNA-mediated repression of FHL2 in KLF8-overexpressing cells reversed the EMT and the proliferative and metastatic phenotypes. In vivo, KLF8 promoted FHL2-mediated proliferation and metastasis via orthotopic implantation. Taken together, this work identified KLF8-induced FHL2 activation as a novel and critical signaling mechanism underlying human breast/colorectal cancer invasion and metastasis.

Vasodilator-stimulated phosphoprotein promotes activation of hepatic stellate cells by regulating Rab11-dependent plasma membrane targeting of transforming growth factor beta receptors.

UNLABELLED: Liver microenvironment is a critical determinant for development and progression of liver metastasis. Under transforming growth factor beta (TGF-ß) stimulation, hepatic stellate cells (HSCs), which are liver-specific pericytes, transdifferentiate into tumor-associated myofibroblasts that promote tumor implantation (TI) and growth in the liver. However, the regulation of this HSC activation process remains poorly understood. In this study, we tested whether vasodilator-stimulated phosphoprotein (VASP) of HSCs regulated the TGF-ß-mediated HSC activation process and tumor growth. In both an experimental liver metastasis mouse model and cancer patients, colorectal cancer cells reaching liver sinusoids induced up-regulation of VASP and alpha-smooth muscle actin (α-SMA) in adjacent HSCs. VASP knockdown in HSCs inhibited TGF-ß-mediated myofibroblastic activation of HSCs, TI, and growth in mice. Mechanistically, VASP formed protein complexes with TGF-ß receptor II (TßRII) and Rab11, a Ras-like small GTPase and key regulator of recycling endosomes. VASP knockdown impaired Rab11 activity and Rab11-dependent targeting of TßRII to the plasma membrane, thereby desensitizing HSCs to TGF-ß1 stimulation. CONCLUSIONS: Our study demonstrates a requirement of VASP for TGF-ß-mediated HSC activation in the tumor microenvironment by regulating Rab11-dependent recycling of TßRII to the plasma membrane. VASP and its effector, Rab11, in the tumor microenvironment thus present therapeutic targets for reducing TI and metastatic growth in the liver.

Sphingosine-1-phosphate mediates a reciprocal signaling pathway between stellate cells and cancer cells that promotes pancreatic cancer growth.

Sphingosine-1-phosphate (S1P) is produced by sphingosine kinase 1 and is implicated in tumor growth, although the mechanisms remain incompletely understood. Pancreatic stellate cells (PSCs) reside within the tumor microenvironment and may regulate tumor progression. We hypothesized that S1P activates PSCs to release paracrine factors, which, in turn, increase cancer cell invasion and growth. We used a combination of human tissue, in vitro, and in vivo studies to mechanistically evaluate this concept. Sphingosine kinase 1 was overexpressed in human pancreatic tissue, especially within tumor cells. S1P activated PSCs in vitro and conditioned medium from S1P-stimulated PSCs, increased pancreatic cancer cell migration, and invasion, which was dependent on S1P2, ABL1 (alias c-Abl) kinase, and matrix metalloproteinase-9. In vivo studies showed that pancreatic cancer cells co-implanted with S1P2 receptor knockdown PSCs led to less cancer growth and metastasis in s.c. and orthotopic pancreatic cancer models compared with control PSCs. Pancreatic cancer cell-derived S1P activates PSCs to release paracrine factors, including matrix metalloproteinase-9, which reciprocally promotes tumor cell migration and invasion in vitro and cancer growth in vivo.

PDGF receptor-α promotes TGF-ß signaling in hepatic stellate cells via transcriptional and posttranscriptional regulation of TGF-ß receptors.

Platelet-derived growth factor (PDGF) and transforming growth factor-ß (TGF-ß) signaling are required for hepatic stellate cell (HSC) activation under pathological conditions such as liver metastatic tumor growth. These two signaling pathways are functionally divergent; PDGF signaling promotes proliferation and migration of HSCs, and TGF-ß induces transdifferentiation of quiescent HSCs into myofibroblasts. Although PDGF signaling is implicated in TGF-ß-mediated epithelial mesenchymal transition of tumor cells, the role of PDGF receptors in TGF-ß activation of HSCs has not been investigated. Here we report that PDGF receptor-α (PDGFR-α) is required for TGF-ß signaling of cultured human HSCs although HSCs express both PDGF-α and -ß receptors. PDGFR-α knockdown inhibits TGF-ß-induced phosphorylation and nuclear accumulation of SMAD2 with no influence on AKT or ERK phosphorylation associated with noncanonical TGF-ß signaling. PDGFR-α knockdown suppresses TGF-ß receptor I (TßRI) but increases TßRII gene transcription. At the protein level, PDGFR-α is recruited to TßRI/TßRII complexes by TGF-ß stimulation. PDGFR-α knockdown blocks TGF-ß-mediated internalization of TßRII and induces accumulation of TßRII at the plasma membrane, thereby inhibiting TGF-ß phosphorylation of SMAD2. Functionally, knockdown of PDGFR-α reduces paracrine effects of HSCs on colorectal cancer cell proliferation and migration in vitro. In mice and patients, colorectal cancer cell invasion of the liver induces upregulation of PDGFR-α of HSCs. In summary, our finding that PDGFR-α knockdown inhibits SMAD-dependent TGF-ß signaling by repressing TßRI transcriptionally and blocking endocytosis of TGF-ß receptors highlights a convergence of PDGF and TGF-ß signaling for HSC activation and PDGFR-α as a therapeutic target for liver metastasis and other settings of HSC activation.

Up-regulation of Krüppel-like factor 8 promotes tumor invasion and indicates poor prognosis for hepatocellular carcinoma.

BACKGROUND & AIMS: The transcription factor Krüppel-like factor 8 (KLF8) has a role in tumor development, growth, and metastasis, but its role in hepatocellular carcinoma (HCC) is not clear. METHODS: KLF8 expression in human HCC cell lines and tumor tissues was measured by quantitative real-time polymerase chain reaction, immunoblot, and immunochemical analyses. The effects of KLF8 depletion or overexpression in HCC cells were observed in cultured cells and in mice. Changes in gene expression patterns in HCC cells in which levels of KLF8 were reduced using small interfering RNA were investigated by microarray analysis. The clinical significance of KLF8 expression levels were validated using tissue microarray analysis of surgical samples from 314 HCC patients. RESULTS: KLF8 was overexpressed in highly metastatic HCC cell lines and in samples from patients with recurrent HCC. In cultured cells, KLF8 up-regulation promoted cell proliferation and invasion; inhibited apoptosis; down-regulated N-cadherin, vimentin, and fibronectin; and up-regulated E-cadherin. In mice, overexpression of KLF8 increased HCC progression and metastasis. Microarray analysis showed that reduction of KLF8 in HCC cells down-regulated expression of multiple genes involved in tumor progression and metastasis. KLF8 expression was a significant predictor of overall survival (P = .040) and time to HCC recurrence (P = .006) and was associated with early tumor recurrence (P = .001). CONCLUSIONS: KLF8 promotes HCC cell proliferation and invasion, inhibits apoptosis, and induces the epithelial-to-mesenchymal transition. KLF8 up-regulation might be used to indicate poor prognosis or early recurrence of cancer in patients who have had surgery for HCC.

Small interference RNA targeting Krüppel-like factor 8 inhibits the renal carcinoma 786-0 cells growth in vitro and in vivo.

PURPOSE: Krüppel-like factor 8 (KLF8) plays an important role in oncogenic transformation and is highly overexpressed in several types of human cancer. We investigated the expression of KLF8 in renal cell carcinoma (RCC) tissues and the role of small interference RNA targeting KLF8 on growth, cell cycle, and apoptosis of human renal carcinoma cell line 786-0 in vitro and in vivo. METHODS: The expression of KLF8 protein and mRNA in human renal carcinoma samples was detected by immunochemistry and reverse transcription polymerase chain reaction (RT-PCR). The effects of small interference RNA (siRNA) targeting KLF8 on growth, invasiveness, cell cycle, and apoptosis of 786-0 cells were evaluated by MTT assay, Matrigel Invasion Assay, and flow cytometry in vitro. We also investigated effect of siRNA targeting KLF8 on growth of 786-0 cells in nude mice in vivo. RESULTS: Immunohistochemistry and RT-PCR results showed the expression of KLF8 protein and mRNA in RCC specimens was significantly higher than that in the adjacent non-tumorous renal tissues (P < 0.001). KLF8-siRNA depressed the cellular growth and invasion of 786-0 cells in vitro. The flow cytometry results revealed that KLF8-siRNA could induce an increase in G0/G1 phase cells and induce cell apoptosis. Intratumor injection of siRNA targeting KLF8 inhibited the growth of 786-0 cells in vivo in nude mice tumor model. CONCLUSIONS: KLF8 possibly involved in regulating the cell growth, invasion, apoptosis, and proliferation of renal carcinoma cancer cells. Blocking the KLF8 channel might be a potential therapeutic strategy for RCC.

Upregulation of GRP78 in renal cell carcinoma and its significance.

OBJECTIVES: To investigate the expression of GRP78 in human renal cell carcinoma (RCC) and its significance. METHODS: We studied RNA and tissue section of a tumor and adjacent nontumorous renal tissues obtained from radical nephrectomy specimens of 42 patients and RCC cell lines. We used reverse transcriptase-PCR and immunohistochemistry to detect the GRP78 mRNA and protein expression, respectively. RESULTS: Reverse transcriptase-PCR revealed that GRP78 mRNA is positively expressed in RCC cell lines (786-0, OS-RC-2, and Caki-1); the GRP78 mRNA expression in the RCC and adjacent nontumorous renal tissues was 0.88 +/- 0.34 and 0.44 +/- 0.15, respectively (P < .001). The GRP78 protein was found in RCC cell lines. Immunohistochemistry results also showed that the level of GRP78 protein expression of RCC tissues was significantly higher than that of the adjacent nontumorous renal tissues (P < .001). The high levels of GRP78 mRNA and protein expression were related to the large tumor size and high clinical stage (P < .001) but not to sex, age, and cell differentiate (P > .05). CONCLUSIONS: To our knowledge, the present study is the first to report the upregulated expression of GRP78 and that it is possibly involved in pathogenesis of RCC.

CD24 is a novel predictor for poor prognosis of hepatocellular carcinoma after surgery.

PURPOSE: To investigate the role of CD24 in tumor invasion and prognostic significance in hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN: CD24 expression was measured in stepwise metastatic HCC cell lines, tumor, peritumoral tissues, and normal liver tissues by quantitative real-time PCR and Western blot. The role of CD24 in HCC was investigated by CD24 depletion using small interfering RNA. Tumor tissue microarrays of 314 HCC patients who underwent resection between 1997 and 2000 were used to detect expression of CD24, beta-catenin, and proliferating cell nuclear antigen. Prognostic significance was assessed using Kaplan-Meier survival estimates and log-rank tests. RESULTS: CD24 was overexpressed in the highly metastatic HCC cell line and in tumor tissues of patients with recurrent HCC. Depletion of CD24 caused a notable decrease in cell proliferation, migration, and invasiveness in vitro. Univariate and multivariate analyses revealed that CD24 was a significant predictor for overall survival and relapse-free survival. CD24 expression was correlated with poor prognosis independent of alpha-fetoprotein, tumor-node-metastasis stage, and Edmondson stage. High CD24 expression was significantly associated with cytoplasmic and nuclear accumulation of beta-catenin (P = 0.023), high tumor proliferative status (P = 0.018), and diffused intrahepatic recurrence and distant metastasis (P = 0.026). Adjuvant transcatheter arterial chemoembolization after surgery reduced the rate of early recurrence (

Role of overexpression of CD151 and/or c-Met in predicting prognosis of hepatocellular carcinoma.

UNLABELLED: It has been reported that tetraspanin CD151 acts as a promoter of metastasis in several tumors and plays an important role in c-Met/hepatocyte growth factor signaling. However, the role of CD151 alone and coexpression of CD151/c-Met in hepatocellular carcinoma (HCC) remains unclear. We found that expression of CD151 was positively related to metastatic potential of HCC cell lines, and modified cells with CD151(high) showed higher secretion of matrix metalloproteinase 9 and aggressiveness in vitro and higher metastatic ability in vivo. Furthermore, HCC patients with vascular invasion, large tumors, multiple tumors, high tumor-node-metastasis stage, and undifferentiated tumor were prone to have higher CD151 expression. The postoperative 3-, 5-, and 7-year overall survival (OS) of patients in HCCs with CD151(high) were significantly lower than those in the CD151(low) group, and correspondingly cumulative recurrence rates in HCCs with CD151(high) were significantly higher than those in the CD151(low) group. Both CD151 and c-Met were remarkably overexpressed in HCCs, compared with adjacent nontumorous and normal liver tissues. Pearson correlation analysis showed a slight correlation between CD151 and c-Met in HCCs. Importantly, the 5- and 7-year OS rates in CD151(high)/c-Met(high) patients were 50.5% and 37.8%, respectively, significantly lower than those of CD151(low)/c-Met(low) patients (63.9% and 54.6%, respectively). Five- and 7-year cumulative recurrence rates in CD151(high)/c-Met(high) patients were 53.3% and 71.9%, respectively, markedly higher than those of CD151(low)/c-Met(low) patients (39.0% and 52.5%, respectively). Multivariate analysis revealed that CD151 and combination of CD151/c-Met were independent prognostic indicators for OS and cumulative recurrence. CONCLUSION: CD151 is positively associated with invasiveness of HCC, and CD151 or combination of CD151/c-Met is a novel marker in predicting the prognosis of HCC and a potential therapeutic target.