Purpose:To evaluate the efficacy and safety of hepatic artery infusion chemotherapy with oxaliplatin, fluorouracil, and leucovorin (FOLFOX-HAIC) combined with donafenib and camrelizumab in patients with unresectable hepatocellular carcinoma (uHCC). Patients and Methods:This retrospective study analyzed clinical data from 49 uHCC patients treated with FOLFOX-HAIC, donafenib tablets, and camrelizumab at the First Affiliated Hospital of Chongqing Medical University between November 1, 2021, and November 30, 2024. The primary endpoint was the objective response rate (ORR), assessed according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 and modified RECIST (mRECIST). Secondary endpoints included surgical conversion rate, disease control rate (DCR), progression-free survival (PFS), overall survival (OS), duration of response (DOR), and safety. Results:The median follow-up duration was 20.3 months. Patients had a mean age of 55.9 years and a mean tumor diameter of 9.9 cm, with 25 patients (51%) presenting with extrahepatic metastasis. According to RECIST v1.1, the ORR was 57.1%, and the DCR was 79.6%. Under mRECIST criteria, the ORR increased to 59.2%, while the DCR remained at 79.6%. The median PFS was 12.1 months, and the median OS was 26.0 months. Twelve patients (24.5%) achieved successful conversion, with five patients (10.2%) undergoing surgery achieving R0 resections. Two patients (4.1%) achieved a pathological complete response (pCR). All patients experienced treatment-related adverse events (TRAEs), with Hypoalbuminemia (87.8%) and Hand-foot skin reaction (79.6%) being the most common. Grade 3 or higher TRAEs were observed in 61.2% of patients. Conclusion:For patients with uHCC, the combination therapy of FOLFOX-HAIC with donafenib and camrelizumab demonstrates favorable efficacy and manageable adverse events.

e16134 Background: Rational and effective treatment strategies for patients with unresectable hepatocellular carcinoma (uHCC) presenting portal vein tumor thrombus (PVTT) are lacking, and hepatic artery infusion chemotherapy (HAIC) combined with tyrosine kinase inhibitors and immune checkpoint inhibitors could be an effective therapy. Herein, we evaluated the efficacy and safety of HAIC combined with donafenib and camrelizumab in patients with uHCC presenting PVTT. Methods: This prospective, single-arm, single-center study enrolled patients with uHCC presenting PVTT (distant metastases were allowed), BCLC stage C, Child-Pugh scores ≤ 7, ECOG PS ≤1, and no previous local or systemic treatment. Patients received mFOLFOX6-HAIC (every 3 weeks, no more than 6 cycles), followed by peroral donafenib (200 mg BID every day) and intravenous camrelizumab (200 mg every 3 weeks). The primary endpoint was the objective response rate (ORR; per RECIST1.1 and mRECIST). Secondary endpoints included surgical conversion rate, disease control rate, progression-free survival (PFS), overall survival (OS), duration of response, and safety. Results: Between November 2021 and September 2022, we enrolled 15 eligible patients with a median age of 53 years (range, 36-76), including 12 (80%) males. Considering enrolled patients, 14 (93.3%) were HBV+, 13(86.7%) presented with PVTT invasion into the main portal vein, Child-Pugh scores 5/6/7: 9/5/1, ECOG PS 0/1: 10/5, median tumor size was 8.4 cm (range, 5.2-13.6), and 5 (33.3%) had extrahepatic metastases. The median number of HAIC procedures was 5 (range, 1-6). The median follow-up time was 338.0 days (95% CI, 298.7-377.4). Considering the 15 evaluable patients (completed at least one cycle of treatment), the ORR was 66.7% (10/15) according to RECIST 1.1, with 0 complete response (CR) and 10 partial responses (PR). According to mRECIST, the ORR was 73.3% (11/15), with 2 CR and 9 PR. Three patients (20%) became eligible for surgical resection, with one undergoing surgical resection (two refused surgery for financial reasons). Median PFS and OS were insufficient. All 15 (100%) patients experienced treatment-related adverse events (TRAEs). Common TRAEs included hand-and-foot skin reaction (HFSR; 93.3%), abdominal pain (73.3%), hypoalbuminemia (60.0%), platelet count decreased (60.0%), nausea (60%), aspartate aminotransferase increased (53.3%), and vomiting (53.3%). Grade 3/4 TRAEs included platelet count decreased (13.3%), HFSR (13.3%), hepatic function abnormal (6.7%), and ascites (6.7%). No grade 5 TRAEs were observed. TRAEs led to drug reduction in 8 patients (53.3%). Conclusions: HAIC combined with donafenib and camrelizumab afforded promising efficacy and safety in patients with uHCC presenting PVTT. Longer follow-up is required for further evaluation. Clinical trial information: ChiCTR2100051714 .

Supplementary Data from CD24 Is a Novel Predictor for Poor Prognosis of Hepatocellular Carcinoma after Surgery

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.

放射治疗是恶性肿瘤最重要的治疗手段之一,放射肿瘤科住院医师规范化培训(规培)尤其重要.我国的放射肿瘤科住院医师规培到目前取得了很大的成绩,很多方面能和美国毕业医学教育认证委员会(ACGME)的要求趋于一致.与美国放射肿瘤科住院医师培养制度相比,我国放射肿瘤科住院医师规培项目起步较晚,某些方面如培训时间的长度,培训内容的广度、深度上仍存在较大的差距.该文将美国ACGME对放射肿瘤科住院医师培训的要求与我国《住院医师规范化培训内容与标准(试行)放射肿瘤科培训细则》内容进行对比分析,并结合我国目前放射肿瘤科住院医师规培实际提出思考与建议,有助于进一步完善细化我国的培训制度,提高住院医师规培质量,达到同质化,从而更好地服务于我国广大肿瘤患者.

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-3β (Ser9) and β-catenin protein levels were reduced, the promoter activity of TCF/LEF construction was inhibited after MsrB1 knockdown in cells, suggesting that GSK-3β/β-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.

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.

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.

目的:探讨重组人血管内皮抑素(recombinant human endostatin,rh-ES)联合化放疗治疗晚期恶性肿瘤的疗效及安全性.方法:回顾性分析我科2010年1月至2013年8月收治的27例晚期恶性肿瘤患者病例,27例患者均接受rh-ES联合化疗±放疗的综合治疗,rh-ES用药1周期后评价生活质量变化及不良反应,2周期后评价疗效.结果:23例可评价近期疗效的患者客观有效率30.4％,疾病控制率78.3％.18例非小细胞肺癌(non-small cell lung cancer,NSCLC)中位无进展生存期5.5月,中位总生存期12.0月,1年生存率50.0％,2年生存率28.6％.全组患者生活质量改善或稳定23例(85.2％),Ⅲ～Ⅳ度不良反应8例次.结论:Rh-ES联合化放疗治疗晚期恶性肿瘤的疗效及安全性良好,可改善或稳定患者生活质量.与其他单纯化疗研究结果比较,晚期NSCLC的中位无进展生存期及1年生存率较高,值得临床推广.

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)-β1. 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.

Abstract Introduction: Liver microenvironment is a critical determinant for development and progression of liver metastasis. Under TGF-β stimulation, hepatic stellate cells (HSCs), which are liver specific pericytes, transdifferentiate into myofibroblasts that promote tumor implantation and growth in the liver. The regulation of this HSC activation process in the liver however remains poorly understood. In this study, we tested a hypothesis that actin binding protein vasodilator-stimulated phosphoprotein (VASP), previously implicated in focal adhesion and migration of HSCs, may regulate the TGF-β mediated HSC activation process by interacting with and regulating TGF-β receptors. Methods: VASP shRNA and siRNA were used to knockdown VASP in primary human HSCs and LX2, an immortalized HSC cell line. TGF-β activation of HSCs was quantitated by immunofluorescence staining (IF) for alpha-smooth muscle actin (α-SMA) and Western blot analysis (WB) for α-SMA, fibronectin and P-SMAD2. Total TGF-β receptor levels were determined by WB, and plasma membrane TβRII was quantitated by biotinylation of cell surface proteins approach. Protein subcellular localization was investigated by IF and protein interactions were determined by double IF and co-immunoprecipitation (IP). Regulation of stellate cell VASP by HSC/tumor interactions was investigated by IF on human colorectal liver metastases and the role of stellate cell VASP in tumor growth was studied in a tumor/HSC coimplantation mouse model. Results: TGF-β1 induced more than 60% of human primary HSCs into myofibroblasts as determined by α-SMA IF. Two different VASP shRNAs significantly reduced this effect of TGF-β1 on HSC activation (P<0.05). The effect of VASP knockdown on HSC activation was confirmed in LX2 cells as well as using a VASP siRNA approach. VASP knockdown increased total TGF receptor protein levels with no effects on SMAD2 and 3 protein levels. VASP interacted with TβRII as evidenced by double IF and co-IP. Biotin-labeling of cell surface proteins and IF revealed that VASP knockdown reduced TβRII protein level at the plasma membrane and that VASP overexpression increased TβRII at the plasma membrane. Concurrently, VASP knockdown reduced plasma membrane localization of Rab11a, a small GTPase that marks recycling endosomes and is essential for recycling of plasma membrane receptors. VASP/Rab11 interactions were confirmed by IF and co-IP. Furthermore, we found that VASP knockdown HSCs significantly reduced tumor growth in mice as compared to control HSCs in a HSC/tumor coimplantation model. In patients, metastasis of colorectal cancer cells in liver sinusoids induced upregulation of VASP and α-SMA in adjacent HSCs as revealed by IF on biopsies of human colorectal liver metastases. Conclusions: Our studies demonstrate a requirement of VASP in the TGF-β dependent HSC activation in the tumor microenvironment and ensuing tumor growth. VASP interacts with Rab11 and regulates Rab11 positive recycling endosomal function thereby promoting TβRII recycling back to the plasma membrane in HSCs. VASP in the tumor microenvironment thus presents a therapeutic target for reducing tumor implantation and metastatic growth in the liver. Citation Format: Kangsheng Tu, Jiachu Li, Chunsheng Liu, Vijay H. Shah, Ningling Kang. Vasodilator-stimulated phosphoprotein promotes TGF-beta mediated myofibroblastic activation by regulating recycling of TGF-beta receptor II to the plasma membrane. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A70. doi:10.1158/1538-7445.CHTME14-A70

Liver microenvironment is a critical determinant for development and progression of liver metastasis. Under transforming growth factor beta (TGF‐β) 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‐β‐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‐β‐mediated myofibroblastic activation of HSCs, TI, and growth in mice. Mechanistically, VASP formed protein complexes with TGF‐β receptor II (TβRII) and Rab11, a Ras‐like small GTPase and key regulator of recycling endosomes. VASP knockdown impaired Rab11 activity and Rab11‐dependent targeting of TβRII to the plasma membrane, thereby desensitizing HSCs to TGF‐β1 stimulation. Conclusions: Our study demonstrates a requirement of VASP for TGF‐β‐mediated HSC activation in the tumor microenvironment by regulating Rab11‐dependent recycling of TβRII 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. (Hepatology 2015;61:361–374)

Platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β) 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-β induces transdifferentiation of quiescent HSCs into myofibroblasts. Although PDGF signaling is implicated in TGF-β-mediated epithelial mesenchymal transition of tumor cells, the role of PDGF receptors in TGF-β activation of HSCs has not been investigated. Here we report that PDGF receptor-α (PDGFR-α) is required for TGF-β signaling of cultured human HSCs although HSCs express both PDGF-α and -β receptors. PDGFR-α knockdown inhibits TGF-β-induced phosphorylation and nuclear accumulation of SMAD2 with no influence on AKT or ERK phosphorylation associated with noncanonical TGF-β signaling. PDGFR-α knockdown suppresses TGF-β receptor I (TβRI) but increases TβRII gene transcription. At the protein level, PDGFR-α is recruited to TβRI/TβRII complexes by TGF-β stimulation. PDGFR-α knockdown blocks TGF-β-mediated internalization of TβRII and induces accumulation of TβRII at the plasma membrane, thereby inhibiting TGF-β 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-β signaling by repressing TβRI transcriptionally and blocking endocytosis of TGF-β receptors highlights a convergence of PDGF and TGF-β signaling for HSC activation and PDGFR-α as a therapeutic target for liver metastasis and other settings of HSC activation.

Objective:To investigate the regulating effect of Krüppel-like transcription factor 8 (KLF8) on the expression of vascular endothelial growth factor A (VEGFA) in hepatocellular carcinoma (HCC) and its potential mechanism.Methods:The expressions of KLF8 and VEGFA proteins and their correlation in paired HCC and adjacent paracancerous tissues were detected by immunohistochemistry staining.The KLF8 expression plasmid pcDNA3.1-KLF8 and the control plasmid were transfected into HCC SMMC7721 cells,respectively.The expressions of VEGFA and phosphoinositide-3-kinase (PI3K)/Akt pathway-related molecules including phospho-Akt (p-Akt),phospho-phosphoinositide-dependent kinase 1 (p-PDK1) and phospho-phosphatase and tensin homolog deleted on chromosome 10 (p-PTEN) in SMMC7721 cells were detected by real-time fluorescent quantitative PCR and Western blotting.The effect of PI3K/Akt pathway inhibitor LY294002 on the expression of VEGFA protein in SMMC7721 cells with KLF8 overexpression was detected by Western blotting.Chicken chorioallantoic membrane (CAM) model was used to detect the inducing effect of SMMC7721 cells with up-regulation of KLF8 expression on angiogenesis in chicken embryos.Results:The expression levels of KLF8 and VEGFA proteins were much higher in HCC tissues than those in the adjacent paracancerous tissues (P ＜ 0.01).KLF8 protein expression was positively correlated with VEGFA protein expression in HCC tissues (r =0.622,P ＜ 0.01).The expression levels of KLF8,VEGFA,p-Akt,p-PDK1 and p-PTEN proteins were up-regulated in SMMC7721 cells transfected with pcDNA3.1-KLF8 plasmids (P ＜ 0.05).LY294002 inhibited VEGFA expression in SMMC7721 cells with KLF8 overexpression (P ＜ 0.05).The SMMC7721 cells with KLF8 overexpression had a higher potential in inducing angiogenesis in chick embryos (P ＜ 0.01).Conclusion:KLF8 gene induces VEGFA expression and angiogenesis in HCC via PI3K/Akt signal pathway.

BACKGROUND Primary neuroendocrine tumors of the gallbladder (GB-NET) are uncommon and rare.Due to their rarity, there is limited data on its clinicopathologic characteristics.Also, the data on survival outcomes and the effect of cancer directed therapies for these tumors are limited.The aim of this study was to analyze the survival outcomes and its predictors in GB-NET.METHODS We retrospectively reviewed the Surveillance Epidemiology and End Result data (SEER) that captures tumor data from 18 geographically distinct cancer registries of the United States.Patients with microscopically confirmed GB-NET diagnosed between 1988 and 2010 were included.Patients with a second different primary tumor and unknown tumor stage were excluded.Clinicopathologic variables of the tumor were analyzed and compared using Wilcoxon Rank Sum test for ordinal variable and Chi-Square test for categorical variables.The primary end point of the study was disease specific survival (DSS).Univariate and multivariate Cox proportional hazards modeling were used to analyze the effect of co-variates on DSS.The DSS was derived using Kaplan-Meier method.All associations were considered statistically significant at an alpha error <0.01 (p value 0.01).RESULTS There were 267 patients who met initial inclusion criteria.The median age of diagnosis was 67 years.The male to female ratio (M: F) was 1:3.Of the 267 patients, 106 (40%) had localized tumor stage, 59 (22%) had loco-regional disease and 102 (38%) had metastatic disease.Based on grade of GB-NET, 22 patients had well and moderately differentiated tumor and 102 patients had high grade tumor.In the univariate DSS model, high grade tumor predicted poor outcome (HR=5.51,95%CI: 2.39 -12.66, p=<0.001).Radical surgical resection when compared to simple cholecystectomy as the cancer directed surgery, failed to show improved survival outcome (HR=1.29,95%CI: 0.66-2.52,p=0.46).The 3 year DSS rate for GB-NET was 80% for localized, 28% for loco-regional and 9% for metastatic tumor stages.Also, the 3 year DSS rate was 82% for well and moderately differentiated tumor and 13% for high grade tumors.CONCLUSION In GB-NET, grade and extent of disease highly correlates with outcomes.The clinical and biologic characteristics should be correlated with outcomes to help with planning treatment in this exceedingly rare neoplasm.

Platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) 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-beta induces transdifferentiation of quiescent HSCs into myofibroblasts. Although PDGF signaling is implicated in TGF-beta-mediated epithelial mesenchymal transition of tumor cells, the role of PDGF receptors in TGF-beta activation of HSCs has not been investigated. Here we report that PDGF receptor-alpha (PDGFR-alpha) is required for TGF-beta signaling of cultured human HSCs although HSCs express both PDGF-alpha and -beta receptors. PDGFR-alpha knockdown inhibits TGF-beta-induced phosphorylation and nuclear accumulation of SMAD2 with no influence on AKT or ERK phosphorylation associated with non-canonical TGF-beta signaling. PDGFR-alpha knockdown suppresses TGF-beta receptor I (T beta RI) but increases T beta RII gene transcription. At the protein level, PDGFR-alpha is recruited to T beta RI/T beta RII complexes by TGF-beta stimulation. PDGFR-alpha knockdown blocks TGF-beta-mediated internalization of T beta RII and induces accumulation of T beta RII at the plasma membrane, thereby inhibiting TGF-beta phosphorylation of SMAD2. Functionally, knockdown of PDGFR-alpha 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-alpha of HSCs. In summary, our finding that PDGFR-alpha knockdown inhibits SMAD-dependent TGF-beta signaling by repressing T beta RI transcriptionally and blocking endocytosis of TGF-beta receptors highlights a convergence of PDGF and TGF-beta signaling for HSC activation and PDGFR-alpha as a therapeutic target for liver metastasis and other settings of HSC activation.

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.

Objective:To construct Krüppel-like factor 8(KLF8)expression plasmid pcDNA3.1-KLF8 and KLF8 interference plasmid pGPU6/GFP/Neo-KLF8 and to investigate role of KLF8 in regulating angiogenesis related factors in SMMC7721.Methods:KLF8 expression plasmid pcDNA3.1-KLF8 and KLF8 interference plasmid pGPU6/GFP/Neo-KLF8 targeting four interference sites were constructed and then were screened for the most effective one.Real-time quantitative PCR and Western blot were used to confirm the effects on regulating KLF8 levels.pcDNA3.1-KLF8,pcDNA3.1,pGPU6/GFP/Neo-KLF8 and pGPU6/GFP/Neo-ShNC plasmid were transfected into human SMMC7721 cells separately.mRNA levels of hypoxia-inducible factor(HIF)1-α,vascular endothelial growth factor(VEGF),angiopoietin 1(Ang1),angiopoietin 2(Ang2),angiopoietin receptor Tie2,matrix metallo-proteinase(MMP)2 were investigated by qPCR.Results:Sequencing showed that KLF8 eukaryotic expression plasmids pcDNA3.1-KLF8 was successfully constructed and pcDNA3.1-KLF8 significantly up-regulated KLF8 expressions(relative expression:68.8±6.5).The most efficient KLF8 interference plasmids pGPU6/GFP/Neo-KLF8 was obtained and pGPU6/GFP/Neo-KLF8 down-regulated KLF8 expressions(relative expression:0.31±0.01).Compared with those in control group,mRNA levels of VEGF,Ang2,HIF1-α and MMP2 were increased(P<0.05) while angiopoietin receptor Tie2 and Ang1 had no significant changes(P >0.05)in experiment group(transfected with pcDNA3.1-KLF8).Meanwhile,compared with those in control group(transfected with pGPU6/GFP/Neo-ShNC),mRNA levels of Ang2 and HIF1-α were decreased(P<0.05)while VEGF,angiopoietin receptor Tie2,MMP2 and Ang1 displayed no significant changes(P >0.05) in experiment group(transfected with pGPU6/GFP/Neo-KLF8).Conclusion:KLF8 may regulate angiogenesis in SMMC7721 by regulating several angiogenesis related factors such as VEGF,Ang2,HIF1-α and MMP2.