Nerve growth factor regulates CD133 function to promote tumor cell migration and invasion via activating ERK1/2 signaling in pancreatic cancer

BackgroundPerineural invasion (PNI) is extremely high frequency among the various metastatic routes in pancreatic cancer. Nerve growth factor, secreted by astroglial cells, exerts effects on tumor invasion in some cancer cells, but its function on migration and invasion in pancreatic cancer is still unclear. In the present study, we determined the effects of NGF on modulating tumor cell metastatic potential and invasion activity and explored its mechanisms in pancreatic cancer.MethodsNGF and CD133 expression were detected in tumor tissues using immunohistochemical analysis and Western blotting analysis. The effects of NGF on the regulation of CD133 expression and the promotion of cancer migration and invasion were investigated using wound healing and matrigel transwell assay. A related mechanism that NGF regulates CD133's function via activating ERK1/2 signaling also was observed.ResultsNGF/CD133 is overexpressed in human pancreatic cancer and promotes the migration and invasion of human pancreatic cancer cells through the activation of the ERK/CD133 signaling cascade. NGF/ERK signaling modulates the cancer cell EMT process, migration and invasion through the regulation of CD133 expression and its subcellular localization.ConclusionsNGF/CD133 signaling initiated the migration and invasion of pancreatic cancer cells. NGF/CD133 might be an effective and potent therapeutic target for pancreatic cancer metastasis, particularly in PNI.     

Midkine promotes perineural invasion in human pancreatic cancer

AIM:To investigate midkine(MK)and syndecan-3protein expression in pancreatic cancer by immunohistochemistry,and to analyze their correlation with clinicopathological features,perineural invasion,and prognosis.METHODS:Pancreatic cancer tissues(including adequately sized tumor tissue samples and tissue samples taken from areas less than 2.0 cm around the tumor)were taken from 42 patients who were undergoing a partial duodenopancreatectomy.MK and syndecan-3proteins were detected by immunohistochemistry using a standardized streptavidin-peroxidase method,and analyzed for their correlation with clinicopathological features,perineural invasion,and prognosis.Associations of neural invasion with aggressive characteristics of pancreatic cancer and the presence of perineural invasion were assessed by two independent observers blinded to the patient status.RESULTS:MK and syndecan-3 were found in 26(61.9%)and 24(57.1%)specimens,respectively.MK and syndecan-3 expression was associated with perineural invasion(P=0.018 and 0.031,respectively).High MK expression was closely associated with advanced tumor,node and metastasis stage(P=0.008),lymph node metastasis(P=0.042),and decreased postoperative survival at 3years(51.0%vs 21.8%,P=0.001).Syndecan-3 levels were correlated with tumor size(P=0.028).Patients who were syndecan-3 negative had a higher cumulative survival rate than those who were positive,but the difference was not significant(44.0%vs 23.0%,P>0.05).CONCLUSION:MK and syndecan-3 are frequently expressed in pancreatic cancer and associated with perineural invasion.High expression of MK and syndecan-3may contribute to the highly perineural invasion and poor prognosis of human pancreatic cancer.     

Involvement of Ataxin-3 (ATXN3) in the malignant progression of pancreatic cancer via deubiquitinating HDAC6

BackgroundPancreatic cancer is a common digestive system cancer and one of the most lethal malignancies worldwide. Ataxin-3 (ATXN3) protein is a deubiquitinating enzyme implicated in the occurrence of diverse human cancers. The potential role of ATXN3 in pancreatic cancer still remains unclear.MethodsATXN3 was screened from differentially-upregulated genes of GSE71989, GSE27890 and GSE40098 datasets. The mRNA and protein levels of ATXN3 was evaluated in pancreatic cancer samples and cell lines. Through the gain- and loss-of-function experiments, the effects of ATXN3 on cell proliferation, migration and invasion were evaluated using cell counting kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU) staining, wound healing and Transwell assays. Subsequently, the interaction between ATXN3 and HDAC6 was confirmed using double immunofluorescence staining, co-immunoprecipitation (co-IP) and proximity ligation assay (PLA). The underlying mechanism of ATXN3 was determined by knockdown of HDAC6 in ATXN3-upregulated pancreatic cancer cells. The function of ATXN3 in vivo was verified through xenograft assay.ResultsHigh expression of ATXN3 was found in pancreatic cancer tissues. Increased ATXN3 expression dramatically promoted cell proliferation, migration, and invasion. The malignant phenotypes were suppressed in ATXN3-silenced pancreatic cancer cells. ATXN3 was proved to interact with HDAC6 and regulate its degradation through deubiquitination. Downregulation of HDAC6 inhibited ATXN3-induced development of pancreatic cancer cells through regulating the expression of PCNA, vimentin and E-cadherin. ATXN3 facilitated tumor growth of pancreatic cancer and increased HDAC6 expression in vivo.ConclusionsThis study confirmed that ATXN3 facilitated malignant phenotypes of pancreatic cancer via reducing the ubiquitination of HDAC6.     

The HMGA1-COX-2 axis: A key molecular pathway and potential target in pancreatic adenocarcinoma

ContextAlthough pancreatic cancer is a common, highly lethal malignancy, the molecular events that enable precursor lesions to become invasive carcinoma remain unclear. We previously reported that the high-mobility group A1 (HMGA1) protein is overexpressed in >90% of primary pancreatic cancers, with absent or low levels in early precursor lesions.MethodsHere, we investigate the role of HMGA1 in reprogramming pancreatic epithelium into invasive cancer cells. We assessed oncogenic properties induced by HMGA1 in non-transformed pancreatic epithelial cells expressing activated K-RAS. We also explored the HMGA1-cyclooxygenase (COX-2) pathway in human pancreatic cancer cells and the therapeutic effects of COX-2 inhibitors in xenograft tumorigenesis.ResultsHMGA1 cooperates with activated K-RAS to induce migration, invasion, and anchorage-independent cell growth in a cell line derived from normal human pancreatic epithelium. Moreover, HMGA1 and COX-2 expression are positively correlated in pancreatic cancer cell lines (r² = 0.93; p < 0.001). HMGA1 binds directly to the COX-2 promoter at an AT-rich region in vivo in three pancreatic cancer cell lines. In addition, HMGA1 induces COX-2 expression in pancreatic epithelial cells, while knock-down of HMGA1 results in repression of COX-2 in pancreatic cancer cells. Strikingly, we also discovered that Sulindac (a COX-1/COX-2 inhibitor) or Celecoxib (a more specific COX-2 inhibitor) block xenograft tumorigenesis from pancreatic cancer cells expressing high levels of HMGA1.ConclusionsOur studies identify for the first time an important role for the HMGA1-COX-2 pathway in pancreatic cancer and suggest that targeting this pathway could be effective to treat, or even prevent, pancreatic cancer.     

Notch pathway activation is associated with pancreatic cancer treatment failure

BackgroundPancreatic cancer is resistant to conventional treatment. The aim of the study was to confirm the hypothesis that changes in cancer stem cells (CSCs) and developmental pathway after treatment was responsible for treatment failure in pancreatic cancer.MethodsAfter recovery from a gemcitabine treatment, the percentage of pancreatic cancer CSCs and Notch pathway in BxPC3 and HPAC pancreatic cancer cell lines were analyzed by FACS (CD24 and CD44) and western blot (Notch1, Hes1, β-catenin, and pAKT). The effect of DAPT, a gamma-secretase inhibitor, was similarly investigated. The association between immunohistochemical expression of Hes1 and survival was analyzed.ResultsThe percentage of CD24⁺CD44⁺ cells was higher in gemcitabine-treated BxPC3 and HPAC cells than at pre-treatment. CD24⁺CD44⁺ cells sorted from the gemcitabine-treated cell lines showed higher migration and invasion ability than CD24⁻CD44⁻ or CD24⁻CD44⁺ cells from the same cell lines. Western blot analysis showed an increased expression of Notch1 and Hes1 in gemcitabine-treated cell lines. The overall survival of pancreatic cancer patients with strong expression of Hes1 was shorter than that in patients with no or weak expression (11.1 vs. 21.6 months, P = 0.036). Treatment with DAPT reversed the increase in Hes1, β-catenin, and pAKT expression and the proportion of CD24⁺CD44⁺ cells in gemcitabine-treated cell lines. The treatment also decreased migration and invasion ability.ConclusionOur data suggested that an increase in CSCs and activation of the Notch pathway might contribute to the failure of treatment in pancreatic cancer. Notch pathway can be a potential target to overcome treatment failure.     

Pancreatic Stellate Cells Increase the Invasion of Human Pancreatic Cancer Cells through the Stromal Cell-Derived Factor-1/CXCR4 Axis

Aim: Both pancreatic stellate cells (PSCs) and the stromal cell-derived factor-1(SDF-1)/CXCR4 receptor ligand system have important roles in pancreatic cancer progression. This study set out to detect if PSCs express SDF-1 and promote the invasion of pancreatic cancer through the SDF-1/CXCR4 receptor ligand axis. Methods: RT-PCR was performed to detect the expression of SDF-1 and CXCR4 in PSCs, pancreatic cancer lines and cancer tissue samples. ELISA was used to investigate the concentration of SDF-1 in PSC supernatants. An MTT assay was applied to detect the proliferation of pancreatic cancer cells. A transwell chamber migration assay was employed to detect the migration of AsPC-1 cells. An in vitro invasion assay was used to detect the invasion of AsPC-1 cells. Results: CXCR4 expression was detected in PSCs; AsPC-1, SW1990 and BxPC-3 cancer cells; and cancer tissues. SDF-1 was detected in PSCs and cancer tissues, but not in AsPC-1, SW1990 and BxPC-3 cells. SDF-1α protein was found in PSC supernatants. PSC-conditioned media can promote the proliferation, migration and invasion of pancreatic cancer cells. SDF-1 neutralizing antibody or AMD3100 can significantly inhibit these promotive effects. Conclusion: PSCs can secrete SDF-1 and increase the invasion of pancreatic cancer cells through the SDF-1/CXCR4 axis.     

Expression of fibroblast activation protein in human pancreatic adenocarcinoma and its clinicopathological significance

AIM: To examine fibroblast activation protein (FAP) expression in pancreatic ductal adenocarcinoma (PDAC) and to analyze its relationship with the clinicopathology of PDAC.METHODS: FAP expression was examined in 134 PDAC specimens by immunohistochemistry, and in four pancreatic cancer cell lines (SW1990, Miapaca-2, AsPC-1 and BxPC-3) by Western blotting assay. We also analyzed the association between FAP expression in PDAC cells and the clinicopathology of PDAC patients.RESULTS: The results showed that the FAP was ex-pressed in both stromal fibroblast cells (98/134, 73.1%) and carcinoma cells (102/134, 76.1%). All 4 pancreatic cancer cell lines expressed FAP protein at different levels. Protein bands corresponding to the proteolytically active 170-kDa seprase dimer and its 88-kDa seprase subunit were identified. Higher FAP expression in carcinoma cells was associated with tumor size (P < 0.001), fibrotic focus (P = 0.003), perineural invasion (P = 0.009) and worse clinical outcome (P = 0.0085).CONCLUSION: FAP is highly expressed in carcinoma cells and fibroblasts in PDAC tissues, and its expression is associated with desmoplasia and worse prognosis.     

Mechanism of action of salinomycin on growth and migration in pancreatic cancer cell lines

ObjectivesPancreatic cancer is one of the most aggressive and lethal cancers worldwide and there are few effective treatments. Recently, salinomycin (Sal) was reported to alter proliferation and apoptosis in various tumors. This prompted us to investigate the effect of Sal on pancreatic cancer cells and to explore the possible molecular mechanism in vitro.MethodsAfter treatment with Sal, pancreatic cancer cell viability and apoptosis were analyzed by Hoechst 33342 staining and flow cytometry, respectively. Invasion and metastasis of pancreatic cancer cells were assayed by a Transwell migration assay. Flow cytometry was also used to assessed the fraction of CD133⁺ cell subpopulations. The expression of proliferating cell nuclear antigen (PCNA), Bcl-2, E-cadherin, and Wnt/β-catenin signaling-related proteins were detected by RT-PCR and western blot.ResultsSal inhibited the growth and migration of pancreatic cancer cells in vitro in a dose- and time-dependent manner. We found that the proportion of CD133⁺ cell subpopulations decreased after treatment with Sal in pancreatic cancer cell lines at the same time. The percentage of apoptotic cells was increased after Sal treatment. Compared with control groups, Bax and E-cadherin were significantly upregulated, and Bcl-2 and PCNA were significantly downregulated in Sal-treated cells. The expression of Wnt/β-catenin signaling-related proteins (β-catenin and p-GSK-3β) was inhibited.ConclusionsThese results indicate that Sal could influence the cell growth and migration in pancreatic cancer cells in vitro, which may occur by inhibition of Wnt/β-catenin signaling.     

WASF3 Knockdown Sensitizes Gastric Cancer Cells to Oxaliplatin by Inhibiting ATG12-Mediated Autophagy

BackgroundGastric cancer is one of the most aggressive tumors, usually resulting in metastasis, and therapies for advanced gastric cancer remain limited. Drug resistance is the main reason for chemotherapeutic failure in gastric cancer. Wiskott-Aldrich syndrome protein family member 3 (WASF3) is required for invasion and metastasis of different cancers. However, there has been little study of WASF3 expression involvement in gastric cancer. In this study, we explored the role of WASF3 in the sensitivity of gastric cancer to oxaliplatin, and the underlying mechanisms.MethodsWe silenced WASF3 using WASF3-siRNA in MGC803 cells. Then, CCK-8, flow cytometry and transwell assay were performed to study the effect of WASF3 silencing on proliferation, migration, invasiveness and apoptosis of MGC803 cells. Moreover, we evaluated the potential mechanism in vitro to determine the sensitization to oxaliplatin induced by WASF3.ResultsWASF3 silencing by small interfering RNA inhibited the proliferation, migration and invasiveness of gastric cancer cells. We also observed that WASF3 knockdown promoted cell apoptosis and enhanced oxaliplatin sensitivity. Furthermore, the sensitization to oxaliplatin induced by WASF3 knockdown depended on the inhibition of Atg12-mediated autophagy.ConclusionsOur analysis demonstrates WASF3 targeting is a new potential therapeutic strategy for gastric cancer.     

WNT5A/JNK signaling regulates pancreatic cancer cells migration by Phosphorylating Paxillin

BackgroundExpression of WNT5A associated with aggressive tumor biology and poor clinical outcome of various types of cancer. However its function in the metastasis property of pancreatic cells still needs to be elucidated.MethodsWe detected the expressions of WNT5A, JNK1/p-JNK1 and Paxillin/p-Paxillin in cancer and the para-carcinoma tissues of pancreatic cancer. To understand how WNT5A/JNK signaling affects pancreatic cancer cell migration through the phosphorylation of cellular substrates of Paxillin, In vitro, we knocked down the WNT5A in PANC1, Capan-2 and HT1080 cell lines, and then tested the expression of JNK1. We detected the proteins of phosphorylation of Paxillin after JNK1 was inhibited and then the cells migration assay was evaluated. Moreover, JNK1 functionally phosphorylates serine178 on paxillin in vitro was detected .At last we subsequently observed whether WNT5A/JNK signaling modulates some molecule expressions relevant to focal adhesion (FA) formation and mesenchymal transition (EMT) and cell cycle.ResultsWNT5A, p-JNK1 and p-Paxillin were highly expressed in early stage of tumor tissues. In vitro, WNT5A/JNK signaling promotes cell migration in pancreatic cancer by phosphorylating serine178 on Paxillin, an FA adaptor, which means WNT5A may regulate FA's function.WNT5A up-regulates the molecule's expressions relevant to cell adhesion through the phosphorylation of JNK1, including MMP1, MMP2, ICAM and CD44. In addition, WNT5A/JNK signaling promoted the mRNA expressions of vimentin, but decreased in E-Cadherin expression, which suggested its regulatory effects on the EMT processes. WNT5A/JNK signaling didn't modulate cell proliferation.ConclusionWNT5A/JNK signaling initiate cell migration of pancreatic cancer through activation of Paxillin, which suggested WNT5A has the potency of being an effective therapeutic target for the metastasis of pancreatic cancer.     

HOXA10 Promotes Cell Invasion and MMP-3 Expression Via TGFβ2-Mediated Activation of the p38 MAPK Pathway in Pancreatic Cancer Cells

Background

HOXA10 is closely related to tumor progression in many human cancers. However, the role of HOXA10 in pancreatic cancer remains unclear. The aim of this study was to determine the involvement of HOXA10 in pancreatic cancer cell invasion and migration.

Methods

The effect of HOXA10 on the invasion and migration of pancreatic cancer cells was assessed by invasion and migration assays. The protein of transforming growth factor beta-2 (TGFβ2) was neutralized by TGFβ2 blocking antibody. The activation of p38 was inhibited by SB239063.

Results

HOXA10 could promote the invasion and migration of pancreatic cancer cells. Knockdown of HOXA10 decreased the expressions of TGFβ2 and matrix metallopeptidase-3 (MMP-3) and suppressed the activation of p38. Conversely, overexpression of HOXA10 increased the levels of TGFβ2 and MMP-3. Further experiments identified that TGFβ2 contributed to the HOXA10-promoted invasion and migration and regulated MMP-3 expression and p38 activation. Additionally, inhibition of p38 suppressed cell invasion and MMP-3 expression in pancreatic cancer cells.

Conclusions

HOXA10 promotes cell invasion and MMP-3 expression of pancreatic cancer cells via TGFβ2-p38 MAPK pathway. Thus, HOXA10 could be a useful target for the treatment of pancreatic cancer.     

Par-4 activation restrains EMT-induced chemoresistance in PDAC by attenuating MDM-2

BackgroundWe recently reported prostate apoptosis response 4 (Par-4), a potential tumor suppressor protein restrains epithelial-mesenchymal transition (EMT) properties and promotes mesenchymal-epithelial transition (MET) in invasive cancer cells by repressing Twist-1 promoter activity. Here, we demonstrate that genetic as well as pharmacological modulation of Par-4 by NGD16 (a small molecule antimetastatic agent), limits EMT-induced chemoresistance in aggressive cancer cells by suppressing MDM-2, a downstream effector of Twist-1.MethodsMatrigel invasion assay, gelatin degradation assay, cell scattering assay, MTT assay and colony formation assay were used to study the proliferation and migration abilities of invasive cancer cells. Immunoblotting, immunocytochemistry, and immunoprecipitation analysis were utilized for determining protein expression and protein-protein interaction. 4T1 aggressive mouse carcinoma model was employed to evaluate tumor growth and lung metastasis.ResultsTreatment of gemcitabine (nucleoside analogue anticancer agent) to pancreatic cancer (Panc-1, MiaPaca-2) and breast cancer (MDA-MB-231) cells amplified MDM-2 expression along with increase in EMT properties. Conversely, NGD16 boosted expression of tumor suppressor Par-4 and inhibited invasion and migration abilities of these cells. Moreover, induction of Par-4 effectively diminished MDM-2 along with pro-EMT markers, whereas, augmented the expression of epithelial markers. Furthermore, siRNA-mediated silencing of Par-4 divulged that NGD16 exerts its EMT inhibitory effects in a Par-4-dependent manner. Mechanistically, Par-4 activation provokes p53 by disrupting MDM-2-p53 interaction, which restored epithelial characteristics in cancer cells. Additionally, partial knockdown of MDM-2 through siRNA pronounced the anti-proliferative and anti-invasive effects of NGD16. Finally, NGD16 efficiently inhibited tumor growth and lung metastasis in mouse mammary carcinoma model without showing any undesirable effects.ConclusionOur findings unveil Par-4 as a key therapeutic target and NGD16 (the pharmacological modulator of Par-4) are potential tools to suppress EMT and associated chemoresistance, which could be exploited clinically for the treatment of aggressive cancers.     

Peroxisome proliferator-activated receptor gamma ligand inhibits cell growth and invasion of human pancreatic cancer cells

Summary Background. Peroxisome proliferator-activated receptor γ (PPARγ) is expressed in certain human cancers; ligand-induced PPARγ activation can result in growth inhibition and differentiation in these cells. However, the precise mechanism for the antiproliferative effect of PPARγ ligands is not entirely known. Aim of Study. The purpose of this study was to examine the effect of PPARγ ligands on pancreatic cancer cell growth and invasiveness. Methods. The effect of two PPARγligands, 15 deoxy-Δ^12,14prostaglandin J₂ (15d-PGJ₂) and ciglitazone, on the growth of four human pancreatic cancer cell lines (BxPC-3, MIA PaCa-2, Panc-1, and L3.6) was assessed. Expression of cell-cycle and apoptotic-related proteins was measured. Finally, the effect of 15d-PGJ₂ on pancreatic cancer cell invasiveness and matrix metalloproteinase expression was determined. Results. Both 15d-PGJ₂ and ciglitazone inhibited the growth of all four pancreatic cancer cell lines in a dose- and time-dependent fashion. Treatment of BxPC-3 cells with 15d-PGJ₂ resulted in a time-dependent decrease in cyclin D1 expression associated with a concomitant induction of p21^waf1 and p27^kip1. In addition, 15d-PGJ₂ treatment induced apoptosis through activation of caspase-8, -9, and -3. Moreover, pancreatic cancer cell invasiveness was significantly suppressed after treatment with a nontoxic dose of 15d-PGJ₂, which was associated with a reduction of MMP-2 and MMP-9 protein levels and activity. Conclusion. These results demonstrate that PPARγ ligands have the dual advantage of inhibiting pancreatic cancer cell growth while reducing the invasiveness of the tumor cells, suggesting a potential role for these agents in the adjuvant treatment of pancreatic cancer.     

Long non-coding RNA TP73-AS1 promotes pancreatic cancer growth and metastasis through miRNA-128-3p/GOLM1 axis

BACKGROUNDPrevious studies have suggested that long non-coding RNAs (lncRNA) TP73-AS1 is significantly upregulated in several cancers. However, the biological role and clinical significance of TP73-AS1 in pancreatic cancer (PC) remain unclear. AIMTo investigate the role of TP73-AS1 in the growth and metastasis of PC.METHODSThe expression of lncRNA TP73-AS1, miR-128-3p, and GOLM1 in PC tissues and cells was detected by quantitative real-time polymerase chain reaction. The bioinformatics prediction software ENCORI was used to predict the putative binding sites of miR-128-3p. The regulatory roles of TP73-AS1 and miR-128-3p in cell proliferation, migration, and invasion abilities were verified by Cell Counting Kit-8, wound-healing, and transwell assays, as well as flow cytometry and Western blot analysis. The interactions among TP73-AS1, miR-128-3p, and GOLM1 were explored by bioinformatics prediction, luciferase assay, and Western blot. RESULTSThe expression of TP73-AS1 and miRNA-128-3p was dysregulated in PC tissues and cells. High TP73-AS1 expression was correlated with a poor prognosis. TP73-AS1 silencing inhibited PC cell proliferation, migration, and invasion in vitro as well as suppressed tumor growth in vivo. Mechanistically, TP73-AS1 was validated to promote PC progression through GOLM1 upregulation by competitively binding to miR-128-3p. CONCLUSIONOur results demonstrated that TP73-AS1 promotes PC progression by regulating the miR-128-3p/GOLM1 axis, which might provide a potential treatment strategy for patients with PC.     

Clinical relevance of CD70 expression in resected pancreatic cancer: Prognostic value and therapeutic potential

BackgroundAberrant expression of CD70 in several malignancies is potentially associated with poor patient prognosis and could serve as a therapeutic target. However, the clinical relevance of CD70 expression in pancreatic cancer has not been thoroughly explored.MethodsWe evaluated CD70 expression in 166 surgical specimens obtained from human patients with pancreatic cancer. We analyzed the function of CD70 in proliferation and migration using pancreatic cancer cell lines with silenced CD70 expression.ResultsCD70 expression was positively stained in 42 patients (25%). In the whole cohort, high CD70 expression was not associated with overall survival (OS: 33.1 vs. 40.8 months, P = 0.256), although it was significantly associated with inferior OS in a population of patients that completed adjuvant chemotherapy (OS: 45.4 vs. 63.8 months, P = 0.027). Moreover, the incidence of hematogenous metastasis was significantly higher in patients with high CD70 expression than in those with low CD70 expression (P = 0.020). This finding was also statistically significant in multivariate analyses (P = 0.001). In vitro experiments demonstrated that CD70 expression contributed to cancer cell proliferation independently of gemcitabine treatment as well as cell migration. Furthermore, real-time polymerase chain reaction analysis of frozen surgical tissues showed a correlation between the expression of CD70 and mesenchymal markers.ConclusionsCD70 expression in pancreatic cancer might be involved in hematogenous metastasis. Furthermore, our results imply that CD70 overexpression can serve as a novel prognostic factor and a potential therapeutic target in patients who have completed adjuvant chemotherapy.     

RING finger and WD repeat domain 3 regulates proliferation and metastasis through the Wnt/β-catenin signalling pathways in hepatocellular carcinoma

BACKGROUNDHepatocellular carcinoma (HCC) exhibits high invasiveness and mortality rates, and the molecular mechanisms of HCC have gained increasing research interest. The abnormal DNA damage response has long been recognized as one of the important factors for tumor occurrence and development. Recent studies have shown the potential of the protein RING finger and WD repeat domain 3 (RFWD3) that positively regulates p53 stability in response to DNA damage as a therapeutic target in cancers. AIMTo investigate the relationship between HCC and RFWD3 in vitro and in vivo and explored the underlying molecular signalling transduction pathways. METHODS RFWD3 gene expression was analyzed in HCC tissues and adjacent normal tissues. Lentivirus was used to stably knockdown RFWD3 expression in HCC cell lines. After verifying the silencing efficiency, Celigo/cell cycle/apoptosis and MTT assays were used to evaluate cell proliferation and apoptosis. Subsequently, cell migration and invasion were assessed by wound healing and transwell assays. In addition, transduced cells were implanted subcutaneously and injected into the tail vein of nude mice to observe tumor growth and metastasis. Next, we used lentiviral-mediated rescue of RFWD3 shRNA to verify the phenotype. Finally, the microarray, ingenuity pathway analysis, and western blot analysis were used to analyze the regulatory network underlying HCC. RESULTSCompared with adjacent tissues, RFWD3 expression levels were significantly higher in clinical HCC tissues and correlated with tumor size and TNM stage (P < 0.05), which indicated a poor prognosis state. RFWD3 silencing in BEL-7404 and HCC-LM3 cells increased apoptosis, decreased growth, and inhibited the migration in shRNAi cells compared with those in shCtrl cells (P < 0.05). Furthermore, the in vitro results were supported by the findings of the in vivo experiments with the reduction of tumor cell invasion and migration. Moreover, the rescue of RFWD3 shRNAi resulted in the resumption of invasion and metastasis in HCC cell lines. Finally, gene expression profiling and subsequent experimental verification revealed that RFWD3 might influence the proliferation and metastasis of HCC via the Wnt/β-catenin signalling pathway.CONCLUSIONWe provide evidence for the expression and function of RFWD3 in HCC. RFWD3 affects the prognosis, proliferation, invasion, and metastasis of HCC by regulating the Wnt/β-catenin signalling pathway.     

New high-throughput screening detects compounds that suppress pancreatic stellate cell activation and attenuate pancreatic cancer growth

Background/objectivesPancreatic stellate cells (PSCs) are involved in abundant desmoplasia, which promotes cancer cell aggressiveness and resistance to anti-cancer drugs. Therefore, PSCs are suggested to be a promising therapeutic target by attenuating PSC activation to inhibit tumor-stromal interactions with pancreatic cancer cells. Here, we developed a screen to identify compounds that reduce the activity of PSCs and investigated the effect of candidates on pancreatic cancer.MethodsLipid droplet accumulation in PSCs was used to observe differences in PSC activity and a new high-throughput screening platform that quantified lipid droplets in PSCs was established. A library of 3398 Food and Drug Administration-approved drugs was screened by this platform. Validation assays were performed in vitro and in vivo.ResultsThirty-two compounds were finally selected as candidate compounds by screening. These compounds decreased α-smooth muscle actin expression and inhibited autophagic flux in PSCs in vitro. Among the candidates, three drugs selected for validation assays inhibited the proliferation and migration of PSCs and invasion of cancer cells by disrupting tumor-stromal interactions. Production of extracellular matrix molecules was also decreased significantly by this treatment. In vivo testing in xenograft models showed that dopamine antagonist zuclopenthixol suppressed tumor growth; this suppression was significantly increased when combined with gemcitabine.ConclusionsA new screening platform that focused on the morphological features of PSCs was developed. Candidate drugs from this screening suppressed PSC activation and tumor growth. This screening system may be useful to discover new compounds that attenuate PSC activation.