Establishment of Canine Transitional Cell Carcinoma Cell Lines Harboring BRAF V595E Mutation as a Therapeutic Target

Transitional cell carcinoma (TCC) is the most common malignant tumor of the canine urinary tract and tends to have a poor prognosis due to its invasive potential. Recent studies have reported that up to 80% of canine urothelial carcinoma has the BRAF V595E mutation, which is homologous to the human V600E mutation. Activating the BRAF mutation is an actionable target for developing effective therapeutic agents inhibiting the BRAF/mitogen-activated protein kinase (MAPK) pathway in canine cancer as well as human cancer. We established novel canine TCC cell lines from two tumor tissues and one metastatic lymph node of canine TCC patients harboring the BRAF V595E mutation. Tumor tissues highly expressed the BRAF mutant and phosphorylated extracellular signal-related kinases (ERK)1/2 proteins. The derived cell lines demonstrated activated MAPK pathways. We also evaluated the cell lines for sensitivity to BRAF inhibitors. Sorafenib, a multiple kinase inhibitor targeting RAF/vascular endothelial growth factor receptor (VEGFR), successfully inhibited the BRAF/MAPK pathway and induced apoptosis. The established canine TCC cell lines responded with greater sensitivity to sorafenib than to vemurafenib, which is known as a specific BRAF inhibitor in human cancer. Our results demonstrated that canine TCC cells showed different responses compared to human cancer with the BRAF V600E mutation. These cell lines would be valuable research materials to develop therapeutic strategies for canine TCC patients.     

The GNAQ T96S Mutation Affects Cell Signaling and Enhances the Oncogenic Properties of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC), the most common malignant tumor in the liver, grows and metastasizes rapidly. Despite advances in treatment modalities, the five-year survival rate of HCC remains less than 30%. We sought genetic mutations that may affect the oncogenic properties of HCC, using The Cancer Genome Atlas (TCGA) data analysis. We found that the GNAQ T96S mutation (threonine 96 to serine alteration of the Gαq protein) was present in 12 out of 373 HCC patients (3.2%). To examine the effect of the GNAQ T96S mutation on HCC, we transfected the SK-Hep-1 cell line with the wild-type or the mutant GNAQ T96S expression vector. Transfection with the wild-type GNAQ expression vector enhanced anchorage-independent growth, migration, and the MAPK pathways in the SK-Hep-1 cells compared to control vector transfection. Moreover, cell proliferation, anchorage-independent growth, migration, and the MAPK pathways were further enhanced in the SK-Hep-1 cells transfected with the GNAQ T96S expression vector compared to the wild-type GNAQ-transfected cells. In silico structural analysis shows that the substitution of the GNAQ amino acid threonine 96 with a serine may destabilize the interaction between the regulator of G protein signaling (RGS) protein and GNAQ. This may reduce the inhibitory effect of RGS on GNAQ signaling, enhancing the GNAQ signaling pathway. Single nucleotide polymorphism (SNP) genotyping analysis for Korean HCC patients shows that the GNAQ T96S mutation was found in only one of the 456 patients (0.22%). Our data suggest that the GNAQ T96S hotspot mutation may play an oncogenic role in HCC by potentiating the GNAQ signal transduction pathway.     

The Expression of Connexin 26 Regulates the Radiosensitivity of Hepatocellular Carcinoma Cells through a Mitogen-Activated Protein Kinases Signal Pathway

Connexin 26 (Cx26) is a protein that constitutes a gap junction and is widely expressed in the liver. Abnormal expression of Cx26 is one of the important mechanisms of liver cancer, and is closely related to the transmission of radiation damage signals between cells. In the present study, we investigated the radiosensitivity of hepatocellular carcinoma (HCC) cells HepG2, with low expression of Cx26, and SK-hep-1, with high expression of Cx26 after X-ray irradiation. The cell survival, micronucleus formation and protein expressions of the mitogen-activated protein kinases (MAPK) signaling pathway were detected. The expression level of Cx26 could affect the radiosensitivity of liver cancer cells by affecting the phosphorylation of p38 and ERK proteins and regulating the expression of downstream NF-κB. Cell lines with knock-out and overexpression of Cx26 were also built to confirm the findings. Our results suggested that Cx26 might play an important role in the radiosensitivity of liver cancer and could be a potential target for clinical radiotherapy of liver cancer.     

Plectin Downregulation Inhibits Migration and Suppresses Epithelial Mesenchymal Transformation of Hepatocellular Carcinoma Cells via ERK1/2 Signaling

Plectin, as a cytoskeleton-related protein, is involved in various physiological and pathological processes of many cell types. Studies have found that plectin affects cancer cell invasion and metastasis, but the exact mechanism is not fully understood. In this study, we aim to investigate the role of plectin in the migration of hepatocellular carcinoma (HCC) cells and explore its relevant molecular mechanism. Herein, we found that the expression of plectin in HCC tissue and cells was significantly increased compared with normal liver tissue and cells. After downregulation of plectin, the migration ability of HCC cells was significantly lower than that of the control group. Moreover, the expression of E-cadherin was upregulated and the expression of N-cadherin and vimentin was downregulated, suggesting that plectin downregulation suppresses epithelial mesenchymal transformation (EMT) of HCC cells. Mechanically, we found that plectin downregulation repressed the extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Activation of ERK1/2 recovered the plectin downregulation-inhibited migration and EMT of HCC cells. Taken together, our results demonstrate that downregulation of plectin inhibits HCC cell migration and EMT through ERK1/2 signaling, which provides a novel prognostic biomarker and potential therapeutic target for HCC.     

MafA基因真核表达质粒的构建及其在人肝癌细胞HePG-2中的表达和作用

目的构建胰岛素启动子肌腱膜的纤维肉瘤肿瘤基因同系物A(MafA)基因的真核表达质粒,并观察MafA基因在人肝癌细胞HePG-2中的表达及其作用,为糖尿病基因治疗的研究奠定基础。方法提取小鼠胰腺组织总RNA,经RT-PCR扩增MafA基因片段,克隆至真核表达载体pEGFP-N2和pEGFP-C1中。用脂质体将重组表达质粒转染至人肝癌细胞HePG-2中,荧光显微镜观察绿色荧光蛋白的表达,RT-PCR检测转染细胞中MafA和insulinⅡ基因的转录水平,Western blot检测融合蛋白EGFP-MafA的表达。结果重组表达质粒pEGFP-N2/MafA和pEGFP-C1/MafA经PCR、双酶切和测序证明构建正确;重组质粒转染的HePG-2细胞有绿色荧光蛋白表达;经RT-PCR可检测到MafA基因表达,但未检测到insulinⅡ基因表达;经Westernblot检测,在相对分子质量约70 000处可见特异性反应条带。结论已成功构建了MafA基因真核表达质粒pEGFP-N2/MafA和pEGFP-C1/MafA,以其转染的HePG-2细胞可表达MafA基因,并翻译成蛋白,但未表达insulinⅡ基因。     

Modulating the ERK1/2–MMP1 Axis through Corosolic Acid Inhibits Metastasis of Human Oral Squamous Cell Carcinoma Cells

Corosolic acid (CA; 2α-hydroxyursolic acid) is a natural pentacyclic triterpenoid with antioxidant, antitumour and antimetastatic activities against various tumour cells during tumourigenesis. However, CA’s antitumour effect and functional roles on human oral squamous cell carcinoma (OSCC) cells are utterly unknown. In this study, our results demonstrated that CA significantly exerted an inhibitory effect on matrix metalloproteinase (MMP)1 expression, cell migration and invasion without influencing cell growth or the cell cycle of human OSCC cells. The critical role of MMP1 was confirmed using the GEPIA database and showed that patients have a high expression of MMP1 and have a shorter overall survival rate, confirmed on the Kaplan–Meier curve assay. In the synergistic inhibitory analysis, CA and siMMP1 co-treatment showed a synergically inhibitory influence on MMP1 expression and invasion of human OSCC cells. The ERK1/2 pathway plays an essential role in mediating tumour progression. We found that CA significantly inhibits the phosphorylation of ERK1/2 dose-dependently. The ERK1/2 pathway played an essential role in the CA-mediated downregulation of MMP1 expression and in invasive motility in human OSCC cells. These findings first demonstrated the inhibitory effects of CA on OSCC cells’ progression through inhibition of the ERK1/2–MMP1 axis. Therefore, CA might represent a novel strategy for treating OSCC.     

Expression of Cancer Stem Cell Markers EpCAM and CD90 Is Correlated with Anti- and Pro-Oncogenic EphA2 Signaling in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. Additionally, the efficacy of targeted molecular therapies with multiple tyrosine kinase inhibitors is limited. In this study, we focused on the cellular signaling pathways common to diverse HCC cells and used quantitative reverse phase protein array (RPPA) and statistical analyses to elucidate the molecular mechanisms determining its malignancy. We examined the heterogeneity of 17 liver cancer cell lines by performing cluster analysis of their expression of CD90 and EpCAM cancer stem cell markers. Gaussian mixture model clustering identified three dominant clusters: CD90-positive and EpCAM-negative (CD90+), EpCAM-positive and CD90-negative (EpCAM+) and EpCAM-negative and CD90-negative (Neutral). A multivariate analysis by partial least squares revealed that the former two cell populations showed distinct patterns of protein expression and phosphorylation in the EGFR and EphA2 signaling pathways. The CD90+ cells exhibited higher abundance of AKT, EphA2 and its phosphorylated form at Ser⁸⁹⁷, whereas the EpCAM+ cells exhibited higher abundance of ERK, RSK and its phosphorylated form. This demonstrates that pro-oncogenic, ligand-independent EphA2 signaling plays a dominant role in CD90+ cells with higher motility and metastatic activity than EpCAM+ cells. We also showed that an AKT inhibitor reduced the proliferation and survival of CD90+ cells but did not affect those of EpCAM+ cells. Taken together, our results suggest that AKT activation may be a key pro-oncogenic regulator in HCC.     

Possible Mechanisms of Di(2-ethylhexyl) Phthalate-Induced MMP-2 and MMP-9 Expression in A7r5 Rat Vascular Smooth Muscle Cells

Proliferation and migration of vascular smooth muscle cells (VSMC) are important in the development and/or progression of many cardiovascular diseases, including atherosclerosis. Evidence shows that matrix metalloproteinase (MMP)-2 and MMP-9 are related to the pathogenesis of atherosclerosis. The expressions of MMP-2 and MMP-9 in atherosclerosis are regulated via various pathways, such as p38 mitogen activated protein kinase (MAPK), extracellular signal regulated kinase 1 and 2 (ERK1/2), Akt, and nuclear factor kappa (NF-κB). Di(2-ethylhexyl) phthalate (DEHP) has been shown to induce atherosclerosis by increasing tumor necrosis factor (TNF)-α, interleukin (IL)-6, and intercellular adhesion molecule (ICAM) productions. However, whether DEHP poses any effects on MMP-2 or MMP-9 expression in VSMC has not yet been answered. In our studies, rat aorta VSMC was treated with DEHP (between 2 and 17.5 ppm) and p38 MAPK, ERK1/2, Akt, NF-κB, and MMP-2 and MMP-9 proteins and activities were measured. Results showed that the presence of DEHP can induce higher MMP-2 and MMP-9 expression than the controls. Similar results on MMP-regulating proteins, i.e., p38 MAPK, ERK1/2, Akt, and NF-κB, were also observed. In summary, our current results have showed that DEHP can be a potent inducer of atherosclerosis by increasing MMP-2 and MMP-9 expression at least through the regulations of p38 MAPK, ERK1/2, Akt, and NF-κB.     

The Sphingosine Kinase 2 Inhibitor ABC294640 Restores the Sensitivity of BRAFV600E Mutant Colon Cancer Cells to Vemurafenib by Reducing AKT-Mediated Expression of Nucleophosmin and Translationally-Controlled Tumour Protein

Vemurafenib (PLX4032), small-molecule inhibitor of mutated BRAFV600E protein, has emerged as a potent anti-cancer agent against metastatic melanoma harboring BRAFV600E mutation. Unfortunately, the effect of PLX4032 in the treatment of metastatic BRAF mutated colorectal cancer (CRC) is less potent due to high incidence of fast-developing chemoresistance. It has been demonstrated that sphingolipids are important mediators of chemoresistance to various therapies in colon cancer. In this study, we will explore the role of major regulators of sphingolipid metabolism and signaling in the development of resistance to vemurafenib in BRAF mutant colon cancer cells. The obtained data revealed significantly increased expression levels of activated sphingosine kinases (SphK1 and SphK2) in resistant cells concomitant with increased abundance of sphingosine-1-phosphate (S1P) and its precursor sphingosine, which was accompanied by increased expression levels of the enzymes regulating the ceramide salvage pathway, namely ceramide synthases 2 and 6 and acid ceramidase, especially after the exposure to vemurafenib. Pharmacological inhibition of SphK1/SphK2 activities or modulation of ceramide metabolism by exogenous C6-ceramide enhanced the anti-proliferative effect of PLX4032 in resistant RKO cells in a synergistic manner. It is important to note that the inhibition of SphK2 by ABC294640 proved effective at restoring the sensitivity of resistant cells to vemurafenib at the largest number of combinations of sub-toxic drug concentrations with minimal cytotoxicity. Furthermore, the obtained findings revealed that enhanced anti-proliferative, anti-migratory, anti-clonogenic and pro-apoptotic effects of a combination treatment with ABC294640 and PLX4032 relative to either drug alone were accompanied by the inhibition of S1P-regulated AKT activity and concomitant abrogation of AKT-mediated cellular levels of nucleophosmin and translationally-controlled tumour protein. Collectively, our study suggests the possibility of using the combination of ABC294640 and PLX4032 as a novel therapeutic approach to combat vemurafenib resistance in BRAF mutant colon cancer, which warrants additional preclinical validation studies.