20(S)‐Protopanaxadiol inhibits epithelial‐mesenchymal transition by promoting retinoid X receptor alpha in human colorectal carcinoma cells

Colorectal carcinoma (CRC) recurrence is often accompanied by metastasis. Most metastasis undergo through epithelial‐mesenchymal transition (EMT). Studies showed that retinol X receptor alpha (RXRα) and 20(S)‐Protopanaxadiol (PPD) have anti‐tumour effects. However, the anti‐metastasis effect of 20(S)‐PPD and the effect of RXRα on EMT‐induced metastasis are few studies on. Therefore, the role of RXRα and 20(S)‐PPD in CRC cell metastasis remains to be fully elucidated. RXRα with clinicopathological characteristics and EMT‐related expression in clinical samples were examined. Then, RXRα and EMT level in SW480 and SW620 cells, overexpressed and silenced RXRα in SW620 cells and SW480 cells, respectively, were evaluated. Finally, 20(S)‐PPD effect on SW620 and SW480 cells was evaluated. The results showed that a lower RXRα expression in cancer tissues, and a moderate negative correlation between RXRα and N stage, and tended to higher level of EMT. SW480 and SW620 cells had the highest and lowest RXRα expression among four CRC cell lines. SW480 had lower EMT level than SW620. Furthermore, 20(S)‐PPD increased RXRα and inhibited EMT level in SW620 cell. Finally, 20(S)‐PPD cannot restore SW480 cells EMT level to normal when RXRα silencing. These findings suggest that 20(S)‐PPD may inhibit EMT process in CRC cells by regulating RXRα expression.     

Analysis of epithelial–mesenchymal transition markers in psoriatic epidermal keratinocytes

Psoriasis is similar to endpoints of epithelial–mesenchymal transition (EMT), a process of epithelial cells transformed into fibroblast-like cells. The molecular epithelial and mesenchymal markers were analysed in psoriatic keratinocytes. No obvious alteration of epithelial markers E-cadherin (E-cad), keratin 10 (K10), K14 and K16 was detected in psoriatic keratinocytes. However, significantly increased expression of Vim, FN, plasminogen activator inhibitor 1 (PAI-1) and Slug was seen. IL-17A and IL-13 at 50 ng ml⁻¹ strongly decreased expression of K10, Vim and FN. TGF-β1 at 50 ng ml⁻¹ promoted the production of N-cad, Vim, FN and PAI-1. Slug was decreased by dexamethasone (Dex), but E-cad was upregulated by Dex. Silencing of ERK partially increased E-cad and K16, but remarkably inhibited K14, FN, Vim, β-catenin, Slug and α5 integrin. Moreover, inhibition of Rho and GSK3 by their inhibitors Y27632 and SB216763, respectively, strongly raised E-cad, β-catenin and Slug. Dex decreased Y27632-mediated increase of β-catenin. Dex at 2.0 µM inhibited SB216763-regulated E-cad, β-catenin and slug. In conclusion, EMT in psoriatic keratinocytes may be defined as an intermediate phenotype of type 2 EMT. ERK, Rho and GSK3 play active roles in the process of EMT in psoriatic keratinocytes.     

NOP14 regulates the growth,migration, and invasion of colorectal cancer cells by modulating the NRIP1/GSK-3β/β-catenin signaling pathway

Colorectal cancer (CRC) is the third most common cancer diagnosed worldwide. Recently, nucleolar complex protein 14 (NOP14) has been discovered to play a critical role in cancer development and progression, but the mechanisms of action of NOP14 in colorectal cancer remain to be elucidated. In this study, we used collected colorectal cancer tissues and cultured colorectal cancer cell lines (SW480, HT29, HCT116, DLD1, Lovo), and measured the mRNA and protein expression levels of NOP14 in colorectal cancer cells using qPCR and Western blotting. GFP-NOP14 was constructed and siRNA fragments against NOP14 were synthesized to investigate the importance of NOP14 for the development of colorectal cells. Transwell migration assays were used to measure cell invasion and migration, CCK-8 kits were used to measure cell activity, and flow cytometry was applied to the observation of apoptosis. We found that both the mRNA and protein levels of NOP14 were significantly upregulated in CRC tissues and cell lines. Overexpression of GFP-NOP14 markedly promoted the growth, migration, and invasion of the CRC cells HT19 and SW480, while genetic knockdown of NOP14 inhibited these behaviors. Overexpression of NOP14 promoted the expression of NRIP1 and phosphorylated inactivation of GSK-3β, leading to the upregulation of β-catenin. Genetic knockdown of NOP14 had the opposite effect on NRIP1/GSK-3/β-catenin signals. NOP14 therefore appears to be overexpressed in clinical samples and cell lines of colorectal cancer, and promotes the proliferation, growth, and metastasis of colorectal cancer cells by modulating the NRIP1/GSK-3β/β-catenin signaling pathway.Key words: Colorectal cancer, NOP14, proliferation, migration, invasion     

Role of fucosyltransferase IV in epithelial–mesenchymal transition in breast cancer cells

Epithelial–mesenchymal transition (EMT) is a crucial step in tumor progression and has an important role during cancer invasion and metastasis. Although fucosyltransferase IV (FUT4) has been implicated in the modulation of cell migration, invasion and cancer metastasis, its role during EMT is unclear. This study explores the molecular mechanisms of the involvement of FUT4 in EMT in breast cancer cells. Breast cancer cell lines display increased expression of FUT4, which is accompanied by enhanced appearance of the mesenchymal phenotype and which can be reversed by knockdown of endogenous FUT4. Moreover, FUT4 induced activation of phosphatidylinositol 3-kinase (PI3K)/Akt, and inactivation of GSK3β and nuclear translocation of NF-κB, resulting in increased Snail and MMP-9 expression and greater cell motility. Taken together, these findings indicate that FUT4 has a role in EMT through activation of the PI3K/Akt and NF-κB signaling systems, which induce the key mediators Snail and MMP-9 and facilitate the acquisition of a mesenchymal phenotype. Our findings support the possibility that FUT4 is a novel regulator of EMT in breast cancer cells and a promising target for cancer therapy.     

Akt2 Is Involved in Loss of Epithelial Cells and Renal Fibrosis following Unilateral Ureteral Obstruction

Obstructive nephropathy is an aggressive form of chronic kidney disease (CKD), which is characterized by an epithelial-to-mesenchymal transition (EMT) and interstitial fibrosis. However, the molecular mechanisms of EMT and fibrosis are complex and not fully understood. In this study, we investigated the contribution of Akt2 to experimental renal EMT and fibrosis using the well-established model of unilateral ureteral obstruction (UUO). We found that Akt2 and phosphor (p)-Akt protein levels were increased in the obstructed kidneys. UUO induced activation of transforming growth factor-β1 (TGF-β1) signaling. Importantly, knockout of Akt2 suppressed UUO-induced EMT, kidney fibrosis, increased GSK3β activity, and decreased expression of Snail and β-catenin. Inhibition of GSK3β with LiCl (the inhibitor of GSK3β) increased the expression of Snail and β-catenin in cultured kidney epithelial cells. Our findings suggest that Akt2 partially contributes to interstitial fibrosis following UUO and that inhibition of this signaling pathway may provide a novel approach of prevent progression of renal fibrosis.     

AIF inhibits tumor metastasis by protecting PTEN from oxidation

Apoptosis-inducing factor (AIF) exerts dual roles on cell death and survival, but its substrates as a putative oxidoreductase and roles in tumorigenesis remain elusive. Here, we report that AIF physically interacts with and inhibits the oxidation of phosphatase and tensin homolog on chromosome ten (PTEN), a tumor suppressor susceptible for oxidation-mediated inactivation. More intriguingly, we also identify PTEN as a mitochondrial protein and the ectopic expression of mitochondrial targeting sequence-carrying PTEN almost completely inhibits Akt phosphorylation in PTEN-deficient cells. AIF knockdown causes oxidation-mediated inactivation of the lipid phosphatase activity of PTEN, with ensuing activation of Akt kinase, phosphorylation of the Akt substrate GSK-3β, and activation of β-catenin signaling in cancer cells. Through its effect on β-catenin signaling, AIF inhibits epithelial–mesenchymal transition (EMT) and metastasis of cancer cells in vitro and in orthotopically implanted xenografts. Accordingly, the expression of AIF is correlated with the survival of human patients with cancers of multiple origins. These results identify PTEN as the substrate of AIF oxidoreductase and reveal a novel function for AIF in controlling tumor metastasis.     

Hypoxia-induced Bmi1 promotes renal tubular epithelial cell–mesenchymal transition and renal fibrosis via PI3K/Akt signal

Hypoxia is an important microenvironmental factor in the development of renal fibrosis; however, the underlying mechanisms are not well elucidated. Here we show that hypoxia induces Bmi1 mRNA and protein expression in human tubular epithelial cells. We further demonstrate that Bmi1 expression might be directly regulated by hypoxia-inducible factor-1a (HIF-1a) under low oxygen. Moreover, chromatin immunoprecipitation and reporter gene assay studies reveal cooperative transactivation of Bmi1 by HIF-1α and Twist. Enforced Bmi1 expression induces epithelial–mesenchymal transition (EMT), whereas silencing endogenous Bmi-1 expression reverses hypoxia-induced EMT. Up-regulation of Bmi1 leads to stabilization of Snail via modulation of PI3K/Akt signaling, whereas ablation of PI3K/Akt signaling partially rescues the phenotype of Bmi1-overexpressing cells, indicating that PI3K/Akt signaling might be a major mediator of Bmi1-induced EMT. In a rat model of obstructive nephropathy, Bmi1 expression increases in a time-dependent manner. Furthermore, we demonstrate that increased levels of Bmi1, correlated with HIF-1α and Twist, are associated with patients with chronic kidney disease. We provide in vitro and in vivo evidence that activation of HIF-1a/Twist-Bmi1 signaling in renal epithelial cells is associated with the development of chronic renal disease and may promote fibrogenesis via modulation of PI3K/Akt/Snail signaling by facilitating EMT.     

Dioxin Receptor Expression Inhibits Basal and Transforming Growth Factor β-induced Epithelial-to-mesenchymal Transition

Recent studies have emphasized the role of the dioxin receptor (AhR) in maintaining cell morphology, adhesion, and migration. These novel AhR functions depend on the cell phenotype, and although AhR expression maintains mesenchymal fibroblasts migration, it inhibits keratinocytes motility. These observations prompted us to investigate whether AhR modulates the epithelial-to-mesenchymal transition (EMT). For this, we have used primary AhR^+/+ and AhR^−/− keratinocytes and NMuMG cells engineered to knock down AhR levels (sh-AhR) or to express a constitutively active receptor (CA-AhR). Both AhR^−/− keratinocytes and sh-AhR NMuMG cells had increased migration, reduced levels of epithelial markers E-cadherin and β-catenin, and increased expression of mesenchymal markers Snail, Slug/Snai2, vimentin, fibronectin, and α-smooth muscle actin. Consistently, AhR^+/+ and CA-AhR NMuMG cells had reduced migration and enhanced expression of epithelial markers. AhR activation by the agonist FICZ (6-formylindolo[3,2-b]carbazole) inhibited NMuMG migration, whereas the antagonist α-naphthoflavone induced migration as did AhR knockdown. Exogenous TGFβ exacerbated the promigratory mesenchymal phenotype in both AhR-expressing and AhR-depleted cells, although the effects on the latter were more pronounced. Rescuing AhR expression in sh-AhR cells reduced Snail and Slug/Snai2 levels and cell migration and restored E-cadherin levels. Interference of AhR in human HaCaT cells further supported its role in EMT. Interestingly, co-immunoprecipitation and immunofluorescence assays showed that AhR associates in common protein complexes with E-cadherin and β-catenin, suggesting the implication of AhR in cell-cell adhesion. Thus, basal or TGFβ-induced AhR down-modulation could be relevant in the acquisition of a motile EMT phenotype in both normal and transformed epithelial cells.     

microRNA-211 promotes invasion and migration of colorectal cancer cells by targeting FABP4 via PPARγ

Fatty acid binding protein 4 (FABP4) is a novel tumor regulator that is abnormally expressed in many human cancers. In our study, upregulated microRNA-211 (miR-211) and reduced FABP4 expression were detected in colorectal cancer (CRC) patients and CRC cells. Mimic miR-211 or anti-miR-211 were transfected to investigate the effects of miR-211 on SW480 cells. The results showed that miR-211 promoted but anti-miR-211 inhibited cell migration, invasion, and epithelial–mesenchymal transition (EMT) of SW480 cells. Luciferase activity was decreased after cotransfection with miR-211 and WT-FABP4-UTR in SW480 cells. And reduced FABP4 protein expression by miR-211 indicated that FABP4 was the targeted gene of miR-211. miR-211 inhibited the activation of peroxisome proliferator-activated receptor (PPAR) γ, whereas overexpression of FABP4 reversed that effect. Finally, FABP4 inhibited the migration, invasion, and EMT of SW480 cells, whereas PPARγ agonist reversed the effects of FABP4. Thus, the miR-211/FABP4/PPARγ axis may be a novel target for CRC therapy.     

Global Phosphotyrosine Proteomics Identifies PKCδ as a Marker of Responsiveness to Src Inhibition in Colorectal Cancer

Sensitive and specific biomarkers of protein kinase inhibition can be leveraged to accelerate drug development studies in oncology by associating early molecular responses with target inhibition. In this study, we utilized unbiased shotgun phosphotyrosine (pY) proteomics to discover novel biomarkers of response to dasatinib, a small molecule Src-selective inhibitor, in preclinical models of colorectal cancer (CRC). We performed unbiased mass spectrometry shotgun pY proteomics to reveal the pY proteome of cultured HCT-116 colonic carcinoma cells, and then extended this analysis to HCT-116 xenograft tumors to identify pY biomarkers of dasatinib-responsiveness in vivo. Major dasatinib-responsive pY sites in xenograft tumors included sites on delta-type protein kinase C (PKCδ), CUB-domain-containing protein 1 (CDCP1), Type-II SH2-domain-containing inositol 5-phosphatase (SHIP2), and receptor protein-tyrosine phosphatase alpha (RPTPα). The pY313 site PKCδ was further supported as a relevant biomarker of dasatinib-mediated Src inhibition in HCT-116 xenografts by immunohistochemistry and immunoblotting with a phosphospecific antibody. Reduction of PKCδ pY313 was further correlated with dasatinib-mediated inhibition of Src and diminished growth as spheroids of a panel of human CRC cell lines. These studies reveal PKCδ pY313 as a promising readout of Src inhibition in CRC and potentially other solid tumors and may reflect responsiveness to dasatinib in a subset of colorectal cancers.