Timosaponin AⅢ induces drug-metabolizing enzymes by activating constitutive androstane receptor (CAR) via dephosphorylation of the EGFR signaling pathway

The current study aimed to assess the effect of timosaponin AⅢ(T-AⅢ)on drug-metabolizing enzymes during anticancer therapy.The in vivo experiments were conducted on nude and ICR mice.Following a 24-day administration of T-AⅢ,the nude mice exhibited an induction of CYP2B10,MDR1,and CYP3A11 expression in the liver tissues.In the ICR mice,the expression levels of CYP2B10 and MDR1 increased after a three-day T-AⅢ administration.The in vitro assessments with HepG2 cells revealed that T-AⅢ induced the expression of CYP2B6,MDR1,and CYP3A4,along with constitutive androstane receptor(CAR)activation.Treatment with CAR siRNA reversed the T-AⅢ-induced increases in CYP2B6 and CYP3A4 expression.Furthermore,other CAR target genes also showed a significant increase in the expression.The up-regulation of murine CAR was observed in the liver tissues of both nude and ICR mice.Subsequent findings demonstrated that T-AⅢ activated CAR by inhibiting ERK1/2 phosphorylation,with this effect being partially reversed by the ERK activator t-BHQ.Inhibition of the ERK1/2 signaling pathway was also observed in vivo.Additionally,T-AⅢ inhibited the phosphorylation of EGFR at Tyr1173 and Tyr845,and suppressed EGF-induced phosphorylation of EGFR,ERK,and CAR.In the nude mice,T-AⅢ also inhibited EGFR phosphorylation.These results collectively indicate that T-AⅢ is a novel CAR activator through inhibition of the EGFR pathway.

Circular RNA ciRS-7 promotes the proliferation and metastasis of pancreatic cancer by regulating miR-7-mediated EGFR/STAT3 signaling pathway

Background: Pancreatic ductal adenocarcinoma(PDAC) is the most deadly type of tumor, and its pathogenesis remains unknown. Circular RNAs(circRNAs) may be functional and bind to micro RNAs and consequently, influence the activity of targeted mRNAs. Recent researches indicate that one circRNA, ciRS-7, acts as a sponge of miR-7 and thus, inhibits its activity. It is well known that miR-7 is a cancer suppressor in many cancers. However, the relationship between ciRS-7 and miR-7, and the role of ciRS-7 in PDAC, remains to be elucidated. Methods: miR-7 and ciRS-7 expression in 41 pairs of PDAC tumors and their paracancerous tissues were detected by quantitative reverse transcription polymerase chain reaction(qRT-PCR). The relationships between their expression levels and clinicopathological features in PDAC tissues were assessed. The relationship between miR-7 and ciRS-7 was also assessed by Spearman’s correlation. We also used cell lines to evaluate the role of ciRS-7 in cell line behavior. The ciRS-7 interfere RNA(si RNA) and its empty vector were transfected into PDAC cells. PDAC cells proliferation and invasion abilities were detected by MTT assay and invasion analysis. The expression of proteins was assessed by Western blotting. Results: ciRS-7 expression was significantly higher in PDAC tissues than paracancerous tissues( P = 0.002). However, miR-7 expression showed the opposite trend( P = 0.048). Moreover, ciRS-7 expression was inversely correlated with miR-7 in PDAC( r s =-0.353, P = 0.023). ciRS-7 expression was also significantly elevated in venous invasion(3.72 ± 2.93 vs. 2.14 ± 1.26;P = 0.028) and lymph node metastasis(4.19 ± 2.75 vs. 2.32 ± 1.90;P = 0.016) in PDAC patients. Furthermore, ciRS-7 knockdown suppressed cell proliferation and invasion of PDAC cells( P < 0.05), and the downregulation of ciRS-7 resulted in miR-7 overexpression and subsequent inhibition of epidermal growth factor receptor(EGFR) and signal transducer and activator of transcription 3(STAT3). Conclusions: Circular RNA ciRS-7 plays an oncogene role in PDAC, partly by targeting miR-7 and regulating the EGFR/STAT3 signaling pathway.

Exogenous p27KIP1 expression induces anti-tumour effects and inhibits the EGFR/PI3K/Akt signalling pathway in PC3 cells

p27 is a cyclin-dependent kinase inhibitor that regulates the progression of cells from G1 to S phase of the cell cycle. Loss of p27 has been associated with disease progression and with an unfavourable outcome in prostate cancer. In this study, we investigated whether exogenous p27 expression in the human androgen-independent prostate cancer PC3 cell line had any effect on cell growth, and we studied the molecular mechanisms involved. p27 expression was restored in PC3 cells by plasmid delivery. Cell proliferation and apoptosis were assessed in PC3 cells transfected with p27. We also investigated the effects of p27 on the epidermal growth factor receptor （EGFR）/ phosphatidylinositol 3-kinase （PI3K）/Akt signalling pathway in PC3 cells. By restoring p27 expression in PC3 cells, we observed that p27 reduced proliferation and induced arrest in G0/G1 phase. Moreover, p27-transfected PC3 cells underwent apoptosis, as shown by flow cytometric analysis and western blotting analysis of Bcl-2, Bax, Bad, caspase-3 and poly（ADP-ribose）polymerase expression. Furthermore, the p27-induced anti-tumour action corre- lated with inhibition of the EGFR/PI3K/Akt signalling pathway, as confirmed by western blotting analysis and densitometry of EGFR, PI3K （p85）, Akt and p-Akts473 expression. Our results suggest that exogenous expression of p27 inhibits the proliferation of PC3 cells through induction of G1 arrest and apoptosis, and this process correlates with inhibition of the EGFR/PI3K/Akt signalling pathway.

Circ_0053943 complexed with IGF2BP3 drives uveal melanoma progression via regulating N6-methyladenosine modification of Epidermal growth factor receptor

Numerous studies have characterized the critical role of circular RNAs(circRNAs)as regulatory factors in the progression of multiple cancers.However,the biological functions of circRNAs and their underlying molecular mechanisms in the progression of uveal melanoma(UM)remain enigmatic.In this study,we identified a novel circRNA,circ_0053943,through re-analysis of UM microarray data and quantitative RT-PCR.Circ_0053943 was found to be upregulated in UM and to promote the proliferation and metastatic ability of UM cells in both in vitro and in vivo settings.Mechanistically,circ_0053943 was observed to bind to the KH1 and KH2 domains of insulin-like growth factor 2 mRNA-binding protein 3(IGF2BP3),thereby enhancing the function of IGF2BP3 by stabilizing its target mRNA.RNA sequencing assays identified epidermal growth factor receptor(EGFR)as a target gene of circ_0053943 and IGF2BP3 at the transcriptional level.Rescue assays demonstrated that circ_0053943 exerts its biological function by stabilizing EGFR mRNA and regulating the downstream mitogen-activated protein kinase/extracellular signal-regulated kinase(MAPK/ERK)signaling pathway.Collectively,circ_0053943 may promote UM progression by stabilizing EGFR mRNA and activating the MAPK/ERK signaling pathway through the formation of a circ_0053943/IGF2BP3/EGFR RNA-protein ternary complex,thus providing a potential biomarker and therapeutic target for UM.

Effects of (-)-Epigallocatechin gallate on some protein factors involved in the epidermal growth factor receptor signaling pathway

（-）-Epigallocatechin gallate （EGCG）, a major polyphenolic constituent of green tea, can inhibit activity of specific receptor tyrosine kinases （RTKs） and related downstream signal transduction pathways, resulting in the control of unwanted cell proliferation. The epidermal growth factor receptor （EGFR） signaling pathway is one of the most important pathways that regulates growth, survival,proliferation and differentiation in mammalian cells. This review addresses the effects of EGCG on some protein factors involved in the EGFR signaling pathway in a direct or indirect manner. Based on our understanding of the interaction between EGCG and these factors, and based on their structures, EGCG could be used as a lead compound for designing and synthesizing novel drugs with significant biological activity.

Long noncoding RNA LINC00520 prevents the progression of cutaneous squamous cell carcinoma through the inactivation of the PI3K/Akt signaling pathway by downregulating EGFR

Background: Long noncoding RNAs (lncRNAs) play pivotal roles in various malignant tumors. Epidermal growth factor receptor (EGFR) signaling is associated with the pathogenesis of cutaneous squamous cell carcinoma (cSCC). This study aimed to explore the role of LINC00520 in the development of cSCC via EGFR and phosphoinositide 3-kinase-protein kinase B (PI3K/Akt) signaling pathways. Methods: A microarray analysis was applied to screen differentially expressed lncRNAs in cSCC samples. The A431 cSCC cell line was transfected and assigned different groups. The expression patterns of LINC00520, EGFR, and intermediates in the PI3K/Akt pathway were characterized using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting analysis. Cell proliferation, migration, and invasion were detected using the MTT assay, scratch test, and Transwell assay, respectively. Cell-based experiments and a tumorigenicity assay were conducted to assess the effect of LINC00520 on cSCC progression. This study was ended in September 2017. Comparisons between two groups were analyzed with t-test and comparisons among multiple groups were analyzed using one-way analysis of variance. The nonparametric Wilcoxon rank sum test was used to analyze skewed data. The enumerated data were analyzed using the chi-square test or Fisher exact test. Results: Data from chip GSE66359 revealed depletion of LINC00520 in cSCC. Cells transfected with LINC00520 vector and LINC00520 vector + si-EGFR showed elevated LINC00520 level but decreased levels of the EGFR, PI3K, AKT, VEGF, MMP-2 and MMP-9 mRNAs and proteins, and inhibition of the growth, migration and adhesion of cSCC cells, while the si-LINC00520 group showed opposite trends (all P < 0.05). Compared with the LINC00520 vector group, the LINC00520 vector + si-EGFR group showed decreased levels of the EGFR, PI3K, AKT, VEGF, MMP-2 and MMP-9 mRNAs and proteins, and inhibition of the growth, migration and adhesion of cSCC cells, while the LINC00520 vector+ EGFR vector group showed opposite results (all P < 0.05). Conclusion: Based on our results, LINC00520-targeted EGFR inhibition might result in the inactivation of the PI3K/Akt pathway, thus inhibiting cSCC development.

Crosstalk between the B7/CD28 and EGFR pathways:Mechanisms and therapeutic opportunities

Somatic activating mutations in the epidermal growth factor receptor(EGFR)are one of the most common oncogenic drivers in cancers such as non-small-cell lung cancer(NSCLC),metastatic colorectal cancer,glioblastoma,head and neck cancer,pancreatic cancer,and breast cancer.Molecular-targeted agents against EGFR signaling pathways have shown robust clinical efficacy,but patients inevitably experience acquired resistance.Although immune checkpoint inhibitors(ICIs)targeting PD-1/PD-L1 have exhibited durable anti-tumor responses in a subset of patients across multiple cancer types,their efficacy is limited in cancers harboring activating gene alterations of EGFR.Increasing studies have demonstrated that upregulation of new B7/CD28 family members such as B7-H3,B7x and HHLA2,is associated with EGFR signaling and may contribute to resistance to EGFR-targeted therapies by creating an immunosuppressive tumor microenvironment(TME).In this review,we discuss the regulatory effect of EGFR signaling on the PD-1/PD-L1 pathway and new B7/CD28 family member pathways.Understanding these interactions may inform combination therapeutic strategies and potentially overcome the current challenge of resistance to EGFR-targeted therapies.We also summarize clinical data of anti-PD-1/PD-L1 therapies in EGFR-mutated cancers,as well as ongoing clinical trials of combination of EGFR-targeted therapies and anti-PD-1/PD-L1 immunotherapies.

MFN2 suppresses clear cell renal cell carcinoma progression by modulating mitochondria-dependent dephosphorylation of EGFR

Background:Clear cell renal cell carcinoma(ccRCC)is the most lethal renal cancer.An overwhelming increase of patients experience tumor progression and unfavorable prognosis.However,the molecular events underlying ccRCC tumorigenesis and metastasis remain unclear.Therefore,uncovering the underlying mechanisms will pave the way for developing novel therapeutic targets for ccRCC.In this study,we sought to investigate the role of mitofusin-2(MFN2)in supressing ccRCC tumorigenesis and metastasis.Methods:The expression pattern and clinical significance of MFN2 in ccRCC were analyzed by using the Cancer Genome Atlas datasets and samples from our independent ccRCC cohort.Both in vitro and in vivo experiments,including cell proliferation,xenograft mouse models and transgenic mouse model,were used to determine the role of MFN2 in regulating the malignant behaviors of ccRCC.RNA-sequencing,mass spectrum analysis,co-immunoprecipitation,bio-layer interferometry and immunofluorescence were employed to elucidate the molecular mechanisms for the tumor-supressing role of MFN2.Results:we reported a tumor-suppressing pathway in ccRCC,characterized by mitochondria-dependent inactivation of epidermal growth factor receptor(EGFR)signaling.This process was mediated by the outer mitochondrial membrane(OMM)protein MFN2.MFN2 was down-regulated in ccRCC and associated with favorable prognosis of ccRCC patients.in vivo and in vitro assays demonstrated thatMFN2 inhibited ccRCC tumor growth and metastasis by suppressing the EGFR signaling pathway.In a kidney-specific knockout mouse model,loss of MFN2 led to EGFR pathway activation and malignant lesions in kidney.Mechanistically,MFN2 preferably binded small GTPaseRab21 in its GTPloading form,which was colocalized with endocytosed EGFR in ccRCC cells.Through this EGFR-Rab21-MFN2 interaction,endocytosed EGFR was docked to mitochondria and subsequently dephosphorylated by the OMM-residing tyrosine-protein phosphatase receptor type J(PTPRJ).Conclusions:Our findings uncover an important non-canonicalmitochondriadependent pathway regulating EGFR signaling by the Rab21-MFN2-PTPRJ axis,which contributes to the development of novel therapeutic strategies for ccRCC.

Protective effect of Salvia miltiorrhiza and Carthamustinctorius extract against lipopolysaccharide-induced liverinjury

AIM To investigate biological mechanisms underlyingpyruvate kinase M2 isoform （PKM2） regulation of cellmigration and invasion in hepatocellular carcinomacells.METHODS： HepG2 and Huh-7 hepatocellularcarcinoma cell lines were stably transfected and culturedin DMEM （HyClone, Logan, UT, United States）. Toinvestigate the effects of PKM2 on cellular proliferation,hepatocellular carcinoma cells were subjected tothe Cell Counting Kit-8 （Dojindo, Kamimashiki-gun,Kumamoto, Japan）. And investigate the effects ofPKM2 on cell signal pathway related with migrationand invasion, Western immunoblotting were used tofind out the differential proteins. All the antibody usedwas purchaseed from Cell Signal Technology. In orderto explore cell motility used Transwell invasion andwound healing assays. The transwell plate with 0.5mg/mL collagen type Ⅰ （BD Bioscience, San Jose, CA）-coated filters. The wound-healing assay was performedin 6-well plates. Total RNA was extracted using TRIzolreagent （Invitrogen, CA, United States） and thenreverse transcription was conducted. Quantitativereverse transcription-polymerase chain reaction （PCR）analysis was performed with the ABI 7500 real-timePCR system （Applied Biosystems）. We further usedigital gene expression tag profiling and identificationof differentially expressed genes.RESULTS： The cells seeded in four 96-well plates weremeasured OD450 by conducted Cell Counting Kit-8.From this conduction we observed that both HepG2and Huh-7 hepatocellular carcinoma cells with silencedPKM2 turn on a proliferate inhibition; however, cellmigration and invasion were enhanced compared withthe control upon stimulation with epidermal growthfactor （EGF）. Our results indicate that the knockdownof PKM2 decreased the expression of E-cadherin and enhanced the activity of the EGF/EGFR signalingpathway, furthermore up-regulate the subsequentsignal molecular the PLCγ1 and extracellular signalregulatedkinase 1/2 expression in the hepatocellularcarcinoma cell lines HepG2 and Huh-7, which regulatescell motility. These variations we observed were due tothe activation of the transforming growth factor beta（TGFβ） signaling pathway after PKM2 knockdown. Wealso found that the expression of TGFBRI was increasedand the phosphorylation of Smad2 was enhanced.Taken together, our findings demonstrate that PKM2can regulate cell motility through the EGF/EGFR andTGFβ/TGFR signaling pathways in hepatocellularcarcinoma cells.CONCLUSION： PKM2 play different roles in modulatingthe proliferation and metastasis of hepatocellularcarcinoma cells, and this finding could help to guide thefuture targeted therapies.

Persistent EGFR/K-RAS/SIAH pathway activation drives chemo-resistance and early tumor relapse in triple-negative breast cancer

Triple-negative breast cancer(TNBC)is the most aggressive breast cancer subtype.It disproportionately affects BRCA mutation carriers and young women,especially African American(AA)women.Chemoresistant TNBC is a heterogeneous and molecularly unstable disease that challenges our ability to apply personalized therapies.With the approval of immune checkpoint blockade(ICB)for TNBC,the addition of pembrolizumab to systemic chemotherapy has become standard of care(SOC)in neoadjuvant systemic therapy(NST)for high-risk early-stage TNBC.Pembrolizumab plus chemotherapy significantly increased the pathologic complete response(pCR)and improved event-free survival in TNBC.However,clinical uncertainties remain because similarly treated TNBC partial responders with comparable tumor responses to neoadjuvant therapy often experience disparate clinical outcomes.Current methods fall short in accurately predicting which high-risk patients will develop chemo-resistance and tumor relapse.Therefore,novel treatment strategies and innovative new research initiatives are needed.We propose that the EGFR-K-RAS-SIAH pathway activation is a major tumor driver in chemoresistant TNBC.Persistent high expression of SIAH in residual tumors following NACT/NST reflects that the EGFR/K-RAS pathway remains activated(ON),indicating an ineffective response to treatment.These chemoresistant tumor clones persist in expressing SIAH(SIAH^(High/ON))and are linked to early tumor relapse and poorer prognosis.Conversely,the loss of SIAH expression(SIAH^(Low/OFF))in residual tumors post-NACT/NST reflects EGFR/K-RAS pathway inactivation(OFF),indicating effective therapy and chemo-sensitive tumor cells.SIAH^(Low/OFF) signal is linked to tumor remission and better prognosis post-NACT/NST.Therefore,SIAH is well-positioned to become a novel tumor-specific,therapy-responsive,and prognostic biomarker.Potentially,this new biomarker(SIAH^(High/ON))could be used to quantify therapy response,predict chemo-resistance,and identify those patients at the highest risk for tumor relapse and poor survival in TNBC.

ZiyuglycosideⅡinhibits the growth of digestive system cancer cells through multiple mechanisms

Digestive system cancers,including liver,gastric,colon,esophageal and pancreatic cancers,are the leading cause of cancers with high morbidity and mortality,and the question of their clinical treatment is still open.Previous studies have indicated that ZiyuglycosideⅡ(ZYGⅡ),the major bioactive ingredient extract from Sanguisorba officinalis L.,significantly inhibits the growth of various cancer cells.However,the selective anti-tumor effects of ZYGⅡagainst digestive system cancers are not systemically investigated.In this study,we reported the anti-cancer effect of ZYGⅡon esophageal cancer cells(OE21),cholangiocarcinoma cells(Hu CCT1),gastric cancer cells(BGC-823),liver cancer cells(Hep G2),human colonic cancer cells(HCT116),and pancreatic cancer cells(PANC-1).We also found that ZYGⅡinduced cell cycle arrest,oxidative stress and mitochondrial apoptosis.Network pharmacology analysis suggested that UBC,EGFR and IKBKG are predicted targets of ZYGⅡ.EGFR signaling was suggested as the critical pathway underlying the anti-cancer effects of ZYGⅡand both docking simulation and western blot analysis demonstrated that ZYGⅡwas a potential EGFR inhibitor.Furthermore,our results showed synergistic inhibitory effects of ZYGⅡand chemotherapy 5-FU on the growth of cancer cells.In summary,ZYGⅡare effective anti-tumor agents against digestive cancers.Further systemic evaluation of the anti-cancer activities in vitro and in vivo and characterization of underlying mechanism will promote the development of novel supplementary therapeutic strategies based on ZYGⅡfor the treatment of digestive system cancers.

Research progress on KRAS mutations in colorectal cancer

The RAS gene family,responsible for signal transduction within the mitogen activated protein kinase(MAPK)and phosphatidylinositol 3 kinase(PI3K)pathways,is frequently involved in carcinogenesis,and alterations in its member genes can be detected,with variable frequency,in a wide variety of solid and hematological cancers.These alterations may behave as prognostic-predictive biomarkers and driver mutations,making them an interesting therapeutic target.Since their discovery,many strategies have been pursued to act on their signaling pathways.Indeed,in clinical practice,KRAS,the most prominent member of the RAS gene family,represents an especially elusive target in most malignancies;pathway inhibition is carried out upstream,on the EGFR receptor,or downstream,most frequently on the BRAF/MEK/ERK cascade.Recently,clinically relevant direct RAS inhibition has been successfully achieved with the development of potent and selective covalent inhibitors of KRAS c.34G>T(p.G12C).These latest-generation drugs represent both a new and interesting tool in the therapeutic armamentarium and a symbolic end to the myth of KRAS undruggability.However,their clinical relevance and appropriate place in treatment strategies remain to be determined.