Regulation of EGFR signalling by palmitoylation and its role in tumorigenesis

The epidermal growth factor receptor (EGFR) is an essential driver of oncogenic signalling, and EGFR inhibitors are some of the earliest examples of successful targeted therapies in multiple types of cancer. The tractability of EGFR as a therapeutic target is overshadowed by the inevitable drug resistance that develops. Overcoming resistance mechanisms requires a deeper understanding of EGFR regulation in cancer cells. In this review, we discuss our recent discovery that the palmitoyltransferase DHHC20 palmitoylates EGFR on the C-terminal domain and plays a critical role in signal regulation during oncogenesis. Inhibiting DHHC20 expression or mutating the palmitoylation site on EGFR alters the EGF-induced signalling kinetics from a transient signal to a sustained signal. The change in signalling is accompanied by a decrease in cell proliferation in multiple human cancer cell lines. Our in vivo studies demonstrate that ablating the gene Zdhhc20 by CRISPR/Cas9-mediated inhibition in a mouse model of oncogenic Kras-driven lung adenocarcinoma potently inhibits tumorigenesis. The negative effect on tumorigenesis is mediated by EGFR since the expression of a palmitoylation-resistant mutant form of EGFR also inhibits Kras-driven lung adenocarcinoma. Finally, reducing EGFR palmitoylation increases the sensitivity of multiple cancer cell lines to existing inhibitors of EGFR and downstream signalling effector pathways. We will discuss the implications of these effects and strategies for targeting these new vulnerabilities.     

BMP-6 inhibits cell proliferation by targeting microRNA-192 in breast cancer

Although bone morphogenetic protein-6 (BMP-6) has been identified as a tumor suppressor associated with breast cancer differentiation and metastasis, the potential roles of BMP-6 in regulating cell cycle progression have not been fully examined. In the present study, we provide the novel finding that induction of BMP-6 in MDA-MB-231 breast cancer cells significantly inhibits cell proliferation by decreasing the number of cells in S phase of the cell cycle, resulting in inhibition of tumorigenesis in a nude mouse xenograft model. Further investigation indicated that BMP-6 up-regulates the expression of microRNA-192 (miR-192) in MDA-MB-231 cells. Elevated expression of miR-192 caused cell growth arrest, which is similar to the effect of BMP-6 induction. Importantly, depletion of endogenous miR-192 by miRNA inhibition significantly attenuated BMP-6-mediated repression of cell cycle progression. In breast cancer tissue, miR-192 expression is significantly down-regulated in tumor samples and positively correlates with the expression of BMP-6, demonstrating the inhibitory effect of BMP-6 on cell proliferation through miR-192 regulation. Additionally, using the RT² Profiler PCR Array, retinoblastoma 1 (RB1) was identified as a direct target of the BMP-6/miR-192 pathway in regulating cell proliferation in breast cancer. In conclusion, we have identified an important role for BMP-6/miR-192 signaling in the regulation of cell cycle progression in breast cancer. Furthermore, BMP-6/miR-192 was expressed at low levels in breast cancer specimens, indicating that this pathway might represent a promising therapeutic target for breast cancer treatment.     

miR‐134 inhibits non‐small cell lung cancer growth by targeting the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is frequently activated in a wide range of solid tumours and represents an important therapeutic target. MicroRNAs (miRNAs) have recently been recognized as a rational and potential modality for anti‐EGFR therapies. However, more EGFR‐targeting miRNAs need to be explored. In this study, we identified a novel EGFR‐targeting miRNA, miRNA‐134 (miR‐134), in non‐small‐cell lung cancer (NSCLC) cell lines. Luciferase assays confirmed that EGFR is a direct target of miR‐134. In addition, the overexpression of miR‐134 inhibited EGFR‐related signaling and suppressed NSCLC cells proliferation by inducing cell cycle arrest and/or apoptosis, suggesting that miR‐134 functions as a tumour suppressor in NSCLC. Further mechanistic investigation including RNAi and rescue experiments suggested that the down‐regulation of EGFR by miR‐134 partially contributes to the antiproliferative role of miR‐134. Last, in vivo experiments demonstrated that miR‐134 suppressed tumour growth of A549 xenograft in nude mice. Taken together, our findings suggest that miR‐134 inhibits non‐small cell lung cancer growth by targeting the EGFR.     

Exploring pyrimidine-substituted curcumin analogues: Design,synthesis and effects on EGFR signaling

Epidermal growth factor receptor (EGFR) is an effective molecular target of anti-cancer therapies. Curcumin inhibits cancer cell growth in vitro by suppressing gene expression of EGFR and reduces tumor growth in various animal models. To overcome instable and insoluble properties of curcumin as therapeutics, we designed and synthesized six novel pyrimidine-substituted curcumin analogues with or without a hydroxyl group originally present in curcumin. The cell viability tests indicated that IC₅₀ of the analogues containing hydroxyl group were 3 to 8-fold lower than those of the analogues without hydroxyl group in two colon cancer cell lines tested. Western blot analysis indicates the analogues containing hydroxyl group inhibited expression and tyrosine phosphorylation of EGFR. Further protein analyses showed that the analogues had anti-cellular proliferation, pro-apoptosis, and cell cycle arrest properties associated with suppressed EGFR expression. These results indicate that the hydroxyl groups in curcumin and the analogues were critical for observed biological activities.     

Alien inhibits E2F1 gene expression and cell proliferation

Recently, using a proteomic approach we have identified the corepressor Alien as a novel interacting factor of the cell cycle regulator E2F1. Unclear was whether this interaction influences cell proliferation and endogenous E2F1 target gene expression. Here, we show by chromatin immunoprecipitation (ChIP) that Alien is recruited in vivo to the E2F binding sites present in the E2F1 gene promoter, inhibits the transactivation of E2F1 and represses endogenous E2F1 gene expression. Interestingly, using synchronized cells to assess the expression of Alien profile during cell cycle the levels of endogenous Alien are increased during G1, G1/S and G2 phase. Furthermore, stable transfection of Alien leads to reduction of cell proliferation. Thus, the data suggest that Alien acts as a corepressor for E2F1 and is involved in cell cycle regulation.     

Salvicine triggers DNA double-strand breaks and apoptosis by GSH-depletion-driven H2O2 generation and topoisomerase II inhibition

Glutathione (GSH), as the major small-molecule antioxidant in cells, has been implicated in the regulation of cell proliferation and apoptosis. Salvicine (SAL), a novel diterpenoid quinone compound, exhibits potent antitumor activities both in vitro and in vivo by poisoning topoisomerase II (Topo II) and has entered Phase II clinical trials for cancer therapy. Herein, we provide further evidence that SAL-induced DNA double-strand breaks (DSBs) and apoptosis by GSH depletion drives H2O2 generation and Topo II inhibition. Our data reveal that treatment with SAL results in a pronounced increase in intracellular H2O2 and is accompanied by the occurrence of DNA DSBs and apoptosis in epithelial HeLa cells. Furthermore, SAL was also noted to trigger a dramatic depletion of intracellular GSH via its direct reaction with GSH. Importantly, the introduction of GSH and overexpression of catalase antagonized SAL-mediated DNA DSBs and apoptosis, and the GSH synthesis inhibitor dl-buthionine-[S,R]-sulfoximine reduced SAL-mediated H2O2 generation, indicating that SAL-mediated H2O2 generation is derived from intracellular GSH depletion. Notably, SAL-mediated Topo II inhibition was also concentration-dependently reversed by GSH. Furthermore, we found that Topo II-defective HL-60/MX2 cells were almost completely resistant to SAL-induced DNA DSBs, suggesting that, in addition to its direct inhibitory effect on Topo II, SAL-mediated H2O2 generation may also trigger DNA DSBs via poisoning of Topo II. All these findings together suggest that GSH-depletion-driven H2O2 generation and Topo II inhibition are both critical for SAL-induced DNA DSBs and apoptosis.     

Modulation of ErbB2 Blockade in ErbB2-Positive Cancers: The Role of ErbB2 Mutations and PHLDA1

We set out to study the key effectors of resistance and sensitivity to ErbB2 tyrosine kinase inhibitors, such as lapatinib in ErbB2-positive breast and lung cancers. A cell-based in vitro site-directed mutagenesis lapatinib resistance model identified several mutations, including the gatekeeper ErbB2 mutation ErbB2-T798I, as mediating resistance. ErbB2-T798I engineered cell models indeed show resistance to lapatinib but remain sensitive to the irreversible EGFR/ErbB2 inhibitor, PD168393, suggestive of potential alternative treatment strategies to overcome resistance. Gene expression profiling studies identified a select group of downstream targets regulated by ErbB2 signaling and define PHLDA1 as an immediately downregulated gene upon oncogenic ErbB2 signaling inhibition. We find significant down-regulation of PHLDA1 in primary breast cancer and PHLDA1 is statistically significantly less expressed in ErbB2 negative compared with ErbB2 positive tumors consistent with its regulation by ErbB2. Lastly, PHLDA1 overexpression blocks AKT signaling, inhibits cell growth and enhances lapatinib sensitivity further supporting an important negative growth regulator function. Our findings suggest that PHLDA1 might have key inhibitory functions in ErbB2 driven lung and breast cancer cells and a better understanding of its functions might point at novel therapeutic options. In summary, our studies define novel ways of modulating sensitivity and resistance to ErbB2 inhibition in ErbB2-dependent cancers.     

EGFR在胃癌中的研究进展

表皮生长因子受体（EGFR）通过下游的信号传导途径参与肿瘤细胞增殖、血管生成、侵袭、转移及凋亡抑制等的基因调控,在多种肿瘤组织中有高表达。近几年在胃癌的研究中显示：EGFR在胃癌中有高表达,与胃癌的发生、发展及生物学行为密切相关,被认为是胃癌等肿瘤治疗的理想靶点。当前以EGFR为靶点的生物治疗成为胃癌新的研究热点,本文就其近几年EGFR在胃癌的表达、预后及靶向治疗等研究现状作一综述。     

Preproendothelin-1 expression is negatively regulated by IFNγ during hepatic stellate cell activation

Endothelin-1 (ET-1), a powerful vasoconstrictor peptide, is produced by activated hepatic stellate cells (HSC) and promotes cell proliferation, fibrogenesis, and contraction, the latter of which has been thought to be mechanistically linked to portal hypertension in cirrhosis. Interferon-γ (IFNγ), a Th1 cytokine produced by T cells, inhibits stellate cell proliferation, fibrogenesis, and muscle-specific gene expression. Whether IFNγ-induced inhibitory effects are linked to regulation of ET-1 expression in activated stellate cells remains unknown. Here we examined IFNγ's effects on preproET-1 mRNA expression and the signaling pathways underlying this process. We demonstrated that preproET-1 mRNA expression in HSCs was prominently increased during cell culture-induced activation; IFNγ significantly inhibited both preproET-1 mRNA expression and ET-1 peptide production. Similar results were found in an in vivo model of liver injury and intraperitoneal administration of IFNγ. PreproET-1 promoter analysis revealed that IFNγ-induced inhibition of preproET-1 mRNA expression was closely linked to the AP-1 and Smad3 signaling pathways. Furthermore, IFNγ reduced JNK phosphorylation, which tightly was associated with decreased phosphorylation of downstream factors c-Jun and Smad3 and decreased binding activity of c-Jun and Smad3 in the preprpET-1 promoter. Importantly, IFNγ reduced both c-Jun mRNA and protein levels. Given the important role of ET-1 in wound healing, our results suggest a novel negative signaling network by which IFNγ inhibits preproET-1 expression, highlighting one potential molecular mechanism for IFNγ-induced host immunomodulation of liver fibrogenesis.     

Acridine yellow G blocks glioblastoma growth via dual inhibition of epidermal growth factor receptor and protein kinase C kinases

Amplification of the epidermal growth factor receptor (EGFR), frequently expressed as a constitutively active deletion mutant (EGFRvIII), occurs commonly in glioblastoma multiformes (GBM). However, blockade of EGFR is therapeutically disappointing for gliomas with PTEN deletion. To search for small molecules treating this aggressive cancer, we have established a cell-based screening and successfully identified acridine yellow G that preferentially blocks cell proliferation of the most malignant U87MG/EGFRvIII cells over the less malignant U87MG/PTEN cells. Oral administration of this compound markedly diminishes the brain tumor volumes in both subcutaneous and intracranial models. It directly inhibits EGFR and PKCs with IC(50) values of ~7.5 and 5 μM, respectively. It dually inhibits EGFR and PKCs, resulting in a blockade of mammalian target of rapamycin signaling and cell cycle arrest in the G(1) phase, which leads to activation of apoptosis in the tumors. Hence, combinatorial inhibition of EGFR and PKCs might provide proof of concept in developing therapeutic agents for treating malignant glioma and other human cancers.     

EGFR inhibition by pentacyclic triterpenes exhibit cell cycle and growth arrest in breast cancer cells

Aims

Pentacyclic triterpenes are a group of molecules with promising anticancer potential, although their precise molecular target remains elusive. The current work aims to investigate the antiproliferative and associated mechanisms of triterpenes in breast cancer cells in vitro.

Main methods

Effect of triterpenes on cell cycle distribution, ROS and key regulatory proteins were analyzed in three breast cancer cells in vitro. Growth inhibition, new DNA synthesis, colony formation assays and Western blot analysis were performed to assess the EGFR inhibitory effect of triterpenes. Molecular docking was performed to study the interaction between EGFR and triterpenes.

Key findings

We have demonstrated the ability of dimethyl melaleucate (DMM), a pentacyclic triterpene to exhibit cell cycle arrest at G0/G1 phase by down-regulation of cyclin D1 through PI3K/AKT inhibition. Further, to identify the upstream target of DMM, potential EGFR inhibitory activity of DMM and three structurally related pentacyclic triterpenes, ursolic acid, 18α-glycyrrhetinic acid and carbenoxolone was investigated. Interestingly, pentacyclic triterpenes limit EGF mediated breast cancer proliferation through sustained inhibition of EGFR and its downstream effectors STAT3 and cyclin D1 in breast cancer lines. We also show pentacyclic triterpenes to bind at the ATP binding pocket of tyrosine kinase domain of EGFR leading to the hypothesis that pentacyclic triterpenes could be a novel class of EGFR inhibitors. In conclusion, pentacyclic triterpenes inhibit EGFR activation through binding with tyrosine kinase domain thereby suppressing breast cancer proliferation.

Significance

Pentacyclic triterpenes may serve as a potential platform for development of novel drugs against breast cancer.     

Anti-MUC1 antibody inhibits EGF receptor signaling in cancer cells

MUC1 is a type I transmembrane glycoprotein aberrantly overexpressed in various cancer cells. High expression of MUC1 is closely associated with cancer progression and metastasis, leading to poor prognosis. We previously reported that MUC1 is internalized by the binding of the anti-MUC1 antibody, from the cell surface to the intracellular region via the macropinocytotic pathway. Since MUC1 is closely associated with ErbBs, such as EGF receptor (EGFR) in cancer cells, we examined the effect of the anti-MUC1 antibody on EGFR trafficking. Our results show that: (1) anti-MUC1 antibody GP1.4, but not another anti-MUC1 antibody C595, triggered the internalization of EGFR in pancreatic cancer cells; (2) internalization of EGFR by GP1.4 resulted in the inhibition of ERK phosphorylation by EGF stimulation, in a MUC1 dependent manner; (3) inhibition of ERK phosphorylation by GP1.4 resulted in the suppression of proliferation and migration of pancreatic cancer cells. We conclude that the internalization of EGFR by anti-MUC1 antibody GP1.4 inhibits the progression of cancer cells via the inhibition of EGFR signaling.     

Ganglioside GM3 blocks the activation of epidermal growth factor receptor induced by integrin at specific tyrosine sites

The epidermal growth factor receptor (EGFR) can be activated by both direct ligand binding and cross-talk with other molecules, such as integrins. This integrin-mediated cross-talk with growth factor receptors participates in regulating cell proliferation, survival, migration, and invasion. Previous studies have shown that ligand-dependent EGFR activation is inhibited by GM3, the predominant ganglioside of epithelial cells, but the effect of GM3 on ligand-independent, integrin-EGFR cross-talk is unknown. Using a squamous carcinoma cell line we show that endogenous accumulation of GM3 disrupts the ligand-independent association of the integrin beta1 subunit with EGFR and results in inhibition of cell proliferation. Consistently, endogenous depletion of GM3 markedly increases the association of EGFR with tyrosine-phosphorylated integrin beta1 and promotes cell proliferation. The ligand-independent stimulation of EGFR does not require focal adhesion kinase phosphorylation or cytoskeletal rearrangement. Stimulation of EGFR and mitogen-activated protein kinase signaling by GM3 depletion involves the phosphorylation of EGFR at tyrosine residues 845, 1068, and 1148 but not 1086 or 1173. The specific blockade of phosphorylation at Tyr-845 with Src family kinase inhibition and at Tyr-1148 with phosphatidylinositol 3-kinase inhibition suggests that GM3 inhibits integrin-induced, ligand-independent EGFR phosphorylation (cross-talk) through suppression of Src family kinase and phosphatidylinositol 3-kinase signaling.