Young-Kwang Yoon

The growth inhibitory effect of lapatinib, a dual inhibitor of EGFR and HER2 tyrosine kinase, in gastric cancer cell lines ☆

HER2 overexpression is observed in 5-25% of gastric cancers. Lapatinib is a dual inhibitor of the epidermal growth factor receptor and HER2 tyrosine kinase. We examined the antitumor effect of lapatinib in gastric cancer cell lines. Lapatinib induced selective and potent growth inhibition in two HER2-amplified gastric cancer cell lines (SNU-216 and NCI-N87). Lapatinib inhibited the phosphorylation of HER2, EGFR and downstream signaling proteins, resulting in G1 arrest in both cell lines with down-regulation of cMyc and induction of p27kip1. Lapatinib also induced apoptosis in NCI-N87 which has high HER2 amplification ratio. Lapatinib combined with 5-fluorouracil, cisplatin, oxaliplatin or paclitaxel showed an additive or synergistic effect. These results provide a rationale for the future clinical trials of lapatinib combined with cytotoxic drugs in the treatment of HER2-positive gastric cancer.

Lapatinib, a Dual EGFR and HER2 Tyrosine Kinase Inhibitor, Downregulates Thymidylate Synthase by Inhibiting the Nuclear Translocation of EGFR and HER2

Background Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) has been shown to exert a synergistic antitumor effect when combined with fluoropyrimidine. This synergy may be attributable to the downregulation of thymidylate synthase (TS), which is frequently overexpressed in fluoropyrimidine-resistant cancer cells. However, the molecular mechanism underlying the downregulation of TS has yet to be clearly elucidated. Methodology and Principal Findings In this study, we demonstrate that lapatinib, a dual TKI of EGFR and HER2 downregulates TS via inhibition of the nuclear translocation of EGFR and HER2. From our cDNA microarray experiments, we determined that a variety of nucleotide synthesis-related genes, including TS, were downregulated with lapatinib, and this was apparent in HER2-amplified cells. Targeted and pharmacologic inhibition assays confirmed that the dual inhibition of EGFR and HER2 is required for the more effective reduction of TS as compared to what was observed with gefitinib or trasutuzumab alone. Additionally, we determined that co-transfected EGFR and HER2 activate the TS gene promoter more profoundly than do either EGFR or HER2 alone. The translocation of EGFR and HER2 into the nucleus and the subsequent activation of the TS promoter were inhibited by lapatinib. Conclusions and Significance These results demonstrate that lapatinib inhibits the nuclear translocation of EGFR and HER2 and downregulates TS, thus sensitizing cancer cells to fluoropyrimidine.

KRAS mutant lung cancer cells are differentially responsive to MEK inhibitor due to AKT or STAT3 activation: Implication for combinatorial approach

KRAS is frequently mutated in nonsmall cell lung cancer (NSCLC), resulting in the activation of the MAPK/ERK kinase (MEK)/ERK pathway. High‐throughput mutation profile has shown that lung cancer frequently harbors comutation of cancer‐related genes. Therefore, given that cancer cells have multiple genetic alterations, combinatorial therapeutic strategy is demanded for effective cancer therapy. To address this, we first characterized MEK dependence in four NSCLC cells. Two cells (H358, A549) carried KRAS mutation only, and the other two (H23, H157) harbored comutation of KRAS/PTEN. H358 cells with KRAS mutation only were sensitive to MEK inhibition. However, the other KRAS mutant A549 cells were resistant to MEK inhibition. Previously, we have shown that dual inhibition of EGFR and MEK signaling shows a synergistic effect on KRAS mutant gastric cancer cells by suppressing compensatory activation of AKT. Here we also observed that this combination was effective in KRAS mutant A549 cells. However, the combination was ineffective in H23 and 157 cells with comutation of KRAS/PTEN. Compared to KRAS mutant/PTEN wild‐type cells, signal transducer and activator of transcription 3 (STAT3) was significantly activated following MEK inhibition in KRAS/PTEN comutant cells. Combined STAT3 inhibition by a JAK2 inhibitor or gene knockdown with MEK inhibition blocked STAT3 activation, synergistically suppressed cell growth, and induced apoptosis in comutant cells. Taken together, our study provides molecular insights that help explain the heterogeneous response to MEK inhibition in KRAS mutant lung cancers, and presents a rationale for the clinical investigation of combination of MEK and EGFR inhibitor or MEK and JAK2 inhibitor depending on PTEN status.

Combination of EGFR and MEK1/2 inhibitor shows synergistic effects by suppressing EGFR/HER3-dependent AKT activation in human gastric cancer cells

EGFR tyrosine kinase inhibitors have shown promising efficacy in the treatment of tumors with EGFR mutations and amplifications. However, tyrosine kinase inhibitors have also proven ineffective against most tumors with EGFR wild-type (WT) alleles. Although some genetic changes, including the KRAS mutation, have been shown to confer resistance to tyrosine kinase inhibitors, novel strategies for the treatment of cancer patients with tumors harboring EGFR WT alleles have yet to be thoroughly delineated. The principal objective of this study was to improve our current understanding of drug interactions between EGFR and MAP/ERK kinase (MEK) inhibitors in an effort to gain insight into a novel therapeutic strategy against EGFR WT tumors. Using a panel of human EGFR WT gastric cancer cell lines, we showed that gastric cancer cells harboring the KRAS mutation were selectively sensitive to MEK inhibition as compared with those cells harboring KRAS and PI3K mutations and KRAS WT alleles. However, all cell lines were found to be resistant to EGFR inhibition. The results from Western blots and phosphoprotein arrays showed that, in MEK inhibitor resistant cell lines, AKT was activated through the EGFR/HER3/PI3K pathway following AZD6244 (ARRY-142886) treatment. Blockade of this feedback mechanism through the targeting of MEK and EGFR resulted in detectable synergistic effects in some cell lines in vitro and in vivo. Our results provide the basis for a rational combination strategy against human EGFR WT gastric cancers, predicated on the understanding of cross-talk between the MEK and EGFR pathways. [Mol Cancer Ther 2009;8(9):2526–36]

RAD51C-Deficient Cancer Cells Are Highly Sensitive to the PARP Inhibitor Olaparib

A PARP inhibitor is a rationally designed targeted therapy for cancers with impaired DNA repair abilities. RAD51C is a paralog of RAD51 that has an important role in the DNA damage response. We found that cell lines sensitive to a novel oral PARP inhibitor, olaparib, had low levels of RAD51C expression using microarray analysis, and we therefore hypothesized that low expression of RAD51C may hamper the DNA repair process, resulting in increased sensitivity to olaparib. Compared with the cells with normal RAD51C expression levels, RAD51C-deficient cancer cells were more sensitive to olaparib, and a higher proportion underwent cell death by inducing G2–M cell-cycle arrest and apoptosis. The restoration of RAD51C in a sensitive cell line caused attenuation of olaparib sensitivity. In contrast, silencing of RAD51C in a resistant cell line enhanced the sensitivity to olaparib, and the number of RAD51 foci decreased with ablated RAD51C expression. We also found the expression of RAD51C was downregulated in cancer cells due to epigenetic changes and RAD51C expression was low in some gastric cancer tissues. Furthermore, olaparib significantly suppressed RAD51C-deficient tumor growth in a xenograft model. In summary, RAD51C-deficient cancer cells are highly sensitive to olaparib and offer preclinical proof-of-principle that RAD51C deficiency may be considered a biomarker for predicting the antitumor effects of olaparib. Mol Cancer Ther; 12(6); 865–77. ©2013 AACR.

RAD001 shows activity against gastric cancer cells and overcomes 5-FU resistance by downregulating thymidylate synthase

We evaluated RAD001, an inhibitor of the mammalian target of rapamycin (mTOR) in human gastric cancer cell lines and determined the molecular mechanisms. RAD001 has marked growth inhibitory activity against the SNU-1 and SNU-216 cells. It inhibited phosphorylation of mTOR and S6K, and induced G1 cell cycle arrest. Synergistic growth-inhibitory effects in combination with 5-fluorouracil (5-FU) was identified. Furthermore, RAD001 conferred sensitivity to 5-FU-resistant cell lines by downregulating thymidylate synthase (TS). In conclusion, RAD001 showed growth inhibitory activity against gastric cancer cells and acted synergistically with cytotoxic agents such as 5-FU by downregulating TS.

Antitumor Activity of Saracatinib (AZD0530), a c-Src/Abl Kinase Inhibitor, Alone or in Combination with Chemotherapeutic Agents in Gastric Cancer

Src is a nonreceptor tyrosine kinase involved in the cross-talk and mediation of many signaling pathways that promote cell proliferation, adhesion, invasion, migration, and tumorigenesis. Increased Src activity has been reported in many types of human cancer, including gastric cancer. Therefore, this factor has been identified as a promising therapeutic target for cancer treatments, and targeting Src in gastric cancer is predicted to have potent effects. We evaluated the antitumor effect of a c-Src/Abl kinase inhibitor, saracatinib (AZD0530), alone or combined with chemotherapeutic agents in gastric cancer cell lines and a NCI-N87 xenograft model. Among 10 gastric cancer cell lines, saracatinib specifically inhibited the growth and migration/invasion of SNU216 and NCI-N87 cells. Saracatinib blocked the Src/FAK, HER family, and oncogenic signaling pathways, and it induced G1 arrest and apoptosis in SNU216 and NCI-N87 cells. Apoptosis required induction of the proapoptotic BCL2 family member Bim. Knockdown of Bim using siRNA decreased apoptosis induced by treatment with saracatinib, suggesting that Bim has an important role in saracatinib-induced apoptosis. Saracatinib enhanced the effects of lapatinib, an EGFR/HER2 dual inhibitor, in SNU216 and NCI-N87 cells. Furthermore, combined treatment with saracatinib and 5-fluorouracil (5-FU) or cisplatin exerted synergistic effects in both saracatinib-sensitive and saracatinib-resistant cells. Consistent with our in vitro findings, cotreatment with saracatinib and 5-FU resulted in enhanced antitumor activity in the NCI-N87 xenografts. These data indicate that the inhibition of Src kinase activity by saracatinib alone or in combination with other agents can be a strategy to target gastric cancer. Mol Cancer Ther; 12(1); 16–26. ©2013 AACR.

Heterodimerization of glycosylated insulin-like growth factor-1 receptors and insulin receptors in cancer cells sensitive to anti-IGF1R antibody.

Background Identification of predictive biomarkers is essential for the successful development of targeted therapy. Insulin-like growth factor 1 receptor (IGF1R) has been examined as a potential therapeutic target for various cancers. However, recent clinical trials showed that anti-IGF1R antibody and chemotherapy are not effective for treating lung cancer. Methodology/Principal Findings In order to define biomarkers for predicting successful IGF1R targeted therapy, we evaluated the anti-proliferation effect of figitumumab (CP-751,871), a humanized anti-IGF1R antibody, against nine gastric and eight hepatocellular cancer cell lines. Out of 17 cancer cell lines, figitumumab effectively inhibited the growth of three cell lines (SNU719, HepG2, and SNU368), decreased p-AKT and p-STAT3 levels, and induced G 1 arrest in a dose-dependent manner. Interestingly, these cells showed co-overexpression and altered mobility of the IGF1R and insulin receptor (IR). Immunoprecipitaion (IP) assays and ELISA confirmed the presence of IGF1R/IR heterodimeric receptors in figitumumab-sensitive cells. Treatment with figitumumab led to the dissociation of IGF1-dependent heterodimeric receptors and inhibited tumor growth with decreased levels of heterodimeric receptors in a mouse xenograft model. We next found that both IGF1R and IR were N-linked glyosylated in figitumumab-sensitive cells. In particular, mass spectrometry showed that IGF1R had N-linked glycans at N913 in three figitumumab-sensitive cell lines. We observed that an absence of N-linked glycosylation at N913 led to a lack of membranous localization of IGF1R and figitumumab insensitivity. Conclusion and Significance The data suggest that the level of N-linked glycosylated IGF1R/IR heterodimeric receptor is highly associated with sensitivity to anti-IGF1R antibody in cancer cells.

Antitumor activity of HM781-36B, an irreversible Pan-HER inhibitor, alone or in combination with cytotoxic chemotherapeutic agents in gastric cancer

Trastuzumab, a HER2 directed treatment has shown clinical benefit in HER2 amplified gastric cancer. This study demonstrated the potent antitumor activity of HM781-36B, a quinazoline-based irreversible pan-HER inhibitor, in HER2 amplified gastric cancer cells (SNU216 and N87) in vitro and in vivo. HM781-36B inhibited phosphorylation of HER family and downstream signaling molecules, and induced apoptosis and G1 arrest. Furthermore, HM781-36B exerted synergistic effects with chemotherapeutic agents in both HER2 amplified and HER2 non-amplified gastric cancer cells. Therefore, HM781-36B may be useful for the treatment of HER2 amplified gastric cancer alone or in combination with chemotherapeutic agents.

Gene silencing of EREG mediated by DNA methylation and histone modification in human gastric cancers

Epiregulin (EREG) induces cell growth by binding to the epidermal growth factor receptor (EGFR). Expression of EREG affects sensitivity to cetuximab a chimeric monoclonal antibody that inhibits the EGFR signaling pathway. The mechanism through which EREG is regulated is largely unknown, but a methyl-array study previously performed by our group revealed that EREG is methylated in gastric cancer cells. In this study, we found that EREG gene expression was low in 7 out of 11 gastric cancer cells and this downregulation was mediated by aberrant CpG methylation of the EREG promoter. Treatment with 5-aza-CdR restored EREG expression and demethylated CpG sites in the EREG promoter. Compared with DNA methyltransferase 1 (DNMT1), knock-down of DNA methyltransferase 3b (DNMT3b) significantly increased the expression of EREG and led to the demethylation of specific CpG sites in the EREG promoter, suggesting that DNMT3b primarily regulates CpG methylation and silencing of the EREG gene. EREG methylation was observed in 30% (4/13) of human primary gastric tumor tissues we evaluated. In addition to DNA methylation, results from a chromatin immunoprecipitation assay demonstrated that transcriptional levels of EREG were associated with the enrichment of active histone marks (H3K4me3 and AcH3) and of a repressive mark (H3K27me2). Treatment with 5-aza-CdR dynamically increased the low occupancy of H3K4me3 and AcH3, while decreasing the high enrichment of H3K27me2, indicating that dynamic histone modifications contribute to EREG regulation in addition to DNA methylation. Finally, the combination of 5-aza-CdR and cetuximab exerted a synergistic anti-proliferative effect on gastric cancer cells. Taken together, the results of our study showed for the first time that EREG is epigenetically silenced in gastric cancer cells by aberrant DNA methylation and histone modification.