Hwang-Phill Kim

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.

OPB-31121, a novel small molecular inhibitor, disrupts the JAK2/STAT3 pathway and exhibits an antitumor activity in gastric cancer cells

We investigated the mechanisms of action and antitumor effects of OPB-31121, a novel STAT3 inhibitor, in gastric cancer cells. OPB-31121 downregulated JAK2 and gp130 expression and inhibited JAK2 phosphorylation which leads to inhibition of STAT3 phosphorylation. OPB-31121 inhibited constitutively activated and IL-6-induced JAK/STAT signaling pathway. OPB-31121 decreased cell proliferation in both gastric cancer cells and in a xenograft model, induced the apoptosis of gastric cancer cells, inhibited the expression of antiapoptotic proteins, and showed synergism with 5-fluorouracil and cisplatin. Taken together, our study suggests that STAT3 inhibition with OPB-31121 can be tested in patients with gastric cancer.

Mucoepidermoid carcinoma of lung: Potential target of EGFR-directed treatment

Mucoepidermoid carcinoma (MEC) of lung is a rare malignancy of lung which originates from minor salivary glands of tracheobronchial tree. EGFR targeted therapy by inhibition of EGFR activation with the specific tyrosine kinase inhibitors (TKIs) has shown meaningful anti-tumor activity in patients with EGFR TK mutation and/or amplification, or in patients with adenocarcinoma. In the present study, we find that MEC has EGFR mutation in 40% (2 out of 5) of cases, and all mutations are L858R mutation. In addition, we also observed that a MEC patient well-responded to EGFR TKI in the absence of EGFR mutation or amplification. These data indicate for the first time that MEC of lung is another potential target of EGFR inhibitor, and more extended clinical investigation is warranted.

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]

Inhibition of the PI3K-Akt/PKB survival pathway enhanced an ethanol extract of Rhus verniciflua Stokes-induced apoptosis via a mitochondrial pathway in AGS gastric cancer cell lines

Rhus verniciflua Stokes (RVS) has been used in traditional Eastern Asia medicine for the treatment of gastritis and stomach cancer, although the mechanism for its biological activity remains to be elucidated. We previously established that an ethanol extract of RVS-induced G(1)-cell cycle arrest via accumulation of p27(Kip1) controlled by Skp2 reduction and apoptosis in AGS human gastric cancer cells. Here, we showed that an ethanol extract of RVS-induced apoptosis via caspase-9 activation (mitochondrial death pathway) is mediated by the loss of mitochondrial membrane potential (MMP, Deltapsi(m)) and the release of cytochrome C from the mitochondrial intermembrane space. In addition, an ethanol extract of RVS inactivated PI3K-Akt/PKB kinase in a time-dependent manner. Moreover, combined treatment of an ethanol extract of RVS and LY294002 (a PI3K inhibitor) markedly increased apoptosis compared to treatment with an ethanol extract of RVS alone. The role of PI3K-Akt/PKB in this process was confirmed by constitutive expression of inactive mutants of this kinase in AGS cells. Finally, siRNA-mediated knockdown of Akt/PKB expression resulted in a significant reduction in AGS cell proliferation. Taken together, these results suggest that an ethanol extract of RVS induces apoptosis via a mitochondrial death pathway in human gastric cancer cells, but not in normal cells, and inhibition of the PI3K-Akt/PKB pathway enhanced the mitochondrial death pathway.

Evaluation of the antitumor effects and mechanisms of PF00299804, a pan-HER inhibitor, alone or in combination with chemotherapy or targeted agents in gastric cancer

Recently, HER2-directed treatment, such as trastuzumab, has shown clinical benefit in HER2-amplified gastric cancer. On the basis of recent studies about epidermal growth factor receptor (EGFR) or HER2-targeting agents (including gefitinib, lapatinib, and trastuzumab) in gastric cancer, the potent effects of pan-HER inhibitors targeting the HER family are anticipated. In this study, we evaluated the activity and mechanisms of PF00299804, an irreversible pan-HER inhibitor, in gastric cancer in vitro and in vivo models. PF00299804 showed significant growth-inhibitory effects in HER2-amplified gastric cancer cells (SNU216, N87), and it had lower 50% inhibitory concentration values compared with other EGFR tyrosine kinase inhibitors, including gefitinib, lapatinib, BIBW-2992, and CI-1033. PF00299804 induced apoptosis and G1 arrest and inhibited phosphorylation of receptors in the HER family and downstream signaling pathways including STAT3, AKT, and extracellular signal–regulated kinases (ERK) in HER2-amplified gastric cancer cells. PF00299804 also blocked EGFR/HER2, HER2/HER3, and HER3/HER4 heterodimer formation as well as the association of HER3 with p85α in SNU216 cells. The combination of PF00299804 with clinically relevant chemotherapeutic agents or molecular-targeted agents including trastuzumab (an anti-HER2 monoclonal antibody), CP751871 (an IGF1R inhibitor), PD0325901 (an ERK1/2 inhibitor), and PF04691502 (a PI3K/mTOR inhibitor) produced synergistic effects. These findings indicate that PF00299804 can be used as a targeted therapy for the treatment of HER2-amplified gastric cancer through inhibition of HER family heterodimer formation and may augment antitumor efficacy of chemotherapeutic and/or molecular-targeted agents. Mol Cancer Ther; 11(2); 439–51. ©2011 AACR.

Targeted sequencing of cancer-related genes in colorectal cancer using next-generation sequencing.

Recent advance in sequencing technology has enabled comprehensive profiling of genetic alterations in cancer. We have established a targeted sequencing platform using next-generation sequencing (NGS) technology for clinical use, which can provide mutation and copy number variation data. NGS was performed with paired-end library enriched with exons of 183 cancer-related genes. Normal and tumor tissue pairs of 60 colorectal adenocarcinomas were used to test feasibility. Somatic mutation and copy number alteration were analyzed. A total of 526 somatic non-synonymous sequence variations were found in 113 genes. Among these, 278 single nucleotide variations were 232 different somatic point mutations. 216 SNV were 79 known single nucleotide polymorphisms in the dbSNP. 32 indels were 28 different indel mutations. Median number of mutated gene per tumor was 4 (range 0–23). Copy number gain (>X2 fold) was found in 65 genes in 40 patients, whereas copy number loss (<X0.5 fold) was found in 103 genes in 39 patients. The most frequently altered genes (mutation and/or copy number alteration) were APC in 35 patients (58%), TP53 in 34 (57%), and KRAS in 24 (40%). Altered gene list revealed ErbB signaling pathway as the most commonly involved pathway (25 patients, 42%). Targeted sequencing platform using NGS technology is feasible for clinical use and provides comprehensive genetic alteration data.

Combined lapatinib and cetuximab enhance cytotoxicity against gefitinib-resistant lung cancer cells

Although non-small cell lung cancer (NSCLC) cells with somatic mutations in their epidermal growth factor receptors (EGFR) initially show a dramatic response to tyrosine kinase inhibitor (TKI), these cells eventually develop resistance to TKI. This resistance may be caused by a secondary T790M mutation in the EGFR tyrosine kinase, which leads to the substitution of methionine for threonine in 790. In this study, we show that a combination of lapatinib and cetuximab overcomes gefitinib resistance in NSCLC with the T790M mutation. We observed that T790M lung cancer cells were resistant to gefitinib, and Stat3 was persistently activated in the resistant cells. A reversible EGFR and HER2 TKI, lapatinib, decreased Stat3 activation by blocking heterodimerization of EGFR and HER2, which led to a modest increase in the inhibitory effect on gefitinib-resistant T790M cells. In addition to lapatinib, the anti-EGFR antibody, cetuximab, induced down-regulation of EGFR and apoptotic cell death in T790M cells. Finally, combined lapatinib and cetuximab treatment resulted in significantly enhanced cytotoxicity against gefitinib-resistant T790M cells in vitro and in vivo. Taken together, these data suggest that treatment with a combination of lapatinib and cetuximab, which induces dimeric dissociation and EGFR down-regulation, appears to be an effective strategy for treatment of patients with EGFR TKI-resistant NSCLC. [Mol Cancer Ther 2008;7(3):607–15]