Mutant Gastric Cancer Research Articles

Synergistic induction of autophagy in gastric cancer by targeting CDK4/6 and MEK through AMPK/mTOR pathway

KRAS is a commonly mutated oncogene in human gastric cancer and is often associated with drug resistance and poor prognosis. Co-clinical trial of combined MEK-CDK4/6 inhibition in KRAS mutated cancers demonstrated therapeutic efficacy in patient-derived xenografts and safety in patients. Here, present research focuses on targeting CDK4/6 and MEK synergistically block the proliferation of KRAS-mutated gastric cancer cells in vitro and in vivo and induced autophagy through the AMPK/mTOR pathway. Furthermore, autophagy inhibitor combined with targeting CDK4/6 and MEK therapy had significant antitumor effects on KRAS mutant gastric cancer cells. Clinical trials are needed to determine the mechanism behind this finding and its clinical utility. In conclusion, our results demonstrate autophagy inhibitor combined targeting MEK and CDK4/6 that concurrently block multiple metabolic processes may be an effective therapeutic approach for gastric cancer.

DNMT1 expression partially dictates 5-Azacytidine sensitivity and correlates with RAS/MEK/ERK activity in gastric cancer cells

ABSTRACT Though DNMTs inhibitors were widely used in myelodysplastic syndrome and leukaemia, their application in solid tumours has been limited by low response rate and lack of optimal combination strategies. In gastric cancer (GC), the therapeutic implication of KRAS mutation or MEK/ERK activation for combinational use of DNMTs inhibitors with MEK/ERK inhibitors remains elusive. In this study, stable knockdown of DNMT1 expression by lentiviral transfection led to decreased sensitivity of GC cells to 5-Azacytidine. KRAS knockdown in KRAS mutant GC cells or the MEK/ERK activation by EGF stimulation in GC cells increased DNMT1 expression, while inhibition of MEK/ERK activity by Selumetinib led to decreased DNMT1 expression. 5-Azacytidine treatment, which led to dramatic decline of DNMTs protein levels and increased activity of MEK/ERK pathway, altered the activity of MEK/ERK inhibitor Selumetinib on GC cells. Both RAS-dependent gene expression signature and expression levels of multiple MEK/ERK-dependent genes were correlated with DNMT1 expression in TCGA stomach cancer samples. In conclusion, DNMT1 expression partially dictates 5-Azacytidine sensitivity and correlates with RAS/MEK/ERK activity in GC cells. Combining DNMTs inhibitor with MEK/ERK inhibitor might be a promising strategy for patients with GC.

CBF-Beta Mitigates PI3K-Alpha-Specific Inhibitor Killing through PIM1 in PIK3CA-Mutant Gastric Cancer.

Loss of either NEDD9 or BCL-XL confers hypersensitivity to PI3K-alpha inhibition whereas loss of CBFB confers resistance through a CBFB/PIM1 signaling axis.

Mechanism of RBBP8-mediated homologous recombination repair in gastric cancer synthetic lethal.

It is of great clinical significance to further explore new strategies and potential combined therapeutic targets for gastric cancer. This study aimed to investigate the synthetic lethal effect of RBBP8 molecular intervention combined with a poly ADP ribose polymerase (PARP) inhibitor in non-BRCA mutant gastric cancer and clarify the mechanism by which RBBP8 regulates homologous recombination repair. The role of RBBP8 in DNA damage repair was observed using bioinformatic analysis, western blot analysis, and immunofluorescence. The synthetic lethal effect was verified using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS)and flow cytometry apoptosis experiments. Among the patients with gastric cancer treated with chemotherapy, the prognosis of patients with high RBBP8 expression levels was worse (homologous recombination [HR] = 1.54, p = 0.028). RBBP8 knockdown induced DNA damage and had a synergistic effect with PARP inhibitor treatment on cell viability inhibition and cell apoptosis in AGS (generic code for human gastric adenocarcinoma cells) (t = 11.154, p < 0.001) and N87 (t = 6.362, p < 0.001) cells. RBBP8 knockdown inhibited RAD51 activation and DNA terminal excision in homologous recombination repair. RBBP8 is involved in homologous recombination repair, and molecular intervention into RBBP8 could achieve a synthetic lethal effect with PARP inhibitor treatment in gastric cancer cells.

Identification of FEN1 as a prognostic marker and novel therapeutic target in gastric cancer harboring TP53 mutations.

e16057 Background: As a common tumor suppressor gene, TP53 gene mutation leads to persistent activation of multiple downstream effectors that drive the cancer phenotype. Approximately 50% of gastric cancer (GC) patients harbor TP53 mutations, which confer stronger tumor biology and shorter overall survival (OS). Given that TP53-mutated proteins have proven difficult to target directly, identifying genes that function closely with TP53 and targeting these genes appears to be a promising therapeutic strategy for TP53-mutated GC patients. Methods: TP53 functionally sensitive genes were identified by evaluating the correlation between gene-dependent scores from CRISPR knockout screens and TP53 mRNA expression in TP53 mutant GC cell lines in the Cancer Cell Line Encyclopedia (CCLE) database. If the correlation coefficient is ≥0.6, the gene was considered as TP53 functional sensitive gene. Then TP53 functionally sensitive genes associated with prognosis were screened out in The Cancer Genome Atlas (TCGA). PockDrug-Server was used to predict drug susceptibility of candidate genes. Results: The results showed that in 9 GC cell lines with TP53 mutations and 14 with TP53 wild-type, 4 genes (KLF5, FEN1, CCNA2, CCNB1) were identified as TP53 functional sensitive genes. Among the 4 genes, only FEN1 expression was significantly correlated with OS (HR=1.85, 95CI 1.06−3.26, P=0.029). Multivariate Cox regression analysis showed that high FEN1 expression was an independent predictor of prognosis in TP53-mutated GC. Pocket druggability prediction analysis presented that FEN1 had 8 drug-able pockets, four of which all have a druggability score of 0.8 or higher, including one pocket with a score of 1. Conclusions: These findings suggested that FEN1 was promising prognostic marker and therapeutic target in TP53-mutated GC.

Abstract 4949: Identification of novel regulators of response to PI3Ka inhibition in PIK3CA mutant gastric cancer

Abstract PIK3CA, encoding the active alpha isoform of PI3K, is the second most commonly mutated gene in cancer leading to aberrant PI3K/AKT/mTOR signaling and increased protein translation, glucose metabolism, cellular proliferation, survival and migration. Some 4-25% of gastric cancers (GC) display activating PIK3CA mutations including 80% of the EBV-associated GC subset. Small molecules including pan-PI3K, dual PI3K/mTOR, and pan-AKT inhibitors have shown only moderate clinical success, primarily due to high toxicity and off target effects. Even isoform specific PI3K inhibitors, such as PI3Kα inhibitors, have displayed similar clinical problems. Importantly, PI3Kα mutation-selective inhibitors are on the horizon and will be key for treatment of PIK3CA mutant tumors. However, single agent drug resistance is still an anticipated clinical problem and both intrinsic and acquired resistance will affect success of new drugs. There has been a concerted effort to define mechanisms of resistance as well as identify synergistic drug combinations. In this study we aimed to identify mechanisms of sensitivity and resistance to the PI3Kα-specific inhibitor, BYL719, in PIK3CA mutant gastric cancers by using a CRISPR/Cas9-based screening approach. We found that loss of either NEDD9 or BCL-XL was synergistic with BYL719 and led to increased cell cycle arrest and apoptosis, respectively. In addition to identifying these sensitizer genes, the screening approach allowed us to model intrinsic resistance, and we discovered that knockout of the translation and transcription factor, CBFB, conferred resistance to BYL719. To model acquired resistance, we cultured cells in increasing concentrations of BYL719 over three months and generated clones that were resistant when compared to parental cells. We found that in the clones with acquired resistance, CBFB was significantly down-regulated at the protein and RNA level suggesting a conserved mechanism of drug resistance. We found that CBFB loss led to up-regulation of the serine/threonine protein kinase PIM1, which can phosphorylate and activate several of the same downstream substrates as AKT thereby maintaining pathway activity in the presence of PI3Kα inhibition. Furthermore, a pan-PIM inhibitor re-sensitized both CBFB knockout and acquired resistant cells to BYL719. In the TCGA clinical dataset, we found that CBFB is over-expressed in gastric tumors, specifically in the EBV-associated subset, compared to normal tissue and is significantly over-expressed in PIK3CA mutant compared to wild type tumors. Consistently, we found that PIM1 expression displays the inverse phenotype. These data suggest that while CBFB loss is a key step in the development of PI3Kα inhibitor resistance, PIK3CA mutant gastric tumors may be good candidates for clinical success with BYL719 alone and resistance could be prevented with combination with a PIM kinase inhibitor. Citation Format: Lyla Stanland, Hazel Ang, Jacob Hoj, Yunqiang Chu, Patrick Tan, Kris Wood, Micah Luftig. Identification of novel regulators of response to PI3Ka inhibition in PIK3CA mutant gastric cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4949.

PRMT5 acts as a tumor suppressor by inhibiting Wnt/β-catenin signaling in murine gastric tumorigenesis.

Previous studies have demonstrated the in vitro oncogenic role of protein arginine methyltransferase 5 (PRMT5) in gastric cancer cell lines. The in vivo function of PRMT5 in gastric tumorigenesis, however, is still unexplored. Here, we showed that Prmt5 deletion in mouse gastric epithelium resulted in spontaneous tumorigenesis in gastric antrum. All Prmt5-deficient mice displayed intestinal-type gastric cancer within 4 months of age. Of note, 20% (2/10) of Prmt5 mutants finally developed into invasive gastric cancer by 8 months of age. Gastric cancer caused by PRMT5 loss exhibited the increase in Lgr5+ stem cells, which are proposed to contribute to both the gastric tumorigenesis and progression in mouse models. Consistent with the notion that Lgr5 is the target of Wnt/β-catenin signaling, whose activation is the most predominant driver for gastric tumorigenesis, Prmt5 mutant gastric cancer showed the activation of Wnt/β-Catenin signaling. Furthermore, in human gastric cancer samples, PRMT5 deletion and downregulation were frequently observed and associated with the poor prognosis. We propose that as opposed to the tumor-promoting role of PRMT5 well-established in the progression of various cancer types, PRMT5 functions as a tumor suppressor in vivo, at least during gastric tumor formation.

High Frequency of Juxtamembrane Domain ERBB2 Mutation in Gastric Cancer.

ERBB2 mutation is an emerging therapeutic target in solid tumors; its therapeutic responses depend on the location of mutation. In gastric cancer, the profiles of ERBB2 mutations and their relationship with human epidermal growth factor receptor 2 (HER2) overexpression remain unknown. We aimed to describe the details of ERBB2 mutations in gastric cancer. Comprehensive panel sequencing was performed in 234 advanced gastric cancer patients. We investigated hotspots and clinicopathologic features of ERBB2 mutant gastric cancer in a single institute and evaluated the hotspots of ERBB2 mutation in a public database. Eighteen patients (7.7%) had ERBB2 mutations. The most frequent mutation was p.Arg678Gln (42.1%), which was located in the juxtamembrane domain and was the most common mutation in public databases (20.5%). All 18 ERBB2-mutant patients were negative for HER2 expression. Co-occurring genetic alterations included KRAS, PIK3CA, and ATM mutations. ERBB2 mutations were not associated with HER2 overexpression in gastric cancer patients. The most common mutation was located in the juxtamembrane domain of ERBB2.

Targeting DNA and mutant p53 by a naphthalimide derivative, NA20, exhibits selective inhibition in gastric tumorigenesis by blocking mutant p53-EGFR signaling pathway

Mutations of p53 in cancer cells not only subvert its antiproliferative properties but can also promote various oncogenic responses through a gain-of-function activity. Pharmacological manipulation of the mutant p53 pathway by specific compounds could be an effective strategy for cancer therapy. We show here that gain-of-function p53 mutation in gastric cancer cells promotes tumorigenesis by enhancing p53-EGFR (epidermal growth factor receptor) signaling pathway, and such process can be blocked by small molecule NA20, a naphthalimide derivative that exhibited selective inhibition in p53 mutant gastric cancer cell lines. We found that targeting DNA and blocking the mutant p53-drived carcinogenicity accounted for the primary antitumor effect of NA20 in gastric tumor models. NA20 bound to DNA and p53 identified by a combination of drug tracking, DNA relaxation assay and coimmunoprecipitation-mass spectrometry (CoIP-MS) detection, which led to the p21 activation and the suppression of EGFR signal cascading, thereby evoking cell cycle arrest and cell apoptosis, finally leading to cancer cell inhibition both in vitro and in vivo. Taken together, these results suggest that NA20 may be a potential candidate for gastric cancer therapy.

Maspin subcellular expression in wild-type and mutant TP53 gastric cancers.

BACKGROUNDAlthough the role of p53 in the evolution and prognosis of gastric cancer (GC) has been extensively examined, the exact mechanism of action is incompletely understood. In the last years, p53-target genes were supposed to be involved in the p53 pathway. One of them is the tumor-suppressor gene Maspin, which codifies the protein with the same name. Maspin activity depends on its subcellular localization. To our knowledge, the possible role of TP53 gene in Maspin subcellular localization, in GC cells, has not yet been studied in a large number of human samples.AIMTo evaluate the possible role of wild-type and mutated p53 in Maspin subcellular localization.METHODSThe present study included 266 consecutive patients with GC in which TP53 gene status, and mutations in exons 2 to 11, respectively, were analyzed and correlated with immunohistochemical expression of p53 and Maspin.RESULTSNone of the 266 cases showed mutations in exon 9. The rate of TP53 mutations was 33.83%. The mutation rate was slightly higher in distally-located GCs, with a lower degree (≤ 5 buds/ high power fields) of dyscohesivity (P < 0.01). The wild-type cases had a longer survival, compared with mutant GCs, especially in patients without lymph node metastases, despite the high depth of tumor infiltration (P = 0.01). The Dukes-MAC-like staging system was proved to have the most significant independent prognostic value (P < 0.01). The statistical correlations proved that TP53 gene mutations in exon 7 might induce knockdown of Maspin, but wild-type p53 can partially restore nuclear Maspin expression and decrease the metastatic potential of gastric adenocarcinoma cells.CONCLUSIONDownregulated Maspin might be induced by mutations in exon 7 of the TP53 gene but wild-type p53 can partially restore nuclear Maspin expression. These findings should be proved in experimental studies.

Pharmacogenomic Analysis Reveals CCNA2 as a Predictive Biomarker of Sensitivity to Polo-Like Kinase I Inhibitor in Gastric Cancer.

Despite recent innovations and advances in early diagnosis, the prognosis of advanced gastric cancer remains poor due to a limited number of available therapeutics. Here, we employed pharmacogenomic analysis of 37 gastric cancer cell lines and 1345 small-molecule pharmacological compounds to investigate biomarkers predictive of cytotoxicity among gastric cancer cells to the tested drugs. We discovered that expression of CCNA2, encoding cyclin A2, was commonly associated with responses to polo-like kinase 1 (PLK1) inhibitors (BI-2536 and volasertib). We also found that elevated CCNA2 expression is required to confer sensitivity to PLK1 inhibitors through increased mitotic catastrophe and apoptosis. Further, we demonstrated that CCNA2 expression is elevated in KRAS mutant gastric cancer cell lines and primary tumors, resulting in an increased sensitivity to PLK1 inhibitors. Our study suggests that CCNA2 is a novel biomarker predictive of sensitivity to PLK1 inhibitors for the treatment of advanced gastric cancer, particularly cases carrying KRAS mutation.

Abstract C074: Preclinical study of antitumor effect of alpelisib combined with paclitaxel in gastric caner

Abstract Background: PIK3CA, encoding p110α subunit of PI3K, is the third most frequently mutated gene in gastric cancer (GC): 9–13% of non-hypermutated tumors and 32% of hypermutated tumors. Thus, PI3K pathway could be a potential therapeutic target in GC. Nevertheless, only few studies have been conducted so far. Materials and Methods: This preclinical study aimed to investigate the anti-tumor effects of alpelisib (Novartis, Basel, Switzerland), a PI3K p110α-specific inhibitor, using in vitro and in vivo models of GC. The combinatorial effects of alpelisib and paclitaxel, a commonly used agent for GC, were also evaluated. Results: Among eight GC cell lines (SNU1, SNU16, SNU484, SNU601, SNU638, SNU668, AGS, and MKN1), three harbored PIK3CA hotspot mutations: E542K in SNU601, E545A and E453K in AGS, and E545K in MKN1, while the others were PIK3CA wild-type. These three PIK3CA mutant cells were predominantly sensitive to alpelisib. Alpelisib monotherapy decreased AKT and S6K1 phosphorylation and induced G0/G1 cell cycle arrest regardless of PIK3CA mutational status. Moreover, in PIK3CA mutant cells, GSK3β and BAD phosphorylation was potently abrogated by alpelisib alone, which was not evident in PIK3CA wild-type cells. Alpelisib in combination with paclitaxel demonstrated synergistic anti-proliferative effects, preferentially in PIK3CA mutant cells, by increasing caspase 3/7 activity and resulting in increased apoptosis. Moreover, the alpelisib and paclitaxel combination more potently inhibited the cell migration in wound healing assays and the expression of epithelial-mesenchymal transition (EMT)-associated proteins including Snail and Twist in PIK3CA mutant cells, compared with PIK3CA wild-type cells. In a mouse xenograft model of MKN1 cells, alpelisib combined with paclitaxel significantly enhanced the anti-tumor activity by decreasing Ki-67 expression and increasing TUNEL expression. Moreover, this combination tended to prolong survival of tumor-bearing mice for 4 weeks of treatment period without resulting in significant change in body weight. Conclusions: Alpelisib alone or in combination with paclitaxel demonstrated promising anti-tumor activity and tolerability in in vitro and in vivo models of PIK3CA mutant GC via inactivating PI3K down-stream molecules, decreasing cell migration, and increasing apoptosis. Our data suggest that this novel combination is worthy of further clinical investigations in patients with PIK3CA mutant GC. Keywords: alpelisib; BYL719; paclitaxel; PIK3CA; gastric cancer; combination; chemotherapy Citation Format: Ji-Won Kim, Kui-Jin Kim, Ji Hea Sung, Koung Jin Suh, Ji Yun Lee, Se Hyun Kim, Jin Won Kim, Jeong-Ok Lee, Yu Jung Kim, Soo-Mee Bang, Jee Hyun Kim, Jong Seok Lee, Keun-Wook Lee. Preclinical study of antitumor effect of alpelisib combined with paclitaxel in gastric caner [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C074. doi:10.1158/1535-7163.TARG-19-C074

Prolonged MEK inhibition leads to acquired resistance and increased invasiveness in KRAS mutant gastric cancer.

Gastric cancer (GC) is one of the most common causes of cancer-associated death. However, traditional therapeutic strategies have failed to significantly improve the survival of patient with advanced GC. While KRAS mutations have been found in some patients with gastric cancer, an effective therapy to treat KRAS-driven gastric cancer has not been established yet. To provide a rationale for clinical application of kinase inhibitors targeting RAS pathways, we first determined the sensitivity of GC cell lines harboring KRAS mutations or amplification to RAS pathway inhibitors. We found that MAPK pathway inhibitors (MEKi and ERKi) were more effective than AKT inhibitor, suggesting that KRAS-driven gastric cancer cells are dependent on MAPK pathway for survival. Further, we established a KRAS mutant GC cell line with acquired resistance to MEK inhibitors in order to mimic clinical situation of kinase inhibitor resistance. A comprehensive analysis of tyrosine phosphorylation in receptor tyrosine kinases in combination with small molecule chemical library screening revealed upregulated c-MET phosphorylation in this resistance cell line with elevated sensitivity to c-MET TKI (crizotinib) and PI3K/mTOR dual inhibitor (BEZ235). We also showed that migration and invasion of resistant cells were promoted, and crizotinib and BEZ235 could inhibit this malignant phenotype. Overall, our results indicate that prolonged MAPK pathway inhibition could result in acquired resistance which is associated with increased malignant phenotype in KRAS mutant GC and pharmacological targeting c-MET and PI3K/mTOR could overcome this problem.

Abstract 4791: OMO-1, a potent, highly selective, orally bioavailable, MET kinase inhibitor with a favorable preclinical toxicity profile, shows both monotherapy activity, against MET pathway-driven tumors, and EGFR TKI combination activity in acquired resistance models

Abstract Activation of the MET/HGF pathway has been linked to tumor initiation, metastasis, angiogenesis and resistance to therapeutic agents. Here we present pharmacological characterization of OMO-1 (formerly JNJ-38877618), a potent, highly selective, orally bioavailable MET kinase inhibitor with nM binding affinity (Kd=1.4 nM) and enzyme inhibitory activity against wt and M1268T mutant MET (2 and 3 nM IC50). MET inhibitory effects were assessed in proliferation, colony formation and motility assays. OMO-1 displayed nM potency against MET Ampl/mutant and therapy resistant models. In vivo, OMO-1 induced complete inhibition of tumor growth in 3 models: the SNU5 MET amp gastric, U87-MG HGF autocrine glioblastoma and Hs746T MET exon 14 skipping mutant gastric cancer. OMO-1 induced regression of large MET amplified EBC-1 SqNSCLC where OMO-1 led to dose- and time-dependent inhibition of MET kinase activation, with the duration of target shut down considerably exceeding plasma exposure times. Combination treatments were well tolerated and improved EGFR targeted therapy. Although single agent OMO-1 had no effect on NSCLC HCC827 EGFR, combination with Erlotinib led to delayed onset of tumor recurrence. The acquired EGFR inhibitor resistant model HCC827-ER1 was determined to be MET amplified. OMO-1 and erlotinib both inhibited tumor growth of this model whilst combination induced tumor regression. In an EGFR inhibitor resistant PDX having MET amplification, single agent OMO-1 caused tumour stasis whereas MetMab/erlotinib only led to tumor growth delay. The potent preclinical activity we have observed, supports ongoing clinical development of OMO-1 in patients with MET pathway-driven tumors. Citation Format: Marion Libouban, Eleonora Jovcehva, Desiree De Lange, Boudewijn Janssens, Tinne Verhulst, Souichi Ogata, Berthold Wroblowski, Laurence Mevellec, Tianbao Lu, Glen Clack, Timothy Perera. OMO-1, a potent, highly selective, orally bioavailable, MET kinase inhibitor with a favorable preclinical toxicity profile, shows both monotherapy activity, against MET pathway-driven tumors, and EGFR TKI combination activity in acquired resistance models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4791.

Inhibition enhancer of zeste homologue 2 promotes senescence and apoptosis induced by doxorubicin in p53 mutant gastric cancer cells.

Enhancer of zeste homologue 2 (EZH2) is crucially involved in epigenetic silencing by acting as a histone methyltransferase. Although EZH2 is overexpressed in many cancers and is involved in malignant cell proliferation and invasion, the role of EZH2 in senescence induced by DNA damage has up to now remained largely unknown. In this study, we sought to explore the outcome of EZH2 depletion along with exposure of doxorubicin (DOX), and related mechanisms, in gastric cancer cells. Here, senescence induced by DNA damage was achieved in gastric cancer cells by DOX treatment. EZH2 was downregulated by transfection with siRNA or treated with (-)-epigallocatechin-3-gallate, a targeted inhibitor. Senescence-associated β galactosidase (SA-β-gal) and formation of senescence-associated heterochromatin foci were used to identify cell senescence. To investigate effects of EZH2 depletion on the cell cycle, apoptosis and proliferation, flow cytometry and MTT analysis were employed. Changes in p53-p21 axis activation were detected by Western blotting. We found that cell proliferative arrest caused by DOX could be promoted by EZH2 depletion. Mechanistically, EZH2 depletion not only worked in coordination with DNA damage during the progression of cell senescence but also promoted apoptosis in p53 mutant cells. However, it had no cooperative relationship with DOX in p53 wild-type cells. These data help unravel a crucial role for EZH2 in senescence and apoptosis in gastric cancer cells and that p53 genomic status was associated with different cell responses to EZH2 silencing.

The Combination of RAD001 and MK-2206 Exerts Synergistic Cytotoxic Effects against PTEN Mutant Gastric Cancer Cells: Involvement of MAPK-Dependent Autophagic, but Not Apoptotic Cell Death Pathway

In the current study, we showed that the combination of mammalian target of rapamycin (mTOR) inhibitor RAD001 (everolimus) and Akt inhibitor MK-2206 exerted synergistic cytotoxic effects against low-phosphatase and tensin homolog (PTEN) gastric cancer cells (HGC-27 and SNU-601 lines). In HGC-27 cells, RAD001 and MK-2206 synergistically induced G1/S cell cycle arrest, growth inhibition, cell death but not apoptosis. RAD001 and MK-2206 synergistically induced light chain 3B (LC3B) and beclin-1 expression, two important autophagy indicators. Meanwhile, the autophagy inhibitor 3-methyladenine (3-MA) and chloroquine inhibited the cytotoxic effects by RAD001 and MK-2206, suggesting that autophagic, but not apoptotic cell death was important for the cytotoxic effects by the co-administration. We observed that the combination of RAD001 and MK-2206 exerted enhanced effects on Akt/mTOR inhibition, cyclin D1 down-regulation and ERK/MAPK(extracellular signal-regulated kinase/mitogen-activated protein kinases) activation. Intriguingly, MEK/ERK inhibitors PD98059 and U0126 suppressed RAD001 plus MK-2206-induced beclin-1 expression, autophagy induction and cytotoxicity in HGC-27 cells. In conclusion, these results suggested that the synergistic anti-gastric cancer cells ability by RAD001 and MK-2206 involves ERK-dependent autophagic cell death pathway.

The AKT inhibitor AZD5363 is selectively active in PI3KCA mutant gastric cancer, and sensitizes a patient-derived gastric cancer xenograft model with PTEN loss to Taxotere

IntroductionActivation of the PI3K/AKT pathway is a common phenomenon in cancer due to multiple mechanisms, including mutation of PI3KCA, loss or mutation of PTEN, or over-expression of receptor tyrosine kinases. We recently developed a novel AKT kinase inhibitor, AZD5363, and demonstrated that HGC27, a cell line harboring both PI3KCA mutation and PTEN loss, displayed the greatest sensitivity to this AKT inhibitor in vitro and in vivo.Case preparationTo further elucidate the correlation between AZD5363 response and genetic alterations in gastric cancer (GC) and identify GC patients with both PI3KCA mutations and PTEN loss, we investigated the effects of pharmacological inhibition of AKT on a panel of 20 GC cell lines and genetic aberrations in tumor samples from a cohort of Chinese GC patients. We demonstrated that GC cells with PI3KCA mutations were selectively sensitive to AZD5363. Disease linkage studies showed that PI3KCA activating mutations or PTEN loss were found in 2.7% (4/150) and 23% (14/61) of Chinese GC patients respectively. To further dissect the role of PI3KCA mutation and PTEN loss in response to AKT inhibition, we tested the antitumor activity of AZD5363 in two patient-derived GC xenograft (PDGCX) models harboring either PI3KCA mutation or PTEN loss. Our data indicated that AZD5363 monotherapy treatment led to a moderate response in the PI3KCA mutant PDGCX model. Whilst monotherapy AZD5363 or Taxotere were ineffective in the PTEN negative PDGCX model, significant anti-tumor activity was observed when AZD5363 was combined with Taxotere.ConclusionOur results indicated that PI3KCA mutation is an important determinant of response to AKT inhibition in GC and combination with AZD5363 can overcome innate resistance to Taxotere in a PTEN loss PDGCX model. It is suggested that AKT inhibitor is an attractive option for treatment of a new segment of GC patients with aberrant PI3K/AKT signaling.

KRAS and BRAF mutations are rare and related to DNA mismatch repair deficiency in gastric cancer from the East and the West: Results from a large international multicentre study

Background:Inhibitors of the epidermal growth factor (EGFR) signaling pathway have a major role in the treatment of KRAS wild-type colorectal cancer patients. The EGFR pathway has been shown to be activated in gastric cancer (GC). However, published data on KRAS and BRAF mutation status is limited in GC and has not been compared between GC from different geographic regions.Methods:The prevalence of KRAS and BRAF mutations was established in 712 GC: 278 GC from the United Kingdom, 230 GC from Japan and 204 GC from Singapore. The relationship between KRAS/BRAF mutation status, DNA mismatch repair (MMR) status, clinicopathological variables and overall survival was analysed.Results:Overall, 30 (4.2%) GC carried a KRAS mutation. In total, 5.8% of the UK GC, 4% of Japan GC and 1.5% of Singapore GC were KRAS mutant. KRAS mutant GC had fewer lymph node metastases in the UK cohort (P=0.005) and were more frequent in elderly patients in the Japan cohort (P=0.034). KRAS mutations were more frequent in MMR-deficient GC in the UK and the Japanese cohort (P<0.05). A BRAF mutation was only detected in a single Japanese GC.Conclusions:This large multicentre study demonstrated that KRAS mutations and DNA MMR deficiency have a role in a small subgroup of GC irrespective of country of origin, suggesting that this subgroup of GC may have developed along a common pathway. Further studies need to establish whether concomitant mutations or amplifications of other EGFR signalling pathway genes may contribute to the activation of this pathway in GC.

Pharmacologic synergy between dual phosphoinositide-3-kinase and mammalian target of rapamycin inhibition and 5-Fluorouracil in PIK3CA mutant gastric cancer cells

Phosphoinositide-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitors are an emerging class of anti-cancer agents. Here, we tested the hypothesis that the dual PI3K/mTOR inhibitor, PI103, could synergize with the chemotherapeutic agent, 5-fluorouracil (5-FU) by inhibiting E2F1, thymidylate synthase (TS) and enhancing DNA damage. Drug combination effects were assessed in gastric cancer cells using the median-effect equation. The specific effects of inhibition of E2F1 and PIK3CA were examined by siRNA, and mTOR by rapamycin exposure. Protein expression and apoptosis pre- and post-treatment was measured using standard methods. PI103 and 5-FU was synergistic in 3 out of 5 gastric cancer cell lines tested. Synergy was associated with PI3KCA mutation, reduced TS and E2F1 protein levels, increased H2AX phosphorylation and apoptosis. E2F1 siRNA enhanced sensitivity to 5-FU only in cells displaying synergy. Excess thymidine exposure converted synergism to antagonism in all cells. Inhibition of PI3K and mTOR alone enhanced 5-FU cytotoxicity in only 2 out of 3 cell lines that displayed synergy each. In AGS cells, PI3K inhibition alone enhanced 5-FU sensitivity as much as dual PI3K/mTOR inhibition. In HGC27 cells, dual inhibition increased 5-FU sensitivity more than single PI3K or mTOR inhibition. Combined PI103 and 5-FU treatment reduced in vivo tumor growth more than treatment with single agents. PI3K/mTOR inhibitors can enhance 5-FU cytotoxicity in vitro and in vivo, especially in PIK3CA mutant tumor cells. Dual, rather than single, PI3K/mTOR inhibitors may combine better with 5-FU due to cellular heterogeneity in sensitivity to PI3K and mTOR inhibition.

NVP-BKM120, a novel PI3K inhibitor, shows synergism with a STAT3 inhibitor in human gastric cancer cells harboring KRAS mutations

Aberrations of Phosphoinositide 3-kinase (PI3K)/AKT signaling are frequently observed in many types of cancer, promoting its emergence as a promising target for cancer treatment. PI3K can become activated by various pathways, one of which includes RAS. RAS can not only directly activate the PI3K/AKT pathway via binding to p110 of PI3K, but also regulates mTOR via ERK or RSK independently of the PI3K/AKT pathway. Thus, actively mutated RAS can constitutively activate PI3K signaling. Additionally, in RAS tumorigenic transformation, signal transducer and activator of transcription 3 (STAT3) has been known also to be required. In this study, we examined the efficacy of NVP-BKM120, a pan-class I PI3K inhibitor in human gastric cancer cells and hypothesized that the combined inhibition of PI3K and STAT3 would be synergistic in KRAS mutant gastric cancer cells. NVP-BKM120 demonstrated anti-proliferative activity in 11 human gastric cancer cell lines by decreasing mTOR downstream signaling. But NVP-BKM120 treatment increased p-AKT by subsequent abrogation of feedback inhibition by stabilizing insulin receptor substrate-1. In KRAS mutant gastric cancer cells, either p-ERK or p-STAT3 was also increased upon treatment of NVP-BKM120. The synergistic efficacy study demonstrated that dual PI3K and STAT3 blockade showed a synergism in cells harboring mutated KRAS by inducing apoptosis. The synergistic effect was not seen in KRAS wild-type cells. Together, these findings suggest for the first time that the dual inhibition of PI3K and STAT3 signaling may be an effective therapeutic strategy for KRAS mutant gastric cancer patients.