King Xin Koh

MicroRNA-130b regulates the tumour suppressor RUNX3 in gastric cancer

AIM Accumulating evidence indicates that RUNX3 is an important tumour suppressor that is inactivated in many cancer types. This study aimed to assess the role of microRNA (miRNA) in the regulation of RUNX3. METHODS Four bioinformatic algorithms were used to predict miRNA binding to RUNX3. The correlation between candidate miRNAs and RUNX3 expression in cell lines was determined by real-time reverse transcriptase quantitative PCR (RT-qPCR) and Western blot. Candidate miRNAs were tested for functional effects through transfection of miRNA precursors and inhibitors, and monitoring cell viability, apoptosis and Bim expression. miRNA and RUNX3 expression, RUNX3 methylation and RUNX3 protein levels were assessed in gastric tissue by RT-qPCR, Methylight analysis and immunohistochemistry, respectively. RESULTS Bioinformatics, gene and protein expression analysis in eight gastric cell lines identified miR-130b as the top candidate miRNA for RUNX3 binding. Overexpression of miR-130b increased cell viability, reduced cell death and decreased expression of Bim in TGF-beta mediated apoptosis, subsequent to the downregulation of RUNX3 protein expression. In 15 gastric tumours, miR-130b expression was significantly higher compared to matched normal tissue, and was inversely associated with RUNX3 hypermethylation. CONCLUSION Attenuation of RUNX3 protein levels by miRNA may reduce the growth suppressive potential of RUNX3 and contribute to tumourigenesis.

Meta-analysis of genetic polymorphisms and gastric cancer risk: Variability in associations according to race

The goal of this study was to consolidate information on genetic risk factors for gastric cancer. An additional aim was to investigate the influence of race on these genetic risk associations. Relevant studies were identified from PubMed and references of retrieved articles. Meta-analysis techniques were used to summarise associations between genetic polymorphisms and gastric cancer. A total of 203 relevant studies were identified, assessing 225 polymorphisms across 95 genes. Subgroup analysis indicated that Chinese, Japanese and Korean data were consistent and could be pooled. However, 6 of 13 polymorphisms (ACE I/D, CCND1 870G>A, CDH1 -160C>A, IL1B -511C>T, IL4 -590C>T, IL10 -592A>C) displayed conflicting effects between Asian and Caucasian populations, three of which (ACE I/D, CCND1 870G>A, IL1B -511C>T) had significantly different odds ratios between the two racial groups. In total, 37 polymorphisms across 27 genes were found to be significantly associated with gastric cancer in Asians, and 12 polymorphisms across 11 genes in Caucasians. Consolidated panels of polymorphisms associated with gastric cancer risk were identified in Asians and Caucasians. The results caution against the assumption that genetic risk factors are consistent between races.

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.

Increased drug resistance is associated with reduced glucose levels and an enhanced glycolysis phenotype

The testing of anticancer compounds in vitro is usually performed in hyperglycaemic cell cultures, although many tumours and their in vivo microenvironments are hypoglycaemic. Here, we have assessed, in cultures of tumour cells, the effects of reduced glucose levels on resistance to anticancer drugs and investigated the underlying cellular mechanisms.

Genetic factors associated with intestinal metaplasia in a high risk Singapore-Chinese population: a cohort study

BackgroundIntestinal metaplasia (IM) is an important precursor lesion in the development of gastric cancer (GC). The aim of this study was to investigate genetic factors previously linked to GC risk for their possible association with IM. A total of 18 polymorphisms in 14 candidate genes were evaluated in a Singapore-Chinese population at high risk of developing GC.MethodsGenotype frequencies were compared between individuals presenting with (n = 128) or without (n = 246) IM by both univariate and multivariate analysis.ResultsCarriers of the NQO1 609 T allele showed an association with IM in individuals who were seropositive for Helicobacter pylori (HP+; OR = 2.61, 95%CI: 1.18-5.80, P = .018). The IL-10 819 C allele was also associated with IM in HP+ individuals (OR = 2.32, 95%CI: 1.21-4.43, P = 0.011), while the PTPN11 A allele was associated with IM in HP- individuals (OR = 2.51, 95%CI: 1.16-5.40, P = 0.019), but showed an inverse association in HP+ subjects (OR = 0.46, 95%CI: 0.21-0.99, P = 0.048).ConclusionPolymorphisms in NQO1, IL-10 and PTPN11, in combination with HP status, could be used to identify individuals who are more likely to develop IM and therefore GC.

Acquired resistance to PI3K/mTOR inhibition is associated with mitochondrial DNA mutation and glycolysis

Acquired resistance (AQR) to drug treatment occurs frequently in cancer patients and remains an impediment to successful therapy. The aim of this study was to gain insight into how AQR arises following the application of PI3K/mTOR inhibitors. H1975 lung cancer cells with EGFR T790M mutations that confer resistance to EGFR inhibitors underwent prolonged treatment with the PI3K/mTOR inhibitor, BEZ235. Monoclonal cells with stable and increased resistance to BEZ235 were obtained after 8 months treatment. These AQR clones showed class-specific resistance to PI3K/mTOR inhibitors, reduced G1 cell cycle arrest and impedance of migration following PI3K/mTOR inhibition, reduced PTEN expression and increased Akt and S6RP phosphorylation. Transcriptome analysis revealed the AQR clones had increased expression of the metabolite transporters SLC16A9 and SLC16A7, suggestive of altered cell metabolism. Subsequent experiments revealed that AQR clones possess features consistent with elevated glycolysis, including increased levels of glucose, lactate, glutamine, glucose dependence, GLUT1 expression, and rates of post-glucose extracellular acidification, and decreased levels of reactive oxygen species and rates of oxygen consumption. Combination treatment of BEZ235 with the glycolysis inhibitor 3-bromopyruvate was synergistic in AQR clones, but only additive in parental cells. DNA sequencing revealed the presence of a mitochondrial DNA (mtDNA) MT-C01 variant in AQR but not parental cells. Depletion of mitochondrial DNA in parental cells induced resistance to BEZ235 and other PI3K/mTOR inhibitors, and was accompanied by increased glycolysis. The results of this study provide the first evidence that a metabolic switch associated with mtDNA mutation can be an underlying mechanism for AQR.

Predictors of Hand-Foot Syndrome and Pyridoxine for Prevention of Capecitabine–Induced Hand-Foot Syndrome: A Randomized Clinical Trial

Importance Hand-foot syndrome (HFS) is a common adverse effect of capecitabine treatment. Objective To compare the incidence and time to onset of grade 2 or greater HFS in patients receiving pyridoxine vs placebo and to identify biomarkers predictive of HFS. Design, Setting, and Participants This single-center, randomized double-blind, placebo-controlled phase 3 trial conducted at National Cancer Centre Singapore assessed whether oral pyridoxine could prevent the onset of grade 2 or higher HFS in 210 patients scheduled to receive single-agent capecitabine chemotherapy for breast, colorectal, and other cancers. Interventions Patients were randomized to receive concurrent pyridoxine (200 mg) or placebo daily for a maximum of 8 cycles of capecitabine, with stratification by sex and use in adjuvant or neoadjuvant vs palliative setting. Patients were withdrawn from the study on development of grade 2 or higher HFS or cessation of capecitabine. Main Outcomes and Measures Primary end point was the incidence of grade 2 or higher HFS in patients receiving pyridoxine. Secondary end points included the time to onset (days) of grade 2 or higher HFS and identification of biomarkers predictive of HFS, including baseline folate and vitamin B12 levels, as well as genetic polymorphisms with genome-wide arrays. Results In this cohort of 210 patients (median [range] age, 58 [26-82] years; 162 women) grade 2 or higher HFS occurred in 33 patients (31.4%) in the pyridoxine arm vs 39 patients (37.1%) in the placebo arm (P = .38). The median time to onset of grade 2 or higher HFS was not reached in both arms. In univariate analysis, the starting dose of capecitabine (odds ratio [OR], 1.99; 95% CI, 1.32-3.00; P = .001), serum folate levels (OR, 1.27; 95% CI, 1.10-1.47; P = .001), and red blood cell folate levels (OR, 1.25; 95% CI, 1.08-1.44; P = .003) were associated with increased risk of grade 2 or higher HFS. In multivariate analyses, serum folate (OR, 1.30; 95% CI, 1.12-1.52; P < .001) and red blood cell folate (OR, 1.28; 95% CI, 1.10-1.49; P = .001) were the only significant predictors of grade 2 or higher HFS. Grade 2 or higher HFS was associated with 300 DNA variants at genome-wide significance (P < 5 × 10−8), including a novel DPYD variant (rs75267292; P = 1.57 × 10−10), and variants in the MACF1 (rs183324967, P = 4.80 × 10−11; rs148221738, P = 5.73 × 10−10) and SPRY2 (rs117876855, P < 1.01 × 10−8; rs139544515, P = 1.30 × 10−8) genes involved in wound healing. Conclusions and Relevance Pyridoxine did not significantly prevent or delay the onset of grade 2 or higher HFS. Serum and red blood cell folate levels are independent predictors of HFS. Trial Registration clinicaltrials.gov Identifier: NCT00486213

Acquired resistance to combination treatment through loss of synergy with MEK and PI3K inhibitors in colorectal cancer

Historically, understanding of acquired resistance (AQR) to combination treatment has been based on knowledge of resistance to its component agents. To test whether an altered drug interaction could be an additional factor in AQR to combination treatment, models of AQR to combination and single agent MEK and PI3K inhibitor treatment were generated. Combination indices indicated combination treatment of PI3K and MEK inhibitors remained synergistic in cells with AQR to single agent but not combination AQR cells. Differences were also observed between the models in cellular phenotypes, pathway signaling and drug cross-resistance. Genomics implicated TGFB2-EDN1 overexpression as candidate determinants in models of AQR to combination treatment. Supplementation of endothelin in parental cells converted synergism to antagonism. Silencing of TGFB2 or EDN1 in cells with AQR conferred synergy between PI3K and MEK inhibitor. These results highlight that AQR to combination treatment may develop through alternative mechanisms to those of single agent treatment, including a change in drug interaction.

Abstract B52: Bioenergetic switch confers acquired resistance to BEZ235 in EGFR T790M-mutant non-small cell lung cancer

Introduction: Acquired resistance (AQR) continues to be source of treatment failure in EGFR inhibitor therapy and subsequent second-line strategies. In this study, AQR to the PI3K/mTOR inhibitor BEZ235 was generated in EGFR T790M non-small cell lung cancer (NSCLC) cells refractory to EGFR inhibitors to better understand AQR in this setting. Methods: Acquired resistant clones of H1975 were generated by continual exposure of H1975 cells to parental IC50 concentrations of BEZ235, followed by monoclonal selection. Phenotypic and protein analysis were carried out using standard techniques. Differentially expressed genes were determined using Affymetrix Gene 1.0ST analysis. Combination analysis was carried out using Chou and Talalay9s median-effect equation. Oxygen consumption rates (OCR) were measured using the Seahorse XFp analyzer. Metabolomic analysis was carried out with 1H NMR. Depletion of mitochondrial DNA (mtDNA) was achieved by ethidium bromide treatment. Results: Two resistant clones were generated with stably higher IC50 values (Clone 5.2 μM, Clone 6 7.8μM) to parental H1975 cells (0.32μM) after 6 months. The clones were resistant to other PI3K and mTOR inhibitors but not chemotherapeutic drugs, indicating class-specific resistance. Compared to parental cells, the clones exhibited a reduced G1 block, increased migratory capability, reduced PTEN and increased p-AKT. Gene array analysis revealed monocarboxylate transporters as top differentially expressed genes, prompting an investigation of glycolytic status. Proliferation was inhibited in the absence of glucose for the AQR clones but not parental cells. Compared to parental cells, AQR cells were more sensitive to the glycolytic inhibitor, 3BP. Combination treatment with 3BP and BEZ235 reversed the resistance of AQR cells, while in parental cells the combination was antagonistic. Resistant clones had higher extracellular lactate, and lower reactive oxygen species and ATP levels. Metabolomic analysis indicated that the clones had increased intracellular glucose, lactate, NADH and glutamine levels, consistent with enhanced glycolysis. The AQR clones also had lower OCR, reflecting impaired mitochondrial respiratory function. Depletion of mitochondrial DNA in parental cells led to resistance to PI3K and mTOR inhibitors, but not cisplatin. Conclusion: These results highlight that cells could acquire drug resistance through a bioenergetics switch from mitochondrial respiration to glycolysis. Citation Format: Bhaskar Bhattacharya, King Xin Koh, Mohamed Feroz Mohamed Omar, Sarah Low, Juleen Tan, Nur Sabrina Sapari, Barry Iacopetta, Ross Soo, Mounia Beloueche-Babari, Richie Soong. Bioenergetic switch confers acquired resistance to BEZ235 in EGFR T790M-mutant non-small cell lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B52.

Abstract B02: Determinants of acquired resistance to combination treatment with MEK and PI3K inhibitors

Background: Although combination treatment has been a useful strategy for circumventing acquired resistance (AQR) to single agent treatment, AQR also occurs to combination treatment. Nonetheless, models of AQR have thus far been restricted to single agent treatments. In this study, we tested whether models of AQR to combination treatment can be generated, and whether the determinants of AQR to combination treatment differ to those of AQR to single agent treatment. Methods: HCT116 colorectal cancer (CRC) cells with KRAS and PIK3CA mutations were exposed continuously with combined (1 IC50 dose of each agent) or single agent (2 doses of IC25) concentrations of MEK (AZD6244) and PI3K (BKM120) inhibitors, or DMSO (parental). IC50 measurements were assessed by MTS assay after 72 hours treatment. Combination Indices (CI) was calculated by the method of Chou and Talalay, where CI>1 indicates antagonism, Results: HCT116 cells with AQR to combination treatment were obtained after 6 months (CI@fu0.5 of “HCT116CR” cells 1.8±0.1 vs parental 0.23±009, p=0.04). HCT116 cells with AQR to single agent treatment with AZD6244 (IC50 of “HCT116AR” 56.1±0.04µM vs parental 4.3±0.04µM; p=0.03) and BKM120 (IC50 of “HCT116BR” 3.1±0.21µM vs parental 1.0±0.38µM; p=0.007) were also generated. The AQR in HCT116CR cells was associated with a significant reduction in apoptosis and wound healing response after combination treatment compared to HCT116 parental, HCT116AR and HCT116BR cells. Cell cycle phase distribution analysis revealed an inability of the compounds to elicit a G1 arrest in the single agent or combination AQR cells. Basal level of phosphorylated AKT were elevated in HCT116BR and HCT116CR cells, while phosphorylated ERK levels were increased in all the three resistant lines. In addition, there was a significant decrease in phosphorylated 4EBP1 in HCT116CR cells. In cross-resistance studies, no differences in the IC50s of 5FU or oxaliplatin were observed in any of the cell lines tested, suggesting the absence of a multi-drug resistance phenotype. HCT116AR and HCT116BR cells displayed cross resistance to other MEK or PI3K inhibitors, while combination treatment with MEK and PI3K inhibitors remained synergistic in these cells. HCT116CR cells were also resistant to single agent MEK and PI3K inhibitor treatment and to an alternative combination of a MEK inhibitor (GDC0973) and PI3K inhibitor (BYL719). However, HCT116CR cells were ∼20, 11 and 8-fold more sensitive than HCT116 parental cells to MK2206 (Akt), dual PI3K/mTOR (BEZ235; PI103) and mTOR (KU0063794) inhibitors respectively. Gene expression analysis revealed significant overexpression of KRAS and AKT3 in HCT116AR and HCT116BR cells compared to HCT116 cells, consistent with cross resistance data obtained with MEK and PI3K inhibitors. In HCT116CR cells, KRAS expression remained unchanged, however an overexpression of ET-1 (endothelin 1, 33-fold) was observed that was not observed in HCT116AR and HCT116BR cells. Conclusions: AQR to synergistic combinations can evolve in cancer cells, and the mechanisms of this combination treatment AQR may be distinctly different from those for single agent AQR. ET-1 may be a specific mediator of AQR to combination PI3K and MEK resistance in CRC. Citation Format: Bhaskar Bhattacharya, Sarah Low, Dilys Chia, King Xin Koh, Mei Ling Chong, Touati Benoukraf, Richie Soong. Determinants of acquired resistance to combination treatment with MEK and PI3K inhibitors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B02.