Sai Mun Leong

Mutant nucleophosmin deregulates cell death and myeloid differentiation through excessive caspase-6 and -8 inhibition

In up to one-third of patients with acute myeloid leukemia, a C-terminal frame-shift mutation results in abnormal and abundant cytoplasmic accumulation of the usually nucleoli-bound protein nucleophosmin (NPM), and this is thought to function in cancer pathogenesis. Here, we demonstrate a gain-of-function role for cytoplasmic NPM in the inhibition of caspase signaling. The NPM mutant specifically inhibits the activities of the cell-death proteases, caspase-6 and -8, through direct interaction with their cleaved, active forms, but not the immature procaspases. The cytoplasmic NPM mutant not only affords protection from death ligand-induced cell death but also suppresses caspase-6/-8-mediated myeloid differentiation. Our data hence provide a potential explanation for the myeloid-specific involvement of cytoplasmic NPM in the leukemogenesis of a large subset of acute myeloid leukemia.

Wild-type and mutant p53 mediate cisplatin resistance through interaction and inhibition of active caspase-9.

The p53 gene has been implicated in many cancers due to its frequent mutations as well as mutations in other genes whose proteins directly affect p53’s functions. In addition, high expression of p53 [wild-type (WT) or mutant] has been found in the cytoplasm of many tumor cells, and studies have associated these observations with more aggressive tumors and poor prognosis. Cytoplasmic mis-localization of p53 subsequently reduced its transcriptional activity and this loss-of-function (LOF) was used to explain the lack of response to chemotherapeutic agents. However, this hypothesis seemed inadequate in explaining the apparent selection for tumor cells with high levels of p53 protein, a phenomenon that suggests a gain-of-function (GOF) of these mis-localized p53 proteins. In this study, we explored whether the direct involvement of p53 in the apoptotic response is via regulation of the caspase pathway in the cytoplasm. We demonstrate that p53, when present at high levels in the cytoplasm, has an inhibitory effect on caspase-9. Concurrently, knockdown of endogenous p53 caused an increase in the activity of caspase-9. p53 was found to interact with the p35 fragment of caspase-9, and this interaction inhibits the caspase-9 activity. In a p53-null background, the high-level expression of both exogenous WT and mutant p53 increased the resistance of these cells to cisplatin, and the data showed a correlation between high p53 expression and caspase-9 inhibition. These results suggest the inhibition of caspase-9 as a potential mechanism in evading apoptosis in tumors with high-level p53 expression that is cytoplasmically localized.

Sampling circulating tumor cells for clinical benefits: how frequent?

Circulating tumor cells (CTCs) are cells shed from tumors or metastatic sites and are a potential biomarker for cancer diagnosis, management, and prognostication. The majority of current studies use single or infrequent CTC sampling points. This strategy assumes that changes in CTC number, as well as phenotypic and molecular characteristics, are gradual with time. In reality, little is known today about the actual kinetics of CTC dissemination and phenotypic and molecular changes in the blood of cancer patients. Herein, we show, using clinical case studies and hypothetical simulation models, how sub-optimal CTC sampling may result in misleading observations with clinical consequences, by missing out on significant CTC spikes that occur in between sampling times. Initial studies using highly frequent CTC sampling are necessary to understand the dynamics of CTC dissemination and phenotypic and molecular changes in the blood of cancer patients. Such an improved understanding will enable an optimal, study-specific sampling frequency to be assigned to individual research studies and clinical trials and better inform practical clinical decisions on cancer management strategies for patient benefits.

Improved Discrimination of Patients with Breast Cancer from Healthy Controls Using Paper-Based microRNA Expression Profiling of Plasma, Following Precipitation

To the Editor: Breast cancer is a frequently diagnosed solid tumor in women worldwide (1). MicroRNAs (miRNAs), small noncoding RNAs that regulate protein expression, are potential biomarkers in the diagnosis, prognosis, and prediction of response to treatment in breast cancer (2). We recently reported a paper-based method to extract miRNAs from plasma and circulating tumor cells (3). Here, we applied this method to plasma of patients with breast cancer and age- and gender-matched healthy controls, after treating the plasma with a commercial exosome isolation kit employing a nonspecific precipitation step; we compared the miRNA expression profiles from the plasma with and without the use of the precipitation method. After informed consent and institutional approval, we obtained peripheral blood samples from 9 patients with breast cancer and 9 healthy controls. Plasma was isolated by centrifugation at 3000 g for 10 min and stored at −80 °C. All plasma samples were visually inspected to exclude any discernible hemolysis. Then, 60 μL of Total Exosome Isolation reagent …

Longitudinal monitoring reveals dynamic changes in circulating tumor cells (CTCs) and CTC-associated miRNAs in response to chemotherapy in metastatic colorectal cancer patients

We evaluated the changes in CTC count and CTC-associated miRNAs during the course of chemotherapy in patients with metastatic colorectal cancer. Blood samples were collected from 9 metastatic colorectal cancer patients prior to chemotherapy and at every other chemotherapy session during the course of treatment. CTCs were isolated and enumerated using a size-exclusion method (CellSievo, Singapore). CTC-associated miRNAs were isolated using a paper-based, partitioning method, and analyzed using reverse transcription quantitative real-time PCR (MiRXES, Singapore). CTC count trends generally correlated with disease progression defined by radiological measurements and trends in carcinoembryonic antigen (CEA) levels; hence CTC counts may be useful in cases where CEA is not elevated. CTC-associated miRNAs identified were miR-15b, miR-16, miR-19a, miR-21, miR-25, miR-30d, miR-126, miR-185, miR-221, miR-222, and miR-324-5p. The expression of CTC-associated miRNAs did not appear to correlate with CTC count and exhibited inter-individual heterogeneity. This pilot study suggests that analysis of CTC changes during the course of treatment may be useful in monitoring response to therapy in metastatic colorectal cancer.

Su2063 There Is No Relationship Between the Fructose Transporter, GLUT5 and GLUT2, Protein or mRNA Expression Levels in Irritable Bowel Syndrome With Fructose Malabsorption and Intolerance

Purpose: Poor sleep is commonly reported by patients with IBS. When stressed prior to bedtime patients with IBS show dysregulation of the pituitary-adrenal axis during sleep. Evidence suggests the tryptophan (TRP) metabolic pathways are also altered in patients with IBS. Our purpose was to determine whether alterations in TRP metabolism may be linked to cortisol in women with and without IBS. Methods: Women, ages 18-45, (n=46 IBS [Rome III]; controls [C; n=24]) were studied in a sleep laboratory and serum samples obtained on the third night following the introduction of a social stressor (anticipation of public speaking). Samples were obtained 2 hours following sleep onset (time 1) and 1 hour prior to awakening (time 2). All subjects completed a demographic data sheet, Rome III Diagnostic Questionnaire for Functional GI Disorders, Pittsburgh Quality Sleep Index (PSQI) and 28-day symptom diaries. Serum cortisol levels were determined by ELISA and metabolite levels by targeted liquid chromatography-mass spectrometry (LC-MS). Results: More women with IBS reported symptoms of insomnia (PSQI) as compared to C group (40% versus 24%) and reduced sleep efficiency by polysomnography (p<0.05). As reported previously cortisol/ACTH ratios were higher in IBS as compared to C women. Of the 6 kyneurinine pathway metabolites (primary TRP metabolic route) only niacinamide was significantly higher in women with IBS. However, 5-hydroxytryptophan levels were higher (p<.052) in women with IBS. None of the 3 methylation metabolites were significantly different between IBS and Cs. When ratios of metabolites were calculated significant differences were observed as shown in Table 1. Significant negative correlations between cortisol/ACTH and 5-hydroxytryptophan, methionine, kyneurinine, and betaine were found at one or both time points. TRP levels were significantly correlated with sleep quality (diary) in the IBS group. Conclusion: Nighttime levels of cortisol/ACTH are associated with metabolites in the TRP metabolic pathway particularly those associated with methylation. Whether these associations fully account for the frequent reports of poor sleep in women with IBS requires further study. Table 1