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Hexokinase II Expression Is Correlated with Colorectal Cancer Prognosis

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Hexokinase II Expression Is Correlated with Colorectal Cancer Prognosis Cancer Treatment Communications 6 (2016) 11–16 Contents lists available at ScienceDirect Cancer Treatment Communications journal homepage: www.elsevier.com/locate/ctrc Hexokinase II expression is correlated with colorectal cancer prognosis Nelson Ho, Brenda L. Coomber n Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada N1G 2W1 article info abstract Article history: Background: Enhanced glycolysis is observed during tumorigenesis, with an upregulation in key glyco- Received 17 September 2015 lytic enzymes. Hexokinase II (HKII) catalyzes the first irreversible step of glycolysis and is often over- Received in revised form expressed in tumors. Abnormal vasculature within tumors leads to regional ischemic conditions that 24 January 2016 promote tumor progression. The aims of this study were to assess the expression of HKII in colorectal Accepted 12 February 2016 cancer tissues, and to correlate HKII expression with clinical parameters and patient outcome. Materials and method: Sections from 60 FFPE primary colorectal cancer tumor samples were dual Keywords: fluorescence immunostained for HKII and carbonic anhydrase IX (CAIX; serving as an ischemic marker) FFPE tissue and assessed using semi-quantitative immunofluorescence. Associations of HKII and CAIX levels with Immunofluorescence patient characteristics, tumor pathology and clinical outcome were studied using univariate analysis. Ischemia Results: HKII expression was found in neoplastic cells of non-ischemic regions of tumors and within the Tumor stroma Cancer metabolism tumor stroma. CAIX expression was found primarily in neoplastic cells, and was associated with patient age (OR¼4.04, Z70 vs. o70). Tumor samples scoring lower for HKII were associated with early disease progression (p¼0.0155) and poor overall survival (p¼0.0248). Interestingly, tumor samples that pre- sented with stromal HKII staining were associated with early disease progression (p¼0.0485) and poor overall survival (p¼0.0235). Conclusion: We identified low overall HKII expression to be associated with the outcome of colon cancer. However, the correlation between stromal HKII expression and worse survival in colorectal cancer pa- tients warrant further investigation. MicroAbstract: This study assessed the prognostic value of hexokinase II expression in colorectal cancer. In total, 60 FFPE primary colorectal cancer tumor samples were immunostained and correlated with patient characteristics, tumor pathology and clinical outcome. Interestingly, low overall HKII expression was correlated with worse patient outcome. Separately, stromal HKII staining was correlated with worse patient outcome. & 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction production regardless of oxygen tension in a phenomenon called the Warburg effect [5], and studies have correlated the glycolytic Colorectal cancer (CRC) is the second leading cause of cancer- state of cancer cells with tumor aggressiveness [6]. Therefore, related deaths in the United States and the third most common specific glycolytic enzymes may serve as possible biomarkers of cancer in men and in women. In 2015, it was estimated that CRC CRC aggressiveness, providing useful prognostic tools. would account for 17.6% of all new cancer cases and 25.3% of all The family of hexokinase (HK) enzymes function to catalyze the cancer-related deaths [1]. Decreasing mortality rates have been first irreversible step of glycolysis in which glucose is phosphory- attributed to early screening, reduced prevalence of risk factors, lated to glucose-6-phosphate. HKII expression is normally limited to and/or improved treatments [2,3]. However, outcome for patients insulin-sensitive tissues [7]. However, upregulation of HKII is con- with advanced and metastatic disease remains poor. sidered a consequence of metabolic re-programming in cancer, and In 2011, deregulating cellular energetics was identified as an has been shown in breast carcinoma [8], laryngeal squamous cell emerging hallmark of cancer [4]. Many tumors upregulate key carcinoma [9], glioblastoma multiforme [10], and gastric adeno- glycolytic enzymes of the glycolytic pathway for energy carcinoma [11]. Its assessment in CRC has been limited; im- munohistochemical analyses on 104 surgically resected CRC sam- ples showed a positive correlation between HKII expression with n Corresponding author. E-mail addresses: [email protected] (N. Ho), extensive tumor diameter, advanced tumor depth, the presence of [email protected] (B.L. Coomber). lymph node metastasis, and shorter recurrence-free survival [12]. http://dx.doi.org/10.1016/j.ctrc.2016.02.008 2213-0896/& 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 12 N. Ho, B.L. Coomber / Cancer Treatment Communications 6 (2016) 11–16 The rapid and disorganized proliferation associated with tu- (ab107257; 1:1000; Abcam plc, Cambridge, UK). Bands were de- morigenesis leads to ischemic regions within solid tumors [13]. tected with HRP-conjugated goat anti-rabbit or anti-mouse anti- Cells within these regions are exposed to poor blood flow leading bodies (1:10000; Sigma-Aldrich) using Luminata Forte chemilu- to transient or chronic hypoxic and hypoglycemic conditions. HKII minescent substrate (Millipore, Billerica, MA, USA). expression is upregulated under hypoxic conditions as a result of stabilized HIF-1α transcription factor [14]. Carbonic anhydrase 2.3. Immunofluorescence staining nine (CAIX) catalyzes the hydration of carbon dioxide to bicarbo- nate and proton [15]. CAIX is a HIF-inducible gene used as an is- FFPE histological sections were deparaffinized in xylene, rehy- chemic marker and is overexpressed colorectal cancer [16,17]. drated in isopropanol, and antigen retrieval was performed with In this study, we evaluated the expression of HKII and CAIX in heated sodium citrate buffer (pH 6.0). Sections were blocked with formalin-fixed paraffin-embedded (FFPE) specimens of CRC tu- 1:1 solution of 5% goat serum in PBSþ0.1% Tween (PBST) and mors and assessed its relationship to patient outcome. Based on protein block solution (Dako, Denmark) followed by overnight findings from other studies, we hypothesized that high HKII ex- incubation at 4 °C with rabbit mAb HKII (2867, 1:400, Cell Sig- pression would be associated with shorter progression-free status naling Technology, Davers, MA) and mouse mAb CAIX (ab107257, and worse outcome. 1:400, Abcam plc, Cambridge, UK). Sections were washed then incubated with goat anti-rabbit Alexa 488-conjugated (1:300, Life Technologies, Carlsbad, CA) and goat anti-mouse Cy3-conjugated 2. Materials and methods (1:200, Life Technologies) antibodies. Slides were counterstained with DAPI, mounted with Fluorescence Mounting Media (Dako) 2.1. Samples and stored flat in the dark at 4 °C. Sixty FFPE CRC samples used for this study were gathered by 2.4. Imaging and quantification of immunofluorescence the Ontario Tumor Bank, Ontario Institute for Cancer Research, Toronto, ON. Samples were collected between 2005 and 2011. The Images of slides stained with HKII and CAIX antibodies were patients' medical records were reviewed and general information, captured with a QICAM camera (QImaging, BC, Canada) using a including sex, age range at diagnosis, histological grade, and TNM 40 Â objective lens. Files were captured at 1.4 megapixel resolu- staging [18], are provided in Table 1. Follow-up data were re- tion, using excitation and barrier filter sets for fluorescein (green trieved for survival analysis. The last follow-up was May 2013. emission), rhodamine (red emission) and DAPI (blue emission). In Cases were anonymized and the researchers were blinded to tu- total, 5 images were captured per slide, covering different regions mor and patient details until completion of image capture and of the sample in an unbiased, systematic fashion. Fiji [19] was used analysis. to process and analyze the images to determine HKII and CAIX staining. For CAIX staining in all images, a minimum threshold 2.2. Cell lines and immunoblot analysis value of 20 was used in the software and the percent area stained was subsequently calculated. A variation of the semi-quantitative Human colorectal cancer cell lines HCT116 and DLD-1 were H-Score immunohistochemistry evaluation method [20] was used “ ” obtained from ATCC (Manassas, VA, USA) and were maintained in to assess HKII staining, and given the term F-Score : ¼ ¼ þ high glucose DMEM (Sigma-Aldrich, Oakville, ON, Canada) sup- F-Score (% area stained at threshold 25) (% area stained at ¼ plemented with 10% FBS (Life Technologies, Grand Island, NY, USA) threshold 85). and incubated at 37 °C in a humidified atmosphere containing 5% 2.5. Statistical analysis CO2 in room air. Anoxic conditions were achieved using a Modular Incubator Chamber (Billups-Rothenberg Inc., Del Mar, CA, USA) modified to allow continuous flushing of the chamber with a hu- GraphPad Prism software v6.0 (GraphPad Software Inc., La Jolla, CA, USA) was used for statistical analysis. Correlations between midified mixture of 95% N2 and 5% CO2. Cell lysates were subjected to SDS-PAGE followed by western blotting with rabbit anti-HKII staining parameters and patients' characteristics were analyzed (2867; 1:5000; Cell Signaling Technology) and mouse anti-CAIX with Fisher's exact test. Progression-free survival (PFS)
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