Pudova et al. BMC Genomics 2018, 19(Suppl 3):113 DOI 10.1186/s12864-018-4477-4 RESEARCH Open Access HK3 overexpression associated with epithelial-mesenchymal transition in colorectal cancer Elena A. Pudova1†, Anna V. Kudryavtseva1,2†, Maria S. Fedorova1, Andrew R. Zaretsky3, Dmitry S. Shcherbo3, Elena N. Lukyanova1,4, Anatoly Y. Popov5, Asiya F. Sadritdinova1, Ivan S. Abramov1, Sergey L. Kharitonov1, George S. Krasnov1, Kseniya M. Klimina4, Nadezhda V. Koroban2, Nadezhda N. Volchenko2, Kirill M. Nyushko2, Nataliya V. Melnikova1, Maria A. Chernichenko2, Dmitry V. Sidorov2, Boris Y. Alekseev2, Marina V. Kiseleva2, Andrey D. Kaprin2, Alexey A. Dmitriev1 and Anastasiya V. Snezhkina1* From Belyaev Conference Novosibirsk, Russia. 07-10 August 2017 Abstract Background: Colorectal cancer (CRC) is a common cancer worldwide. The main cause of death in CRC includes tumor progression and metastasis. At molecular level, these processes may be triggered by epithelial-mesenchymal transition (EMT) and necessitates specific alterations in cell metabolism. Although several EMT-related metabolic changes have been described in CRC, the mechanism is still poorly understood. Results: Using CrossHub software, we analyzed RNA-Seq expression profile data of CRC derived from The Cancer Genome Atlas (TCGA) project. Correlation analysis between the change in the expression of genes involved in glycolysis and EMT was performed. We obtained the set of genes with significant correlation coefficients, which included 21 EMT-related genes and a single glycolytic gene, HK3. The mRNA level of these genes was measured in 78 paired colorectal cancer samples by quantitative polymerase chain reaction (qPCR). Upregulation of HK3 and deregulation of 11 genes (COL1A1, TWIST1, NFATC1, GLIPR2, SFPR1, FLNA, GREM1, SFRP2, ZEB2, SPP1, and RARRES1) involved in EMT were found. The results of correlation study showed that the expression of HK3 demonstrated a strong correlation with 7 of the 21 examined genes (ZEB2, GREM1, TGFB3, TGFB1, SNAI2, TWIST1, and COL1A1) in CRC. Conclusions: Upregulation of HK3 is associated with EMT in CRC and may be a crucial metabolic adaptation for rapid proliferation, survival, and metastases of CRC cells. Keywords: Warburg effect, Hexokinase, Glycolysis, Colorectal cancer, Epithelial-mesenchymal transition Background making it the third most common cancer and fourth In 2012, an estimated 1.36 million people were diag- most common cause of death worldwide [2]. nosed with colorectal cancer (CRC), including 746,300 The stage of CRC plays a significant role in survival of men and 614,300 women. The mortality rate of CRC is patients. More than 60% of patients are diagnosed at late reported to be approximately half of its global incidence. stage of CRC (III-IV) and approximately 25% of them In the same year, 694,000 people died from CRC [1], display metastatic disease [3, 4]. Development of new drugs and targeted therapies has dramatically improved the survival of these patients over the last decade [5]. * Correspondence: [email protected] †Equal contributors However, the survival outcome in patients with meta- 1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, static or stage IV CRC still remains poor [6, 7]. For in- Moscow, Russia stance, the 5-year relative survival rate for patients with Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Pudova et al. BMC Genomics 2018, 19(Suppl 3):113 Page 6 of 141 metastatic CRC is about 11%, while that for patients invasion, and metastasis [32–34]. Increased glycolysis with stage III CRC ranges from 53% to 89% (https:// has been suggested as an essential component of the www.cancer.org). CRC is characterized by severe genetic malignant phenotype and hallmark of invasive cancers alterations, including microsatellite instability (MSI), epi- [35]. In CRC, redirection of glucose metabolism is pro- genetic changes such as DNA methylation (CpG Island moted by deregulation of key components involved in Methylator Phenotype [CIMP]), and chromosomal in- glucose transport (GLUT-1) [36], metabolism (hexoki- stability (CIN), which are associated with the develop- nases [HK1, HK2], pyruvate kinase M2 [PKM2], lac- ment of the disease and its aggressive behavior [8, 9]. tate dehydrogenase A [LDHA], aldolase A [ALDOA], Thus, identification of the distinct molecular genetic etc.) [37–39], as well as metabolic regulation [40–42]. changes related to CRC progression and metastasis is In addition, variation in the representation of alterna- extremely important. tive spliced transcripts related to energy metabolism A number of studies have demonstrated that seems to be associated with the prevalence of aerobic epithelial-mesenchymal transition (EMT) is involved in glycolysis in cancer cells [43, 44]. EMT in tumors re- CRC carcinogenesis and metastasis [10, 11]. EMT is the quires the distinct metabolic adaptations for survival, conversion of epithelial cells into cells with a mesenchy- rapid proliferation, and metastasis. However, meta- mal phenotype, which is accompanied by a decrease in bolic changes observed upon EMT induction are un- cell-cell cohesion and fundamental reorganization of the common for various cancer types [45–47]. Recent cytoskeleton. Consequences include enhanced migratory studies showed that the glycolytic enzymes aldolase B as well as invasive capacity and resistance to apoptosis (ALDOB), PKM2, and glyceraldehyde-3-phosphate de- [12, 13]. Based on the biological context, three types of hydrogenase (GAPDH) play a role in EMT and me- EMT have been described as follows: type I observed in tastasis in CRC [48, 49]. embryogenesis [14], type II involved in tissue repair and In this work, using CrossHub software we analyzed fibrosis [15], and type III seen in metastatic transform- RNA-Seq data from The Cancer Genome Atlas (TCGA) ation of cancer cells [16]. A hallmark feature of EMT is project to estimate the association between the expres- the downregulation of E-cadherin, a cell-cell adhesion sion of genes participating in EMT and glycolysis in molecule localized on the surface of epithelial cells [17]. CRC. Hexokinase 3 (HK3) was found to exhibit signifi- E-cadherin is a well-known tumor suppressor protein cant correlation with a set of genes involved in EMT. and loss of its expression in tumor cells is associated with Thus, we focused on the investigation of HK3 gene ex- increased tumor invasiveness and metastasis [18, 19]. In pression in CRC samples and its association with EMT. CRC, EMT is characterized by decreased E-cadherin ex- pression and nuclear accumulation of β-catenin, which is Methods an essential protein for correct positioning and function Bioinformatic analysis of one [20, 21]. Loss of epithelial and gain of dedifferen- Using CrossHub software [50], we have analyzed The Can- tiated mesenchyme-like phenotype enable CRC cells to cer Genome Atlas (TCGA, https://cancergenome.nih.gov) develop invasive and metastatic growth characteristics project RNA-Seq dataset derived from CRC. The dataset [22]. The switch from cytoplasmic to nuclear accumula- contained 287 tumor samples including 26 paired ones. tion of β-catenin is a central mechanism related to EMT The selection of genes participating in glycolysis and EMT in CRC [21]. This process may be mediated by the loss-of- was performed using Gene Ontology database and “glyco- function mutations in the adenomatous polyposis coli lytic process” and “epithelial to mesenchymal transition” as (APC) tumor suppressor gene or mutations that result in keywords. A set of 132 genes was selected for further β-catenin stabilization [23]. In the nucleus, β-catenin asso- analysis. ciates with DNA-binding proteins of the T-cell factor/ The correlation analysis between the expressions of lymphoid enhancer factor (TCF/Lef) family, leading to a target genes was performed using Spearman’s rank cor- constitutive activation of Wnt/β-catenin signaling target relation coefficients (rs). We carried out two tests: one genes, c-Myc [24]andCCND1 [25]. Moreover, β-catenin for paired samples and other for pool of tumor samples. triggers the expression of other genes such as MMP7 Spearman’s rank correlation coefficients were calculated [26, 27], FN [28 ], CD44 [29], and UPAR [30]involved between (1) fold change values, the ratio of CPM (counts in invasive cell growth. Except for this mechanism, per million) in tumor sample to CPM in conditional there are at least ten known signaling pathways and normal tissue, (paired samples) and (2) CPM values (pool many molecules associated with EMT in CRC [31]. of tumor samples). Before calculating correlation coeffi- Metabolic reprogramming characterized by upregu- cients, dropping points (no more than 5%) were elimi- lated glycolysis is another crucial feature of cancer. It nated using approximate generalized linear model. We provides cancer cells with energy and metabolites essen- focused on the genes