A pan-cancer analysis of molecular characteristics and oncogenic role of hexokinase family genes in human tumors

Journal Pre-proof A pan-cancer analysis of molecular characteristics and oncogenic role of hexokinase family genes in human tumors

Mingzhe Jiang, Shuangjie Liu, Jiaxing Lin, Wenjun Hao, Baojun Wei, Ying Gao, Chuize Kong, Meng Yu, Yuyan Zhu PII:

S0024-3205(20)31422-3

DOI:

https://doi.org/10.1016/j.lfs.2020.118669

Reference:

LFS 118669

To appear in:

Life Sciences

Received date:

30 July 2020

Revised date:

18 October 2020

Accepted date:

23 October 2020

Please cite this article as: M. Jiang, S. Liu, J. Lin, et al., A pan-cancer analysis of molecular characteristics and oncogenic role of hexokinase family genes in human tumors, Life Sciences (2018), https://doi.org/10.1016/j.lfs.2020.118669

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© 2018 Published by Elsevier.

Journal Pre-proof A pan-cancer analysis of molecular characteristics and oncogenic role of Hexokinase family genes in human tumors Mingzhe Jianga, Shuangjie Liu，Jiaxing Lina, Wenjun Haoa, Baojun Wei, Ying Gaoa, Chuize Konga, Meng Yua,b *,Yuyan Zhua * a

Department of Urology, The First Hospital of China Medical University, Shenyang

110001, ChinaThe First Hospital of China Medical University, Shenyang 110001,

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Department of Reproductive Biology and Transgenic Animal, China Medical

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University, Shenyang 110001, China

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China

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* Corresponding author: Yuyan Zhu, e-mail: [email protected]

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Meng Yu, e-mail: [email protected]

Yuyan Zhu: Department of Urology, The First Hospital of China Medical University,

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Shenyang 110001, China. Tel: +86 24 83283422; Fax: +86 24 86243433; E-mail: [email protected]

Meng Yu: Department of Reproductive Biology and Transgenic Animals, China Medical University, No.92 Bei’er Road, Heping District, Shenyang 110001, China. Tel: +86 24 23260367; Fax: +86 24 23327835; E-mail: [email protected] ABSTRACT Hexokinase (HK) plays a key role in various biological processes such as glycolysis of tumor cells. However, there is still a lack of systematic understanding of the contribution of HK family genes in different types of cancer. In the present study, we systematically analyzed the molecular changes and clinical correlations of HK family

Journal Pre-proof genes in 33 types of cancer extracted from more than 10000 subjects. As a result, there were extensive genetic changes in HK family genes and the expression levels of HK family were significantly correlated with the activity of cancer marker-related pathways. In addition, HK family genes may be useful in predicting prognosis and therapeutic efficacy. Moreover, HK1 ,HK2 and HK3 may become potential oncogenes across a variety of cancer types. Furthermore, the oncogenic functions of HK1 in bladder cancer have been confirmed in vitro. Collectively, our results provide valuable resources to guide the mechanism and therapeutic analysis concerning the role of HK family genes in cancer.

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Key words

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Hexokinase, pan-cancer analysis，biomarker，therapeutic target，TCGA

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INTRODUCTION

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Aerobic glycolysis (Warburg effect) is one of the most important biological characteristics of malignant tumor [1-3]. Malignant tumor is characterized by low degree of differentiation and rapid proliferation, and can maintain the metabolic activity of cells under sufficient oxygen or hypoxia. Hexokinase (HK), the first

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rate-limiting enzyme in cell glycolysis, is considered as the key molecule to regulate cell energy metabolism and cell fate [4-6]. In addition, HK is closely related to the metabolism, proliferation and apoptosis of tumor cells. The high expression and activity of HK in tumor cells ensure the rapid glycolysis of tumor cells[7], therefore, the investigation into HK would shed novel light on the understanding of carcinogenesis, tumor progression and therapy. Five kinds of HK isozymes, HK1, HK2, HK3, GCK (Glucokinase，HK4) and HKDC1 (Hexokinase domain con-taining 1), have been found in mammals [8]. To be specific, HK1 is mainly distributed in the brain; HK2 is mainly distributed in the heart, muscle, fat and bone; HK3 is mainly distributed in bone marrow, lung and spleen; GCK can regulate insulin secretion in pancreas and modulate glucose uptake, synthesis and decomposition of glycogen in liver, while HKDC1 is a human hexokinase-like gene adjacent to HK1 gene on chromosome. Notably, HK2, is rarely expressed in normal liver tissues, but is highly expressed in liver cancer [9], prostate cancer [10], renal cancer[11], breast cancer , osteosarcoma [12], lung cancer [13] and etc. At present, it is generally believed that HK2 plays a dual role in tumor cells. On the one hand, HK2 can induce glycolysis, and the level of glycolysis is positively correlated with the expression and activity of HK2. on the other hand, HK2 can inhibit apoptosis by binding to voltage-dependent anion channels (Volt-dependent anion channel, VDAC)

Journal Pre-proof in the outer membrane of mitochondria[14]. Because of its important function in tumor, HK2 is considered as an ideal target for tumor therapy. Previous studies on the HK family have focused on the role of HK2 in a small group of tumor, which provides a limited understanding or even bias of this important pathway. Up to now, the comprehensive molecular characterization of the HK family in human cancer has not been described, resulting in the knowledge gap on the application of this pathway in cancer research.

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In this study, we comprehensively analyzed the expression characteristics of all members of HK family in various types of cancer by using the multiple database

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based on The Cancer Genome Atlas（TCGA). In addition, the potential biological functions and common characteristics of HK family members were also analyzed and verified in different aspects of cancer.

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RESULTS

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Extensive genetic changes of HK Family genes in multiple types of cancer

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We first identified the prevalence of HK family gene changes in 33 different types of cancer based on data extracted from integrated cell mutations and copy number changes in TCGA. As a result, the overall average mutation frequency of each gene in HK family was 1.3%-2.9%. There were 150 Missense and 19 Truncating in HK1, and their relatively high mutations, such as D365N, were mainly distributed in Hexokinase-2 domain. Moreover, there were 161 Missense and 16 Truncating in HK2, with relatively high mutations, such as P563Q/S, mainly distributed in the latter half of the domain. There were 181 Missense and 24 Truncating in HK3, and the R513 * truncated mutation was distributed in Hexokinase-1 domain. In terms of GCK, there were 94 Missense, 9 Truncating and 1 Inframe, and the mutation number was the least in the family. There were a total of 177 Missenseand 30 Truncating in HKDC1, and the number of mutations was the most in the family, among which the A560T/D point mutation at amino acid residue 560 was the most abundant, which was distributed in the Hexokinase-1 domain（Figure 1A-1B, Supplementary Figure 1）. We then collected data on mutations in 967 cell lines from 23 cancers from the encyclopedia of cancer cell lines (CCLE), which revealed that the HK family gene had a relatively high mutation frequency in diverse types of cancer (Supplementary Figure 2).We further investigate the frequency of Copy number variations(CNV) changes in all HK family genes, showing common CNV changes. HK family genes showed extensive

Journal Pre-proof CNV amplification in cancer types, while CNV deletion was only found in a few tumors such as DLBC (Figure 1A). There are also common CNV changes in the HK family genes across cell lines (Supplementary Figure 2). These results revealed the highly heterogeneous inheritance and expression changes of HK family genes in different types of cancer, indicating that HK family gene disorders played an important role in different cancer microenvironments（Figure 1B）.

We also discussed the possibility of HK family genes in producing fusion genes. We

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found that in addition to HK3, members of the HK family can produce fusion genes in a variety of tumors (Table 1). For example, HK1 was able to generate fusion genes in BLCA (TSPAN15-HK1), COAD (IFIT5-HK1), MESO (CDC123-HK1), PRAD (HK1-CTNNA3) （ Figure 1C ） , and HK2 can produce fusion genes in OV (HK2-ONECUT3). GCK was capable of generating fusion genes in LIHC (EIF2AK1-GCK) and PRAD (C7orf44-GCK), and HKDC1 can produce fusion genes in BLCA (MIRLET7BHG-HKDC1) and STAD (SUPV3L1-HKDC1).

multiple types of cancer

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HK family genes are genetically，epigenetically and transcriptionally regulated in

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To explore the molecular mechanism of abnormal expression of HK family genes，we firstly explored whether these genetic changes affected the expression of HK family genes by assessing the expression disturbance of HK family genes in 17 types of cancer, including at least five normal controls. Consequently, the change of CNV may be one of the important mechanisms leading to the disturbance of gene expression in HK family (Figure 2A).

We further identified the effects of promoter methylation on HK family gene changes in 33 types of cancer by integrating methylation levels and expression profile data （Figure 2B）. Our results suggested that, overall, the HK family genes were hypomethylated in a variety of tumors. To be specific, HK1 was hypomethylated in BRCA and LUSC, and HK2 was hypomethylated in BLCA, KIRC, PAAD, BRCA, UCEC, KIRP and LUSC. The promoter of HK3 was hypomethylated in KIRC and HKDC1 was hypomethylated in BLCA, KIRC, PAAD, BRCA, UCEC, LUSC, COAD, HNSC, LUAD and LIHC and GCK was hypomethylated in KIRC, KIRP and LUAD. Additionally, the methylation level of HK family gene promoter was negatively correlated with their gene expression.

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On the other hand, we determined the effect of transcriptional activation on HK family gene changes in 33 types of cancer by integrating transcription factor and target gene expression profile data（Figure 2C). In 32 cancers of TCGA, our results showed that various transcription factors may be involved in the transcriptional activation of HK family genes during tumorigenesis and progression ( three transcription factors for HK1,19 transcription factors for HK2 ,one transcription factor for HK3 and three transcription factors for HKDC1 ). Notably, the expression of SPI1 was positively correlated with the expression of HK3 in the whole carcinoma of

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TCGA, and RCOR1 was positively correlated with the expression of HK2 in 23 types of cancer.

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Collectively， our results of pan- cancer analysis suggest that the abnormal expression of HK family genes is closely related to abnormal amplification, hypermethylation of promoter and abnormal transcriptional activation.

Cancer-related pathways regulated by HK family genes

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To further understand the molecular mechanism of HK family genes involved in cancer, we determined the correlation between the expression of single HK family gene and the activity of 10 cancer-related pathways. As a result, the expression of HK family genes was associated with the activation or inhibition of a variety of carcinogenic pathways. The expression of HK3, HK2 and GCK was associated with a larger number of activation pathways, such as PI3K-AKTMTOR, cell cycle and EMT pathway（Figure 3A）. Consistent with the above findings, drug sensitivity data from 22 types of cancer cell lines from the cancer drug sensitivity genomics database (GDSC) suggested that the high expression of HK3 and HK2 attenuated the sensitivity of tumor cells against more than 20 anticancer drugs involving cell cycle inhibition, DNA damage repair inhibition and so on（Figure 3B）. In addition, genes do not function separately, instead, the cooperation between HK1 and HK2 has been validated to exist in the context of cancer. Therefore, we studied the binding / interaction protein network of HK family proteins. Intriguingly, we not only found that there was a highly correlated pattern of interaction between HK family proteins, but also revealed that some receptors, kinases, ubiquitin enzymes, transcription factors and apparent regulators involved in signal transduction and regulation played a role in the protein-protein interaction network with HK family proteins as the core

Journal Pre-proof （Figure 3C）. In summary, these results suggest that crosstalk between HK family proteins and interacting proteins plays a key role in the development and progression of different types of cancer. At present, it has been found that some small molecular compounds can effectively regulate the activities of HK1 and GCK(Figure 3D). In view of the structural correlation of HK family genes, it is expected to find small molecular drugs that can effectively regulate the activities of HK2 and HK3 in the future.

Clinical correlations of HK family genes in cross-cancer types

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The common genetic and expression changes of HK family genes in various types of cancer may provide important insights for the development of transformational medicine solution. Through integration analysis of protein expression data, we found that the expression of HK1, HK2, GCK were significantly up-regulated in 20 types of tumor tissues (Figure 4A, Supplementary Figure 3-4). In addition, we showed that the expression of HK family genes was significantly associated with the survival of patients with various types of cancer（Figure 4B-4C）. Moreover, all HK family genes were associated with the overall survival rate of patients with at least one type of cancer. Several HK family genes harbored carcinogenic characteristics, such as HK1, HK2 and HK3, and the higher expression of these genes was associated with worse survival in patients with different types of cancer. In contrast, we found that several HK family genes also showed tumor suppressor characteristics in a small number of tumors, such as HKDC1. In summary, these results suggest that HK family genes play diverse roles in prognosis of patients with specific types of cancer and the development of new therapeutic strategies.

Identification of the oncogene role of HK1 in bladder cancer Given the abnormal expression of HK1 in a variety of solid tumors, the role of HK1 in bladder cancer was further investigated. Consistent with the above observations, the results suggested that the HK1 protein expression increased significantly in bladder cancer tissues (Fig.5A), and the expression level increased with the increase in tumor grade(Fig.5B). The high expression of HK1 predicted the poor prognosis (Fig.5C), which was also closely related to a variety of signaling pathways that affected the progression of bladder cancer (Fig.5D). After inhibiting the expression of HK1, the division and survival capacities of bladder cancer cells were seriously inhibited (Fig.5E-G), suggesting that HK1 acted as a potential target for the treatment of bladder

Journal Pre-proof cancer. DISCUSSION In consideration of the important role of HK family member HK2 in some cancers, it is of great significance to study the expression and regulation patterns of HK family members in different cancers, which could facilitate the diagnosis and treatment of tumors with abnormal HK family members. Using the latest TCGA multidimensional molecular spectrum analysis data, more than 9000 samples from 33 cancer types were comprehensively characterized by HK family. To sum up, our findings show that: (1)

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the HK family may play an important role in the carcinogenesis and progression of various solid tumors and may be a potential prognostic indicator and therapeutic target; (2) the abnormal expression of HK family is related to amplification, low DNA methylation and transcriptional activation. To the best of our knowledge, it is the first report based on data mining and in-depth bioinformation analysis on the comprehensive molecular characteristics of the HK family in the whole cancer. Our results suggest that it is feasible to use computational biology methods to explain the new molecular biological mechanism of HK family and to assist and promote the discovery of experimental biology in the future.

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Our analysis shows that HK family members might be critically involved in the carcinogenesis and progression of a variety of solid tumors. Although our results confirmed the key role of HK2 as previously reported[15-17], emerging evidence has challenged the core position of HK2 in tumor progression. As suggested in several recent studies, HK1 is also actively involved in the carcinogenesis of some solid tumors. For instance, the c-Src phosphorylation site mutations in HK1 significantly eliminate the stimulating effects of c-Src on glycolysis, cell proliferation, migration and invasion, as well as tumorigenesis and metastasis. HK1 has a low Km to glucose, as a result, when the glucose content in tumor cells is seriously insufficient, the greatest need of tumor cells is to prolong their survival time through HK1, rather than HK2. Further bioinformatics analysis showed that the abnormally increased HK1-Y732 phosphorylation level was involved in the occurrence and metastasis of several cancers, such as bladder, kidney, breast and colon tumors. Therefore, the abnormal HK1-Y732 phosphorylation level can be used as an indicator to predict the risk of multiple primary and metastatic solid tumors[18]. Recent study on colorectal cancer reports that HK1, an effector of KRAS4A, alters its kinase activity with KRAS4A via the GTP-dependent direct interaction. Such interaction is unique to KRAS4A, since the palmitoyl-dealdehyde acidification cycle of this RAS subtype

Journal Pre-proof enables it to co-locate with HK1 on the mitochondrial outer membrane[19]. Consistent with the above observations, our result shows that HK1 is not only abnormally expressed in a variety of solid tumors, but also plays a significant role of oncogene in bladder cancer（Fig5）.Furthermore，through the comprehensive analysis of the crosscancer data based on multigroup data, we further revealed the frequent amplification, high expression heterogeneity and significant prognostic correlation of other members of the HK family. Moreover, we also found that members of HK family can form fusion genes with potential diagnostic and therapeutic significance in a variety of solid tumors. In particular, it should be pointed out that HK3, as a less concerned member of the HK family, shows obvious characteristics of cancerous genetic

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mutation and great potential in suggesting poor prognosis, targeted therapeutic effect . In view of the strong selective pressure of changes in glucose metabolism in these cancers, abnormal signal transduction mediated by members of the HK family is likely to play a major role in shaping tumor progression, which is expected to become a potential prognostic indicator and therapeutic target in the future.

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Another finding of this study is that the high activity of HK family is related to the amplification, low DNA methylation and abnormal transcriptional activation. In spite of the obvious biological characteristics of carcinogenic carcinoma of the HK family, the overall mutation rate was not high. Therefore, we systematically constructed cancer type-specific HK family regulatory networks by integrating various types of molecular data. Based on the comprehensive analysis of multigroup data, we found that the abnormal amplification, low DNA methylation and abnormal transcriptional activation is very likely to be involved in regulating the activity of HK family. It is worth noting that in addition to the known Myc [20] and HIF-1 [21], we found that some important carcinogenic and immunomodulatory transcription factors such as STAT3, NFYA and SPI1/PU.1 were also involved in the abnormal transcription of HK family. Thus, it is necessary to further explore the precise regulatory mechanism and functional effects of the above pathways on the HK family in the future, so as to provide inspiration to search active regulatory drugs of the HK family.

In short, our concentrative and systematic analysis of the HK family, an important metabolic kinase family, will be valuable resource for understanding its disorders in cancer and how to maximize its clinical utility. Future research will further investigate the molecular mechanism by which the HK family promotes cancer progression.

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MATERIALS AND METHODS Mutation analysis

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Analysis of Mutual Exclusivity. of HK Family members in pan-cancer was performed by Cbioportal (http://www.cbioportal.org/). The TCGA mutation mutect data of each member of the HK family were downloaded from TCGAbiolinks GUI for ACC, BLCA, BRCA,CESC,CHOL, COAD, DLBC, ESCA, GBM, HNSC, KICH, KIRC, KIRP, LAML, LGG, LIHC,LUAD, LUSC, MESO, OV, PAAD, PCPG, PRAD, READ, SARC, SKCM, STAD, TGCT, THCA, THYM, UCEC, UCS and UVM. By removing mutations such as silent, 3'UTR, 5'Flank, Splice_Region, the non-synonymous mutations were examined in the coding region of the gene. Here we classified Frame_Shift_Del, Nonsense_Mutation, Frame_Shift_Del, Frame_Shift_Ins, Splice_Site as Truncating mutations. The mutations of HK family members were downloaded from CCLE (https://portals.broadinstitute.org/ccle/about) and mapped by Excel statistics.

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Fusion gene analysis

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The fusion gene data of each member of the NTN family was downloaded through

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TCGA Fusion Gene Database (http://www.tumorfusions.org/). The database is a fusion gene predicted by analyzing the RNA sequencing data of 33 types of TCGA cancers by PRADA. Among them, Tier 1 and tier 2 had high credit rating. The fusion gene of HK family was downloaded from Atlas of Genetics and Cytogenetics in Oncology and Haematology (http://www.atlasgeneticsoncology.com). Methylation and clinical analysis. The differential methylation bubble map of the genes of HK family members and their downstream related pathways between cancer tissues and adjacent tissues in 33 types of TCGA cancers was downloaded from GSCALite (http://bioinfo.life.hust.edu.cn/web/GSCALite/). The diagram of correlation between methylation and expression, and diagram between high and low methylation and total survival were plotted. Transcriptional modification analysis The transcription factors and chromatin remodeling factors between upstream and downstream 1kb of family members were downloaded from CHIPBASE V2.0 (http://rna.sysu.edu.cn/chipbase/). And the co-expression of various factors and HK family genes were evaluated in 32 types of TCGA cancers. Afterwards, the absolute

Journal Pre-proof value of correlation was set at greater than 0.2, p < 0. 05 as a meaningful cut off. Immunohistochemical staining. The immunohistochemical patterns of HK family in 20 kinds of cancers were searched through The Human Protein Atlas (https://www.proteinatlas.org). Expression and clinical analysis The median expression and survival curve of HK family genes in 33 types of TCGA cancers and adjacent tissues were downloaded by GEPIA

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(http://gepia.cancer-pku.cn/index.html). Through Kaplan-Meier Plotter (http://kmplot.com/analysis/) on Bladder Carcinoma (BLCA), Breast cancer (BRCA), Cervical squamous cell carcinoma (CESC), Esophageal carcinoma (ESCA), Head-neck squamous cell carcinoma (HNSC), Kidney renal clear cell carcinoma (KIRC), Kidney renal papillary cell carcinoma (KIRP), Liver hepatocellular carcinoma (LIHC), Lung adenocarcinoma (LUAD), Lung squamous cell carcinoma (LUSC), Ovarian cancer (OV), Pancreatic ductal adenocarcinoma (PAAD), Pheochromocytoma and Paraganglioma (PCPG), Rectum adenocarcinoma (READ), Sarcoma (SARC), Stomach adenocarcinoma (STAD), Testicular Germ Cell Tumor (TGCT), Thymoma (THYM), Thyroid carcinoma (THCA), Uterine corpus

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endometrial carcinoma (UCEC), the optimal cut off value was selected to represent the survival curve (minimum p value). Drug and pathway analysis

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The Global percentage and Hetmap percentage maps of HK family genes in 10 cancer-related pathways were downloaded from GSCALite (http://bioinfo.life.hust.edu.cn/web/GSCALite/). Additionally, all the drugs related to the HK family were downloaded from PharmacoDB (https://pharmacodb.pmgenomics.ca/). The absolute value of the correlation was set at more than 0.1, p < 0. 05 indicated statistical significance. Protein-protein interaction (PPI) The genes and proteins interacting with the HK family were searched from BioGRID, followed by visualization by Cytoscape. Bladder cancer tissues Human bladder cancer samples were collected from the Department of Urology at the First Affiliated Hospital of China Medical University. Our study protocols were approved by the Ethics Committee of the hospital (Institutional Review Board). The age of all patients (including both

Journal Pre-proof men and women) ranged from 40 to 76 years. No cancer patient received any neoadjuvant chemoradiotherapy prior to the surgical removal of the tumors. Cell culture The human bladder cancer UMUC3 and 5637 cell lines were purchased from the Chinese Academy of Sciences Committee on Culture Collection Cell Bank, Shanghai Institutes for Biological Sciences (Shanghai, China). Then, the cells were cultured in the RPMI 1640 medium (HyClone, Logan, Utah, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Invitrogen) and 1% glutamine under 37℃ and 5% CO2 conditions.

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Western blotting The lysis buffer (consisting of 150 mM NaCl, 50 mM Tris-HCl and 0.5% NP-40, pH 7.5)

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containing the protease inhibitor cocktail (Sigma, Houston, TX, USA) was used for cell lysis. Thereafter, equivalent amounts of protein were separated by SDS-PAGE and transferred onto the

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PVDF membranes (Millipore, Billerica, MA, USA). Then, the membranes were probed with the primary antibody against HK1 (1:1000, Cell Signaling Technology) for 1 h at room temperature.

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Subsequently, the membranes were further incubated with the horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Cell Signaling Technology) for 1 h at room temperature.

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Finally, an enhanced chemiluminescence system (ECL, Amersham Biosciences, Piscataway, NJ, USA) was utilized to detect the immuno-reactive signals.

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siRNA-mediated knockdown

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The siRNA against HK1 was purchased from GenePharma (Shanghai, RiboBio, China). Afterwards, siRNAs were transfected into the indicated cells using the Lipofectamine 2000 in accordance with the manufacturer’s instructions (Invitrogen). Colony formation assay

In the colony formation assay, 2 × 105 cells were transfected with the indicated siRNAs. At 48 h after the transfection, cells were harvested and counted. Then, both UMUC3 (1.5 × 105 cells) and 5637 cells (1 × 105 cells) were reseeded into the 6-well plates in triplicate, maintained for additional 24 h, and incubated with the normal medium for 7 days. Later, the plates were fixed and stained with Giemsa’s solution.

Cell proliferation assay In the growth curve assay, 1 × 105 cells were seeded into the 6-well plates. Cells in each cell line were prepared in triplicate at each time point. At 24 h later, cells were harvested and counted using

Journal Pre-proof a hemocytometer at the intervals of 24 h until 96 h. Thereafter, the cell counting Kit-8 (Dojindo, Japan) was employed to assess the cell proliferation. For the EdU assay, both the UMUC3 and 5637 cell lines were transfected with HK1 siRNAs in the 24-well plates for 48 h, and EdU (BeyoClickTM, EDU-488, China) was added into the medium (1:1000) according to the manufacturer's protocols. Later, cells were cultured for 2 h at 37 °C, after labeling, the culture medium was removed, and then 1 ml fixation solution (4% paraformaldehyde) was added to fix the cells at room temperature for 20 min. Thereafter, cells were incubated with 1 ml permeate (0.3% Triton X-100) at room temperature for 15 min, and the click reaction buffer was added following the manufacturer's instructions. At last, a fluorescence

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microscope (Olympus Corporation, Japan) was utilized to obtain the images.

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Statistical Analysis

All the in vitro experiments were repeated for at least thrice. Data were expressed as mean ± SD

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and compared by t-test. The Kaplan–Meier method and log-rank test were adopted for survival analysis using the Statistical Package for the Social Sciences (SPSS) (SPSS Inc, Chicago, IL). A

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difference of P< 0.05 indicated statistical significance for all experiments.

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ACKNOWLEDGMENTS

We want to thank Cbioportal, GSCALite, GEPIA and TISDIB for free use.

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CONFLICTS OF INTEREST

FUNDING

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The authors declare that there are no conflicts of interest.

This work was supported in part by National Natural Science Foundation of China (81672523, 81472404, 81472403, 81272834 and 31000572)，2018 Support Plan for innovative talents in Colleges and Universities of Liaoning Province，2018 "million talents Project" funded Project of Liaoning Province，2019 Key R & D projects of Shenyang. REFERENCES: 1. Sanderson SM, Locasale JW. Revisiting the Warburg Effect: Some Tumors Hold Their Breath. Cell Metab. 2018; 28: 669-70. 2. Zhang D, Tang Z, Huang H, Zhou G, Cui C, Weng Y, Liu W, Kim S, Lee S, Perez-Neut M, Ding J, Czyz D, Hu R, et al. Metabolic regulation of gene expression by histone lactylation. Nature. 2019; 574: 575-80.

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18. Zhang J, Wang S, Jiang B, Huang L, Ji Z, Li X, Zhou H, Han A, Chen A, W u Y, Ma H, Zhao W, Zhao Q, Xie C, Sun X, Zhou Y, Huang H, Suleman M, Lin F, Zhou L, Tian F, Jin M, Cai Y, Zhang N, Li Q. c-Src phosphorylation and activation of hexokinase promotes tumorigenesis and metastasis.Nat Commun. 2017;8:1-16. 19.Amendola CR, Mahaffey JP, Parker SJ, Ahearn IM, Chen WC, Zhou M, Court H, Shi J, Mendoza SL, Morten MJ, Rothenberg E, Gottlieb E, Wadghiri YZ, Poss emato R, Hubbard SR, Balmain A, Kimmelman AC, Philips MR.KRAS4A directly regulates hexokinase 1.Nature. 2019 ;576:482-486. 20. Yu P, Wilhelm K, Dubrac A, Tung JK, Alves TC, Fang JS, Xie Y, Zhu J, Chen Z, De Smet F, Zhang J, Jin SW, Sun L, et al. FGF-dependent metabolic control of vascular development. Nature. 2017; 545: 224-8. 21. Zhou L, Wang Y, Zhou M, Zhang Y, Wang P, Li X, Yang J, Wang H, Ding Z. HOXA9 inhibits HIF-1α-mediated glycolysis through interacting with CRIP2 to repress cutaneous squamous cell carcinoma development. Nat Commun. 2018; 9: 1480.

Journal Pre-proof FIGURE LEGEND Figure 1. Pancancerous genetic changes of HK family genes. Genomic aberration landscape of HK family genes in cancer. (A)Waterfall maps of gene mutations and copy number changes in five HK family genes. Each row represents a gene and each column represents a sample. (B)The landscape of HK family gene mutations in cancer. Mutations that cause substitution are represented by single-letter amino acid codes separated by slashes. (C)A schematic diagram of the fusion gene formed by HK1 and CTNNA3 in PRAD.

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Figure 2. Regulatory mechanism of HK Family Gene expression and its role in Cancer. (A)The relationship between the structural variation of HK family genes and the expression level in different cancers. (B) The relationship between promoter methylation and expression level of HK family genes in different cancers . The above figure shows the relationship between promoter methylation and expression levels of HK family genes in different cancers. The following figure shows the difference in promoter methylation of HK family genes in different cancers. (C) The main transcriptional regulator of HK family genes, which is ubiquitous in human cancer. The horizontal axis represents the assumed major transcriptional regulators, and the vertical axis represents the types of cancer.

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Figure. 3. The role of HK family genes in cancer signaling pathways and drug responses. (A) HK family genes are associated with the activation and inhibition of 10 cancer pathways. The pie chart shows the correlation between HK family genes and cancer pathways. Red indicates positive correlation and blue indicates negative correlation. (B)Association of HK2 and HK3 with targeted drugs across different cancer signal transduction pathways. Purple indicated that gene expression was negatively correlated with drug sensitivity. The size of the node corresponds to the value of-log (FDR). (C) Protein-protein interaction based on HK family interaction protein network. (D) A network of drugs that may act on HK1 and GCK, as well as other targets for these drugs. Figure 4. Clinical correlation of HK family genes across cancer types. (A)Abnormal expression of HK1 protein in human cancer. The expression level distribution of HK1 protein in different cancer tissues is shown above. The following figure shows the representative immunohistochemical staining results of HK1 protein in different cancer tissues. (B)Summary of the relationship between HK family gene expression and patient survival. Red indicates that the higher expression of HK family genes is associated with poor survival rate, and blue is associated with better survival rate. Only p values of < 0.05 are displayed. (C) The Kaplan-Meier survival curve of cancer

Journal Pre-proof patients was divided according to the expression level of HK family genes Figure 5. The clinical and functional relevance of HK1 in bladder cancer (BC). A) HK1 was significantly up-regulated in bladder tumors relative to normal tissues. Representative images and expression patterns of HK1 in the 28 primary tumor and matched normal tissues. B) Representative images and expression patterns of HK1 in bladder tumor tissues at different

grades obtained from the Human Protein Atlas (HPA) database. C) Kaplan–Meier analysis on TCGA gene expression datasets of 412 BCC patients stratified according to HK1 expression for overall survival (OS). D) GSEA on TCGA gene expression datasets of BC patients with high or low HK1 expression. E-G) HK1 promoted the proliferation and survival of BC cells. UMUC3 and 5637 cells were transfected with two different anti-HK1 siRNAs for 24 h; E&F) cell

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proliferation and G) survival were determined by CCK-8 assay, EdU experiment and colony

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formation assay, respectively. Bars, SD; **, P < 0.01; ***, P < 0.001.

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Table 1: HK family cancer-related fusion genes identified based on sequencing data. Fusion_pair

HK1

TSPAN15-HK1(BLCA) IFIT5-HK1(COAD) CDC123-HK1(MESO) HK1-CTNNA3(PRAD) CYB561-HK1 HK1-EPN2 HK1-GNL1 HK1-HK1 HK1-MTOR TMEM135-HK1 HK2-ONECUT3(OV) FAM50A-HK2 HK2-TET3 POLE4-HK2 EIF2AK1-GCK(LIHC) EIF2AK1-GCK(LIHC) C7orf44-GCK(PRAD) GCK -FRA10AC1 MIRLET7BHG-HKDC1(BLCA) HKDC1-SUPV3L1(STAD)

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HKDC 1

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HK2 GCK

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Gene

BLCA:Bladder Urothelial Carcinoma;COAD:Colon adenocarcinoma;MESO:Mesothelioma; PRAD:Prostate adenocarcinoma;OV:Ovarian serous cystadenocarcinoma;LIHC:Liver hepatocellular carcinoma;PRAD:Prostate adenocarcinoma;STAD:Stomach adenocarcinoma

Journal Pre-proof Conflict of interest statement We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing

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the position presented in, or the review of, the manuscript entitled。

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CRediT author statement

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Yuyan Zhu: Conceptualization, Methodology, Software Wenjun Hao.: Data curation.Jiaxing Lin: Visualization, Investigation.Chuize Kong: Supervision.: Jieping Yang；Ying Gao: Software, Validation.: Mingzhe Jiang: Writing- Original draft preparation，Writing- Reviewing and Editing,

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