HOXC10 up-regulation promotes gastric cancer cell proliferationand metastasis through MAPK pathway

Chen Guo1*, Jianing Hou2*, Sheng Ao2, Xingming Deng2, Guoqing Lyu2

1Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Peking University, Beijing 100191, China; 2Department of

Gastrointestinal Surgery, Peking University Shenzhen Hospital, Shenzhen 518036, China

*These authors contributed equally to this work.

Correspondence to: Guoqing Lyu. Department of Gastrointestinal Surgery, Peking University Shenzhen Hospital, No. 1120 Lianhua Road, Shenzhen

518036, China. Email: lvguoqing1001@163.com.

Abstract

Objective: As an important regulator of embryonic morphogenesis, homeodomain-containing gene 10

(HOXC10) has been found to promote progression of human cancers and its expression indicates poor survivaloutcome. However, very few studies are available on the role of HOXC10 in gastric carcinoma. Therefore, the aim

of this study was to determine the role of HOXC10 in gastric cancer and the potential mechanism underlying its

function for cancer biology.

Methods: A primary gastric cancer mouse model was obtained via intra-gastric wall injection of gastric cancer

cells and was used to evaluate the function of HOXC10 during gastric cancer progression in vivo.Immunohistochemistry was performed to visualize and measure HOXC10 protein expression in gastric cancer

tissue. Cells were transfected with plasmids to increase the expression of HOXC10, and siRNA transfection wasperformed to suppress HOXC10 expression. Reverse transcription polymerase chain reaction (RT-PCR) andwestern blotting were utilized to measure mRNA and protein expression, respectively. Proliferation, migration, and

invasion were investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay,

wound healing assay, and matrigel invasion assay in vitro, respectively.Results: HOXC10 expression was significantly increased in gastric cancer tissues compared to matched normal

tissues. HOXC10 up-regulation significantly increased tumor volumes in nude mice. Plasmid transfection

significantly increased HOXC10 protein and mRNA expressions and effectively promoted cell proliferation.

Moreover, HOXC10 up-regulation significantly promoted migration and invasion of gastric cancer cells.

Mechanistic investigation showed that HOXC10 up-regulation significantly increased mRNA and protein

expression of mitogen-activated protein kinase (MAPK) signaling related genes, including c-myc, c-jun and p53,while also modulating the phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase

(ERK) and P38 but not their total protein levels.

Conclusions: This study demonstrated the tight link between HOXC10 and gastric cancer cell proliferation and

metastasis via involvement of the MAPK pathway.

Keywords: HOXC10; proliferation; metastasis; gastric cancer

Submitted Oct 10, 2017. Accepted for publication Dec 01, 2017.

doi: 10.21147/j.issn.1000-9604.2017.06.12

View this article at: https://doi.org/10.21147/j.issn.1000-9604.2017.06.12

Introduction

Although the incidence has been declining for several

decades, gastric cancer was the leading cause of cancer-related deaths worldwide until the mid-1990s (1). In China,gastric cancer was the most common type of cancer and the

Original Article

Chinese Journal of Cancer Research. All rights reserved. www.cjcrcn.org Chin J Cancer Res 2017;29(6):572-580

https://doi.org/10.21147/j.issn.1000-9604.2017.06.12

second leading cause of cancer-related deaths after lungcancer until now (2). Therefore, there is an urgent need toidentify novel prognostic and predictive biomarkers toimprove the diagnosis and clinical management of patientswith gastric cancer, which in turn may help to developmore effective treatment strategies.

In humans, 39 homeobox (HOX) genes are present, andorganized into four different genomic clusters (HOX AD)located on four chromosomes (7, 17, 12 and 2) (3). HOXgenes are highly conserved at the genomic level and havebeen well-described as important players in regulatingnumerous processes including apoptosis, receptorsignaling, differentiation, motility, angiogenesis, andmetastasis (4,5). Aberrations in HOX gene expression havebeen reported in numerous malignancies, including ovariancancer, esophageal squamous cell carcinoma, breast cancer,lung cancer, colorectal cancer, and hematologicalmalignancies, and have been associated with significantlyenhanced invasiveness, proliferation, and colony formationof tumor cells (6-12). In gastric cancer, previous datashowed that approximately 2025 HOX genes are expressedat least two-fold higher than in normal tissues (13),especially homeobox gene HOXB5 whose up-regulationinduces invasion and migration through direct transcriptionand up-regulation of -catenin in human gastric carcinoma(14). These results indicate that HOX genes can beconsidered as new targets for future tumor therapies.

HOXC10 is a member of the HOX gene family andsignificantly enhances proliferation, invasion, andmetastasis of cancer cells, thus it might be useful as amarker for cancer diagnosis or progression. In cervicalsquamous cell carcinomas, elevated HOXC10 expression isassociated with increased invasiveness as identified by usinghigh-density oligonucleotide microarrays (15). In theCancer Genome Atlas (TCGA) datasets, HOXC10expression is significantly increased in human thyroidcancer tissues compared to normal human thyroid tissues.Furthermore, HOXC10 promotes migration and invasionof thyroid cancer cells, suggesting a role as a novelbiomarker for human thyroid cancer prognosis (16), andpromotes the metastasis of human lung adenocarcinomaand indicates poor survival outcome (17). However, therole of HOXC10 in breast cancer remains controversial(18-20). Remarkably, the latest research about the functionof miR-136 in the gastric cancer cell line GC-9811 showsthat HOXC10 can work as a direct target of miR-136, whichinhibits gastric cancer-specific peritoneal metastasis. Thisindicates that HOXC10 functions as a metastasis promoter

in gastric cancer peritoneal metastasis (21). However, thereis no definite data to confirm the role of HOXC10 in thecarcinogenesis and progression of gastric cancer.

In the present study, we discovered that HOXC10expression was significantly increased in human gastriccancer tissues compared with normal tissues. In addition,HOXC10 could significantly promote the tumorigenicityof gastric cancer cells in vivo. Furthermore, HOXC10knockdown or over-expression further confirmed thatHOXC10 was required for proliferation, migration, andinvasion of gastric cancer cells. With regard to itsmechanism of action, our study showed that HOXC10exerted its action on cancer through the mitogen-activatedprotein kinase (MAPK) pathway. Thus, HOXC10 might beconsidered as a potential target in the treatment of gastriccancer and as a marker in the prognosis of gastric cancer.

Materials and methods

Tissue samples and cell cultures

The biospecimens used in this study were provided by thePeking University Shenzhen Hospital. Gastric tissue fromgastric cancer patients was divided into cancer tissue andadjacent normal tissue. Human gastric cancer cell linesAGS and SNU638 were purchased from American TypeCulture Collection (ATCC) and were cultured in RPMI1640 medium (Lonza, Walkersville, MD, USA) orDulbeccos modified Eagles medium (DMEM) (Lonza)supplemented with 10% (V/V) heat-inactivated fetal bovineserum (FBS) (Gibco-BRL) and 1% penicillin/streptomycin(Gibco-BRL). Cells were incubated at 37 C in a 5% CO2humidified atmosphere.

Animal experiments

A total of 20 eight-week-old male BALB/c nude miceweighing 1820 g were purchased from the Department ofLaboratory Animal Science, Peking University and, theprotocols used in this study were approved by the AnimalCare and Use Committee of Peking University (No. LA2010-066). Plasmid pcDNA3-HOXC10 and control vectorpcDNA3 were separately transfected into AGS cells toestablish stable HOXC10-overexpressing cells (pcDNA3-HOXC10) and control cells (pcDNA3). Mice wereanesthetized with isoflurane, the area on the midside of theabdomen was sterilized and the stomach was exposed vialaparoscopy. A density of (5106) cells per cell line(pcDNA3-HOXC10 or pcDNA3 control cells) were

Chinese Journal of Cancer Research, Vol 29, No 6 December 2017 573

Chinese Journal of Cancer Research. All rights reserved. www.cjcrcn.org Chin J Cancer Res 2017;29(6):572-580

suspended in 50 L of Hanks balanced salt solution andplaced into a sterile 31-gauge needle. The gauge tip wasintroduced 1.5 mm into the lesser curvature side of thegastric low body with an approximate 10 angle and the cellsuspension was slowly injected (the procedure wasperformed under a binocular lens). Afterward, slightpressure was applied at 5 mm from the injection pointusing a gauze. The syringe needle was removed and thearea around the injection was cleaned with 70% ethanol toavoid seeding of unlikely refluxed tumor cells into theabdominal cavity. After injection, the stomach was placedback into the abdominal cavity and the abdomen was closedwith surgical drapes. Animals were sacrificed 5 weeks aftersurgery, and stomachs were examined. Tumors weremeasured at the indicated time points using calipers, andtumor v