Open Access

TMEM165, a Golgi transmembrane protein, is a novel marker for hepatocellular carcinoma and its depletion impairs invasion activity

  • Authors:
    • Jee‑San Lee
    • Mi‑Yeun Kim
    • Eun‑Ran Park
    • Yan Nan Shen
    • Ju‑Yeon Jeon
    • Eung‑Ho Cho
    • Sun‑Hoo Park
    • Chul Ju Han
    • Dong Wook Choi
    • Ja June Jang
    • Kyung‑Suk Suh
    • Jungil Hong
    • Sang Bum Kim
    • Kee‑Ho Lee
  • View Affiliations

  • Published online on: July 12, 2018     https://doi.org/10.3892/or.2018.6565
  • Pages: 1297-1306
  • Copyright: © Lee et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Transmembrane protein 165 (TMEM165), a Golgi protein, functions in ion homeostasis and vesicular trafficking in the Golgi apparatus. While mutations in TMEM165 are known to cause human ‘congenital disorders of glycosylation’, a recessive autosomal metabolic disease, the potential association of this protein with human cancer development has not been explored to date. In the present study, we revealed that TMEM165 is overexpressed in HCC and its depletion weakens the invasive activity of cancer cells through suppression of matrix metalloproteinase‑2 (MMP‑2) expression. Levels of TMEM165 mRNA and protein were clearly increased in HCC patient tissues and cell cultures. Quantitative real‑time RT‑PCR analysis of fresh HCC tissues (n=88) revealed association of TMEM165 overexpression with more frequent macroscopic vascular invasion, microscopic serosal invasion and higher α‑fetoprotein levels. Notably, depletion of TMEM165 led to a marked decrease in the invasive activity of two different HCC cell types, Huh7 and SNU475, accompanied by downregulation of MMP‑2. Our collective findings clearly indicated that TMEM165 contributed to the progression of HCC by promoting invasive activity, supporting its utility as a novel biomarker and therapeutic target for cancer.

Introduction

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer in adults and the third leading cause of cancer-related mortality worldwide (1). HCC occurs most frequently in patients with chronic liver disease and cirrhosis (2). The common risk factors of HCC include hepatitis B and C virus infection, heavy consumption of alcohol, exposure to aflatoxin B1 and non-alcoholic steatohepatitis (3). Despite significant advances in diagnosis and treatment, liver resection and transplantation are considered the only curative options at present (3). To date, no effective treatment strategies have been developed for patients with advanced HCC, including the approved therapeutic agent, sorafenib (4), owing to chemoresistance and excessive cytotoxicity resulting in dismal prognosis (5). Therefore, identification of early and effective diagnostic markers and therapeutic targets for this disease remains an urgent unmet medical need.

The discovery of genes involved in tumor progression has prompted research into various methods of therapeutic intervention. Based on advances in genome-wide technology in cancer research, we established a microarray dataset to identify the genes responsible for dismal prognosis in earlier studies (68). In the present study, transmembrane protein 165 (TMEM165), a Golgi protein (9), was shown to be associated with aggressive characteristics of HCC. TMEM165 is a highly conserved hydrophobic protein of 324 amino acids that contains 7 transmembrane-spanning domains according to topology predictions (10). Recent research has revealed that mutations in TMEM165 were associated with a rare autosomal recessive disease designated ‘congenital disorders of glycosylation’ leading to defects in metabolic processes through impairment of galactosylation and sialylation of N-glycoproteins (1014). The majority of congenital disorders of glycosylation are caused by defects in glycosylation machinery components, such as SLC35A1, B4GALT1 and MGAT2 (1517). In contrast to the glycosylation-governing proteins, TMEM165 maintains Golgi ion homeostasis and vesicular Golgi trafficking (18). While TMEM165 has been extensively characterized in association with congenital disorders of glycosylation, its relationship with cancer remains to be determined. HCC-related proteins have been identified in multiple cellular compartments, including the nucleus, cytoplasm, mitochondria and plasma membrane. However, association of Golgi proteins with HCC has rarely been documented in the literature to date. To the best of our knowledge, GP73 (a 73-kDa Golgi transmembrane protein) is the protein that has been relatively well documented as a useful serum biomarker for HCC and shown to be involved in the progression of benign liver disease (17,19,20). In the present study, we identified a Golgi transmembrane protein, TMEM165, that is overexpressed in HCC patient tissues. TMEM165 expression was significantly associated with high levels of α-fetoprotein as well as presence of macroscopic vascular and serosal invasion. Consistently, TMEM165 depletion attenuated invasion activities of HCC cells via decrease in MMP-2 expression. Our findings revealed that TMEM165 could serve as a novel HCC marker associated with cancer aggressiveness and presents a potential therapeutic target.

Materials and methods

Patients and tissue samples

HCC tissues were acquired from patients with HCC subjected to hepatic surgery between September 1992 and December 2004 at Korea Cancer Center Hospital. A total of 88 HCCs used in the present study included 33 pair-matched HCC and adjacent liver tissues, and 55 HCC tissues. Normal liver tissues were obtained from patients with colon or rectal cancer, metastasized into liver, who underwent surgical resection at Seoul National University Hospital and Korea Cancer Center Hospital. The adjacent liver tissues around these metastatic cancers were regarded and used as a normal liver (n=15) when liver fibrosis as well as HCC were not observed in the patients. Our study was approved by the Institutional Review Boards of Korea Cancer Center Hospital and Seoul National University Hospital. Written informed consent was either waived by the Institutional Review Board of Korea Cancer Center Hospital or received from patients at Seoul National University Hospital, respectively. Clinicopathological information collected retrospectively was utilized to examine associations between TMEM165 expression patterns and HCC characteristics. Fibrosis was graded on a scale of 0–4 (stage 0, not present; stage 1, portal; stage 2, periportal; stage 3, septal; and stage 4, cirrhosis).

Cell culture and siRNA transfection

Cell lines were obtained from two institutions. The cell lines, SNU354, SNU398, SNU449, SNU475, SNU739 and Huh7 were purchased from the Korean Cell Line Bank (Seoul, South Korea) and the cell lines, Hep3B, HepG2, BJ, IMR90, WI38, normal Small Airway (PCS-301-010) and Prostate (PCS-440-010) epithelial cells from the American Type Culture Collection (ATCC; Manassas, VA, USA). Huh7, SNU354, SNU398, SNU449, SNU475 and SNU739 cells were cultured in RPMI-1640 medium (LM 011–01; Welgene, Daegu, Korea) and BJ, IMR90, WI38, HepG2 and Hep3B were cultured in MEM (LM 007–07; Welgene) supplemented with 10% (w/v) fetal bovine serum (FBS; JR Scientific, Inc., Woodland, CA, USA) and 1% (w/v) penicillin/streptomycin (Welgene) at 37°C. Normal small Airway and Prostate epithelial cells were cultured in Airway Epithelial Basal media (ATCC PCS 300–030) supplemented with Bronchial Epithelial Cell Growth kit (ATCC PCS-300-040) and Prostate Epithelial Cell Basal Medium (ATCC PCS-440-030) supplemented with Prostate Epithelial Cell Growth kit (ATCC PCS-440-040), respectively. For knockdown of TMEM165, siRNA transfection was performed using Lipofectamine RNA iMAX reagent (cat. no. 13778030; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. The following siRNA sequences were used: TMEM165 siRNA#1, 5′-AGCCAUCAUGGCAAUGCGCUAUU-3′ (sense) and 5′-UAGCGCAUUGCCAUGAUGGCUUU-3′ (antisense); and TMEM165 siRNA#2, 5′-UUGGGUAGGACACCCAAUAUAUU-3′ (sense) and 5′-UAUAUUGGGUGUCCUACCCAAUU-3′ (antisense).

RNA extraction and cDNA synthesis

Total RNA was extracted from cultured cells and fresh tissues using the RNeasy Mini Kit (cat. no. 74106; Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's protocol. The quality and concentration of isolated total RNA were determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop; Thermo Fisher Scientific, Inc., Wilmington, DE, USA). RNA samples were stored at −80°C until gene expression analysis. cDNA was synthesized using the iScript reverse transcriptase kit (cat. no. 170890; Bio-Rad Laboratories, Inc., Hercules, CA, USA) in keeping with the manufacturer's protocol.

Quantitative and semi-quantitative RT-PCR

Quantitative RT-PCR was performed with the KAPA SYBR FAST Universal qPCR kit (kit code: KK4301; Kapa Biosystems, Inc., Boston, MA, USA) on the CFX96 Real-Time RT-PCR Detection System (Bio-Rad Laboratories, Inc.). The primer sequences used for real-time RT-PCR were: TMEM165 forward, 5′-GGCAGTAATTGGAGGAAGAATGATAGC-3′ and reverse, 5′-ACCAGAATCAGGGCTTATAAATAGTGC-3′; and β2-microglobulin forward, 5′-AAGGACTGGTCTTTCTATCTCTTGTA-3′ and reverse, 5′-ACTATCTTGGGGTGTGACAAAGTC-3′. Median Ct values from triplicate experiments were used for statistical analyses and TMEM165 levels were normalized to median β2-microglobulin expression. Relative TMEM165 expression in HCC and adjacent liver tissues, compared to that in normal liver tissues, was analyzed using the comparative threshold cycle 2−ΔΔCt method. Semi-quantitative RT-PCR was performed using the Maxime PCR PreMix kit (cat. no. 25185; iNtRON Biotechnology, Kyungi-do, Korea) and the following primers: TMEM165 forward, 5′-GGCAGTAATTGGAGGAAGAATGATAGC-3′ and reverse, 5′-ACCAGAATCAGGGCTTATAAATAGTGC-3′; β2-microglobulin forward, 5′-GTGCTCGCGCTACTCTCTCT-3′ and reverse, 5′-CGGCAGGCATACTCATCTTT-3′; MMP-1 forward, 5′-ACAAAGTTGATGCAGTTTTCA-3′ and reverse, 5′-GAATCCATAAGCCACAAACTT-3′; MMP-2 forward, 5′-ATCTTTGCTGGAGACAAATTC-3′ and reverse, 5′-AACTTCACGCTCTTCAGACTT-3′; MMP-9 forward, 5′-AACTTTGACAGCGACAAGAA-3′ and reverse, 5′-TTGAACAAATACAGCTGGTTC-3′; MMP-13 forward, 5′-CCCAAAATTTTCTACCTCTGA-3′ and reverse, 5′-TTTTGATGATGATGAAACCTG-3′; β-actin forward, 5′-GGACTTCGAGCAAGAGATGG-3′ and reverse, 5′-AGCACTGTGTTGGCGTACAG-3′; RPS26 forward, 5′-CCAAGGACAAGGCCATTAAG-3′ and reverse, 5′-AGCACCCGCAGGTCTAAATC-3′; and GAPDH forward, 5′-GTCAGTGGTGGACCTGACCT-3′ and reverse, 5′-TGCTGTAGCCAAATTCGTTG-3′. The optimal PCR cycle for each primer set was determined by monitoring PCR products harvested at 2-cycle intervals with at least 3 different cycles. PCR amplification was performed with an initial 2 min of denaturation at 94°C, followed by cycling a 20 sec denaturation at 94°C, 10 sec annealing at 55°C and 30 sec extension at 72°C with a final 5 min extension at 72°C. The final PCR products were loaded on agarose gels and mRNA levels quantitated using NIH ImageJ software (http://rsb.info.nih.gov/ij).

Western blot analysis

Total tissue samples were homogenized in lysis buffer [50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% (v/v) Nonidet P-40, 0.1% (w/v) sodium dodecyl sulfate, 0.5% (w/v) sodium deoxycholate] including protease inhibitor cocktail (cat. no. P3100-010; GenDEPOT, Inc., Barker, TX, USA) and placed on ice for 20 min. After incubation, tissues were centrifuged for 20 min at 13,000 × g at 4°C and the supernatant fractions were collected. Quantified and sampled proteins were separated on a 12.0% (w/v) SDS gel and transferred to nitrocellulose membranes (cat. no. 10401396; Whatman, Maidstone, UK). Next, the membranes were blocked with 5% (w/v) skim milk in TBS with Tween-20 (TBS-T) buffer [25 mM Tris-HCl, pH 7.4, 140 mM NaCl, 2.7 mM KCl, 0.05% (w/v) Tween-20] for 1 h at room temperature, followed by incubation with primary antibodies against TMEM165 (diluted to 1:1,000; cat. no. 20485–1-AP; ProteinTech Group, Rosemont, IL, USA) and GAPDH (diluted to 1:2,000; cat. no. sc-25778; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 1 h at room temperature. After washing 3 times with TBS-T, the membranes were treated with horseradish peroxidase-conjugated secondary antibody (diluted to 1:3,000; cat. nos. A120-101P and A90-116P; Bethyl Laboratories, Inc., Montgomery, TX, USA) at room temperature for 1 h. Following 3 further washes with TBS-T, the membranes were reacted with Luminol chemiluminescent reagent (cat. no. sc-2048; Santa Cruz Biotechnology, Inc.) according to the manufacturer's protocol. The intensities of the band were analyzed using the NIH ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Clonal survival analysis

Huh7 and SNU475 cells were transiently transfected with control and TMEM165 siRNAs. At 24 h after transfection, 1,000 cells were seeded on a 6-well plate. Cells were fixed with 10% (w/v) formaldehyde and stained with 0.1% (w/v) crystal violet 10 days after seeding. Colonies were counted using digital images obtained with ImageJ. All experiments were performed in triplicate.

Invasion analysis

Polycarbonate membrane Transwell inserts (cat. no. 354480; Corning Inc., Corning, NY, USA) were coated with 20 µg/ml Matrigel. Following transient transfection, Huh7 and SNU475 cells (2×104) were seeded on the upper compartment of the chamber in serum-free medium. As a chemoattractant, complete medium containing 10% (v/v) FBS was placed in the bottom compartment. After 24 and 48 h of incubation, the cells were fixed, stained with Hemacolor® solution (Merck KGaA, Darmstadt, Germany) and counted under a light microscope in 4 different fields. All experiments were performed in triplicate.

Statistical analysis

Statistical analysis was performed using SPSS v.23.0 software (IBM Corp., Armonk, NY, USA). Receiver operating characteristic (ROC) curves were generated to assess the optimal cut-off point for TMEM165 expression. A one-way analysis of variance (ANOVA) with post hoc Tukey's HSD test was conducted to compare the expression levels of TMEM165 in normal, non-tumor and tumor samples. Correlations between TMEM165 expression and clinicopathological parameters were determined using a 2-sample t-test, Pearson's correlation test, Chi-squared (χ2) and Fisher's exact tests. For the comparison of invasion and clonal survival, ANOVA with post hoc Dunnett's test was performed. The data were considered significant at P<0.05.

Results

TMEM165 is overexpressed in HCC

To establish whether TMEM165 is involved in the pathogenesis of HCC, we examined its expression patterns in tumor and adjacent liver tissue specimens obtained from HCC patients subjected to surgical resection. Semi-quantitative RT-PCR disclosed higher TMEM165 expression in HCC than adjacent liver tissues (P=0.027) (Fig. 1A). To confirm TMEM165 expression, we performed quantitative real-time RT-PCR on an extended HCC sample set (n=88) (Table I) comprised of background liver cirrhosis (n=29) and fibrosis (n=3:7:16:24:29 for grades 0:1:2:3:4, respectively). Similar to semi-quantitative RT-PCR findings, real-time RT-PCR revealed significantly elevated TMEM165 expression in HCC, compared to adjacent non-tumor liver (P<0.001) and normal liver tissues (P=0.034) (Fig. 1B). In contrast to tumor tissues, adjacent liver tissues did not exhibit a significant difference in TMEM165 expression, compared to normal liver tissues (P=0.942). When expression levels were adjusted and compared with those of normal liver tissues, the mean increases in TMEM165 expression in HCC and adjacent liver tissues were 2.40- and 1.02-fold (median, 1.89- and 0.99-fold, respectively). Consistent with the mRNA expression data, the protein levels were higher in HCC than adjacent liver tissues (P=0.002) (Fig. 1C). The collective findings clearly demonstrated TMEM165 overexpression in HCC.

Table I.

Patient demographics and pathological data (n=88).

Table I.

Patient demographics and pathological data (n=88).

VariablesClassificationDistribution
SexMale : Female77 : 11
Age (years)Year, mean ± SD (range)52.05±10.79 (25–74)
EtiologyHepatitis B : Hepatitis C85 : 03
Total bilirubin<1 mg/dl : ≥1 mg/dl59 : 29
AFP<20 ng/dl : ≥20 ng/dl38 : 49
<50 ng/dl : ≥50 ng/dl47 : 40
<200 ng/dl : ≥200 ng/dl55 : 32
ASTIU/l, mean ± SD (range)51.92±31.07 (15–182)
ALTIU/l, mean ± SD (range)46.25±25.21 (10–130)
Child-Pugh classificationA : B and C70 : 07
Tumor sizecm, mean ± SD (range)5.8±3.25 (1.0–24.0)
Tumor numberSingle : Multiple59 : 17
Macroscopic vascular invasionNo : Yes78 : 5
Microscopic vascular invasionNo : Yes34 : 39
Serosal invasionNo : Yes68 : 12
Edmondson gradeaІ : II : III : VI11 : 48 : 27 : 1
Fibrosis0 : 1 : 2 : 3 : 43 : 7 : 16 : 24 : 29

a Edmondson-Steiner histological grade. In our present patient cohort, the clinicopathological information collected retrospectively has missing data, since some parameters were not obtained from all patients. AFP, α-fetoprotein; AST, aspartate aminotransferase; ALT, alanine transaminase.

TMEM165 overexpression is clinically associated with macroscopic vascular invasion, microscopic serosal invasion, and high α-fetoprotein levels of HCC

Next, we examined the potential association of TMEM165 mRNA levels with clinicopathological parameters to ascertain whether overexpression has a clinical impact on HCC. Variations in TMEM165 expression determined based on real-time analysis were mostly <2.0-fold between normal and adjacent liver tissues. To further verify an optimal cut-off point for TMEM165 expression, we performed ROC analysis using SPSS software. In the analysis, the clinical parameters, AFP level (<50 vs. ≥50 ng/ml), macroscopic invasion (no and yes) and serosal invasion (no and yes) were associated with TMEM165 (P<0.05). The optimal cut-off points of TMEM165 expression for these parameters were 1.98- (P=0.009), 3.03- (P=0.032), and 2.32-fold (P=0.018), respectively, indicating a 2.0-fold is suitable for comparison with clinicopathological parameters. Accordingly, we subdivided HCCs into two groups based on TMEM165 expression employing 2.0-fold as a cut-off point. Among the 88 tumor tissues examined, 41 (46.6%) exhibited ≥2.0-fold increase in TMEM165 transcript expression. Consistent with the ROC analysis, higher α-fetoprotein levels (≥50 ng/ml, P=0.022; ≥200 ng/ml, P=0.024) and presence of macroscopic vascular invasion (P=0.023) and serosal invasion (P=0.006) were significantly associated with TMEM165 overexpression (≥2.0-fold) (Table II). In contrast, HCC-based parameters such as sex (P=0.187), age (P=0.479), aspatate transaminase (P=0.204), alanine transaminase (P=0.386), Child-Pugh classification (P=0.273), tumor size (P=0.113), tumor number (P=0.212), microscopic vascular invasion (P=0.377), Edmondson grade (P=0.274) and cirrhosis (P=0.334), were not associated with TMEM165 overexpression. Furthermore, HCCs classified based on significant parameters (Table II) exhibited marked differences in TMEM165 expression (Fig. 2). Specifically, the TMEM165 expression level was positively correlated with the AFP level (Fig. 2A) (n=87, r=0.213 and P=0.048). Additionally, median increases in TMEM165 expression were 1.90- and 3.26-fold according to macroscopic vascular invasion (no and yes, P=0.034) (Fig. 2B) and 1.87- and 2.95-fold according to serosal invasion (no and yes, P=0.03), respectively (Fig. 2C). The clinicopathological association data indicated that TMEM165-associated parameters influence aggressiveness of HCCs, based on the finding that overexpression of this protein is clinically associated with the invasive characteristics of this tumor type.

Table II.

Correlation between TMEM165 expression and clinicopathological parameters (n=88).

Table II.

Correlation between TMEM165 expression and clinicopathological parameters (n=88).

TMEM165 expression

Variables<2.0 fold≥2.0 fold P-valuea
Sex
  Male43340.187
  Female47
Age (years)
  <5220190.479
  ≥522622
AFP (ng/ml) 0.149
  <202315
  ≥202326
AFP (ng/ml)
  <5030170.022
  ≥501624
AFP (ng/ml) 0.024
  <2003421
  ≥2001220
AST (U/l)
  <4020220.204
  ≥402719
ALT (U/l)
  <3517170.386
  ≥353024
Child-Pugh classification
  A36340.273
  B,C52
Tumor size (cm)
  <526160.113
  ≥52124
Tumor number
  Single33260.212
  Multiple710
Macroscopic vascular invasion
  No43350.023
  Yes05
Microscopic vascular invasion
  No17170.377
  Yes2217
Serosal invasion
  No41270.006
  Yes210
Edmondson grade
  I,II33260.274
  III, IV1315
Cirrhosis
  No28220.334
  Yes1415

a Significance of TMEM165 overexpression in association with clinicopathological parameters was calculated using the Chi-Square test

b Bold indicates P<0.05. AFP, α-fetoprotein; AST, aspartate aminotransferase; ALT, alanine transaminase.

TMEM165 depletion decreases invasion of HCC cells without affecting clonal survival

In view of the clinical association of TMEM165 overexpression with macroscopic vascular and serosal invasion, we further investigated the possibility of regulatory effects on the invasion of HCC cells. Prior to evaluation, we determined the expression patterns of TMEM165 in normal and cancer cell lines to select suitable cells. For this experiment, we employed the established HCC cell lines Hep3B, Huh7, SNU354, SNU398, SNU449, SNU475 and SNU739 and hepatoblastoma cell line HepG2. Consistent with data from HCC specimens, most cancer cell lines exhibited greater expression than normal human fibroblast (BJ, Wi38, IMR90) and epithelial cells, including those of the Small Airway and Prostate (Fig. 3A).

Next, we examined the effect of TMEM165 depletion on invasion of Huh7 cells that express high levels of TMEM165. Notably, TMEM165 depletion achieved via transfection of siRNA induced a significant decrease in the invasive activity of Huh7 cells (Fig. 3B). Two siRNAs (#1 and #2) recognizing different regions on the coding sequence induced a noticeable level of suppression of invasive activity (51.3 and 72.1%, respectively, compared to the control siRNA). Significantly reduced invasion was additionally observed in the SNU475 cell line (Fig. 3C). The rates of inhibition of invasion by TMEM165 siRNA #1 and #2 were 88.2 and 95.7%, respectively, clearly indicating that knockdown of TMEM165 in HCC suppresses invasive activity to a significant extent. To further confirm this finding, we assessed the effects of TMEM165 depletion on clonal growth that can detect cell proliferation and death. Notably, clonal growth was hardly inhibited in both cell lines after transfection of TMEM165 siRNAs (P>0.1, Fig. 4A and B). Thus, the TMEM165 depletion-mediated suppression of HCC cell invasion activity was not attributable to decreased clonal survival.

TMEM165 affects matrix metalloproteinase-2 (MMP-2) expression

To further determine the mechanisms underlying TMEM165-mediated regulation of invasion, we examined the potential involvement of MMPs, well-known invasion activators. Specifically, we assessed the expression levels of MMP-1, −2 and −9 in HCC cells depleted of TMEM165. As expected from the cell invasion data, MMP-2 levels were severely decreased under conditions of TMEM165 depletion in both Huh7 and SNU475 cell lines (Fig. 5A and B). In contrast, MMP-1 and −9 levels were not affected. To determine whether TMEM165 controls cancer cell invasion in a similar manner to Golgi phosphoprotein 2, we examined the expression of MMP-13 that are regulated by Golgi phosphoprotein 2 (21). Notably, TMEM165 depletion had no effect on the expression patterns of this molecule, indicating that the mechanism by which TMEM165 promotes the invasive activity of cancer cells is distinct from that of Golgi phosphoprotein 2.

Discussion

The TMEM165 protein, mainly characterized in association with congenital disorders of glycosylation type 2 disease, has recently attracted significant research attention (13,14,22,23). Based on cohort studies, 3 different mutations in TMEM165 have been reported to date, specifically, homozygous point mutation in the deep intronic splice region, homozygous missense mutation and heterozygous missense mutation, all of which culminate in loss or decrease in function of the protein (11,14). Recent in vitro studies have disclosed that TMEM165 is associated with Golgi homeostasis sensitive to manganese concentrations (18), indicating that mutation and loss of function of the protein can trigger defects in manganese-sensitive Golgi homeostasis. The importance of Golgi proteins in promoting cancer progression has been highlighted in previous studies. Golgi phosphoprotein 2 (GP73) is aberrantly expressed in HCC and knockdown of this gene inhibits cancer cell invasiveness via altering expression of E-cadherin, N-cadherin and MMP-13 (24,25). Moreover, Golgi phosphoprotein 3 (GOLPH3) is overexpressed in various cancer types, in turn, increasing invasion and migration of cancer cells (2628). GOLPH3 interacts with phosphatidylinositol-4-phosphate and myosin18A to facilitate Golgi to plasma membrane trafficking and regulates the directional migration of cells (29). Despite the recent focus on Golgi proteins, the specific function of TMEM165 in cancer remains to be determined.

We previously established an integrative analysis tool to determine the gene expression patterns between non-tumor and tumor regions in correlation with clinical outcomes (6). Based on these experiments, we demonstrated for the first time that the TMEM165 protein is significantly upregulated in tumor compared to non-tumor tissues of HCC patients. Notably, overexpression of TMEM165 (using the 2-fold cut-off criterion) was positively correlated with aggressiveness of HCC characterized by high AFP levels as well as presence of macroscopic vascular and microscopic serosal invasion. In keeping with this finding, depletion of TMEM165 significantly reduced invasiveness of human HCC cell lines in vitro, accompanied by a decrease in expression of MMP-2, an endopeptidase that degrades the extracellular matrix to stimulate cancer invasion and metastasis. However, other MMPs (MMP-1, −9, and −13) were not affected by depletion of TMEM165. These results support a role of TMEM165 as a key molecule that directs cancer cell invasion, consequently promoting aggressiveness. Clonal survival analysis can be effectively used to detect several phenotypic changes, including proliferation, death and senescence. TMEM165 depletion decreased cancer cell invasion without affecting clonal survival, indicative of a negligible effect on these phenotypes. Inhibition of cellular proliferation, death or senescence is reported to trigger a decrease in the invasion rate of cancer cells (30). Therefore, TMEM165 regulation of cancer cell invasion is credible, since the possibility that phenotypic changes, such as cellular proliferation, death, and senescence, can affect cancer cell invasion was eliminated.

Despite the significant correlation between TMEM165 overexpression and aggressiveness of the tumor phenotype, TMEM165 was not associated with patient prognosis in the current study (data not shown). This may be attributable to the small number of samples used for our analysis. Studies on larger cohorts are therefore required to validate the expression patterns of TMEM165 in relation to patient prognosis. Furthermore, while decreased invasiveness of cancer cells upon TMEM165 knockdown was evident, the precise mechanisms require elucidation in future studies. Considering the relationship between TMEM165 mutation and type II congenital disorder of glycosylation, it is plausible that its overexpression in HCC is associated with abnormalities in galactosylation and sialylation of N-glycoproteins observed in the disease. These protein modifications influence the invasive activity of cancer cells (31). Several invasion-related proteins (CD44, integrin and E-cadherin) have been revealed to be markedly affected by glycosylation (3234). In summary, TMEM165 is both transcriptionally and translationally overexpressed in HCC and associated with invasive activity.

Acknowledgements

Not applicable.

Funding

The present study was supported by grants from the National Research Foundation of Korea (nos. 2012M3A9B6055346, 2017M3A9A8033561 and 2017R1A2B4008805) and the Korea Institute of Radiological and Medical Sciences (nos. 50531–2018 and 50542–2018) funded by the Ministry of Science, ICT and Future Planning, Republic of Korea.

Availability of data and materials

The datasets used during the present study are available from the corresponding author upon reasonable request.

Authors' contributions

KHL, SBK and JH conceived and supervised the study. KHL, JSL and MYK wrote the manuscript. SBK and ERP reviewed and edited manuscript. MYK, JSL, ERP, YNS and JYJ prepared material and/or performed experiment. JSL and MYK performed statistical analysis. EHC, SHP, CJH, DWC, JJJ, KSS and SBK generated clinical data. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Ethics approval and consent to participate

The present study was approved by the Institutional Review Boards of Korea Cancer Center Hospital and Seoul National University Hospital. Written informed consent was either waived by the Institutional Review Board of Korea Cancer Center Hospital or received from patients at Seoul National University Hospital, respectively.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

Abbreviations:

HCC

human hepatocellular carcinoma

AFP

α-fetoprotein

AST

aspartate aminotransferase

ALT

alanine transaminase

MMP

matrix metalloproteinase

References

1 

Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J and Jemal A: Global cancer statistics, 2012. CA Cancer J Clin. 65:87–108. 2015. View Article : Google Scholar : PubMed/NCBI

2 

El-Serag HB: Hepatocellular carcinoma. N Engl J Med. 365:1118–1127. 2011. View Article : Google Scholar : PubMed/NCBI

3 

Bruix J and Sherman M: American Association for the Study of Liver Diseases: Management of hepatocellular carcinoma: An update. Hepatology. 53:1020–1022. 2011. View Article : Google Scholar : PubMed/NCBI

4 

Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, de Oliveira AC, Santoro A, Raoul JL, Forner A, et al: Sorafenib in advanced hepatocellular carcinoma. N Engl J Med. 359:378–390. 2008. View Article : Google Scholar : PubMed/NCBI

5 

Cheng AL, Kang YK, Chen Z, Tsao CJ, Qin S, Kim JS, Luo R, Feng J, Ye S, Yang TS, et al: Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: A phase III randomised, double-blind, placebo-controlled trial. Lancet Oncol. 10:25–34. 2009. View Article : Google Scholar : PubMed/NCBI

6 

Kim BY, Suh KS, Lee JG, Woo SR, Park IC, Park SH, Han CJ, Kim SB, Jeong SH, Yeom YI, et al: Integrated analysis of prognostic gene expression profiles from hepatitis B virus-positive hepatocellular carcinoma and adjacent liver tissue. Ann Surg Oncol. 19 Suppl 3:S328–S338. 2012. View Article : Google Scholar : PubMed/NCBI

7 

Kim BY, Choi DW, Woo SR, Park ER, Lee JG, Kim SH, Koo I, Park SH, Han CJ, Kim SB, et al: Recurrence-associated pathways in hepatitis B virus-positive hepatocellular carcinoma. BMC Genomics. 16:2792015. View Article : Google Scholar : PubMed/NCBI

8 

Park ER, Kim SB, Lee JS, Kim YH, Lee DH, Cho EH, Park SH, Han CJ, Kim BY, Choi DW, et al: The mitochondrial hinge protein, UQCRH, is a novel prognostic factor for hepatocellular carcinoma. Cancer Med. 6:749–760. 2017. View Article : Google Scholar : PubMed/NCBI

9 

Potelle S, Morelle W, Dulary E, Duvet S, Vicogne D, Spriet C, Krzewinski-Recchi MA, Morsomme P, Jaeken J, Matthijs G, et al: Glycosylation abnormalities in Gdt1p/TMEM165 deficient cells result from a defect in Golgi manganese homeostasis. Hum Mol Genet. 25:1489–1500. 2016. View Article : Google Scholar : PubMed/NCBI

10 

Rosnoblet C, Legrand D, Demaegd D, Hacine-Gherbi H, de Bettignies G, Bammens R, Borrego C, Duvet S, Morsomme P, Matthijs G, et al: Impact of disease-causing mutations on TMEM165 subcellular localization, a recently identified protein involved in CDG-II. Hum Mol Genet. 22:2914–2928. 2013. View Article : Google Scholar : PubMed/NCBI

11 

Foulquier F, Amyere M, Jaeken J, Zeevaert R, Schollen E, Race V, Bammens R, Morelle W, Rosnoblet C, Legrand D, et al: TMEM165 deficiency causes a congenital disorder of glycosylation. Am J Hum Genet. 91:15–26. 2012. View Article : Google Scholar : PubMed/NCBI

12 

Bammens R, Mehta N, Race V, Foulquier F, Jaeken J, Tiemeyer M, Steet R, Matthijs G and Flanagan-Steet H: Abnormal cartilage development and altered N-glycosylation in Tmem165-deficient zebrafish mirrors the phenotypes associated with TMEM165-CDG. Glycobiology. 25:669–682. 2015. View Article : Google Scholar : PubMed/NCBI

13 

Schulte Althoff S, Grüneberg M, Reunert J, Park JH, Rust S, Mühlhausen C, Wada Y, Santer R and Marquardt T: TMEM165 deficiency: Postnatal changes in Glycosylation. JIMD Rep. 26:21–29. 2016. View Article : Google Scholar : PubMed/NCBI

14 

Dulary E, Potelle S, Legrand D and Foulquier F: TMEM165 deficiencies in Congenital Disorders of Glycosylation type II (CDG-II): Clues and evidences for roles of the protein in Golgi functions and ion homeostasis. Tissue Cell. 49:150–156. 2017. View Article : Google Scholar : PubMed/NCBI

15 

Jaeken J, Schachter H, Carchon H, De Cock P, Coddeville B and Spik G: Carbohydrate deficient glycoprotein syndrome type II: A deficiency in Golgi localised N-acetyl-glucosaminyltransferase II. Arch Dis Child. 71:123–127. 1994. View Article : Google Scholar : PubMed/NCBI

16 

Peters V, Penzien JM, Reiter G, Körner C, Hackler R, Assmann B, Fang J, Schaefer JR, Hoffmann GF and Heidemann PH: Congenital disorder of glycosylation IId (CDG-IId) - A new entity: Clinical presentation with Dandy-Walker malformation and myopathy. Neuropediatrics. 33:27–32. 2002. View Article : Google Scholar : PubMed/NCBI

17 

Martinez-Duncker I, Dupré T, Piller V, Piller F, Candelier JJ, Trichet C, Tchernia G, Oriol R and Mollicone R: Genetic complementation reveals a novel human congenital disorder of glycosylation of type II, due to inactivation of the Golgi CMP-sialic acid transporter. Blood. 105:2671–2676. 2005. View Article : Google Scholar : PubMed/NCBI

18 

Potelle S, Dulary E, Climer L, Duvet S, Morelle W, Vicogne D, Lebredonchel E, Houdou M, Spriet C, Krzewinski-Recchi MA, et al: Manganese-induced turnover of TMEM165. Biochem J. 474:1481–1493. 2017. View Article : Google Scholar : PubMed/NCBI

19 

Marrero JA, Romano PR, Nikolaeva O, Steel L, Mehta A, Fimmel CJ, Comunale MA, D'Amelio A, Lok AS and Block TM: GP73, a resident Golgi glycoprotein, is a novel serum marker for hepatocellular carcinoma. J Hepatol. 43:1007–1012. 2005. View Article : Google Scholar : PubMed/NCBI

20 

Wei H, Li B, Zhang R, Hao X, Huang Y, Qiao Y, Hou J and Li X and Li X: Serum GP73, a marker for evaluating progression in patients with chronic HBV infections. PLoS One. 8:e538622013. View Article : Google Scholar : PubMed/NCBI

21 

Jin D, Tao J, Li D, Wang Y, Li L, Hu Z, Zhou Z, Chang X, Qu C and Zhang H: Golgi protein 73 activation of MMP-13 promotes hepatocellular carcinoma cell invasion. Oncotarget. 6:33523–33533. 2015. View Article : Google Scholar : PubMed/NCBI

22 

Krzewinski-Recchi MA, Potelle S, Mir AM, Vicogne D, Dulary E, Duvet S, Morelle W, de Bettignies G and Foulquier F: Evidence for splice transcript variants of TMEM165, a gene involved in CDG. Biochim Biophys Acta. 1861:737–748. 2017. View Article : Google Scholar : PubMed/NCBI

23 

Morelle W, Potelle S, Witters P, Wong S, Climer L, Lupashin V, Matthijs G, Gadomski T, Jaeken J, Cassiman D, et al: Galactose supplementation in patients with TMEM165-CDG rescues the glycosylation defects. J Clin Endocrinol Metab. 102:1375–1386. 2017. View Article : Google Scholar : PubMed/NCBI

24 

Chen X, Wang Y, Tao J, Shi Y, Gai X, Huang F, Ma Q, Zhou Z, Chen H, Zhang H, et al: mTORC1 up-regulates GP73 to promote proliferation and migration of hepatocellular carcinoma cells and growth of xenograft tumors in mice. Gastroenterology. 149:741–752. 2015. View Article : Google Scholar : PubMed/NCBI

25 

Yang Y, Liu Q, Zhang H, Zhao H, Mao R, Li Z, Ya S, Jia C and Bao Y: Silencing of GP73 inhibits invasion and metastasis via suppression of epithelial-mesenchymal transition in hepatocellular carcinoma. Oncol Rep. 37:1182–1188. 2017. View Article : Google Scholar : PubMed/NCBI

26 

Ma Y, Wang X, Wu Y, Sun B, Lv H, Rong F and Zheng X: Overexpression of GOLPH3 protein is associated with worse prognosis in patients with epithelial ovarian cancer. Tumour Biol. 35:11845–11849. 2014. View Article : Google Scholar : PubMed/NCBI

27 

Li Q, Ma Y and Xu W: High GOLPH3 expression is associated with poor prognosis and invasion of hepatocellular carcinoma. Mol Med Rep. 11:4315–4320. 2015. View Article : Google Scholar : PubMed/NCBI

28 

Li W, Guo F, Gu M, Wang G, He X, Zhou J, Peng Y, Wang Z and Wang X: Increased expression of GOLPH3 is associated with the proliferation of prostate cancer. J Cancer. 6:420–429. 2015. View Article : Google Scholar : PubMed/NCBI

29 

Taft MH, Behrmann E, Munske-Weidemann LC, Thiel C, Raunser S and Manstein DJ: Functional characterization of human myosin-18A and its interaction with F-actin and GOLPH3. J Biol Chem. 288:30029–30041. 2013. View Article : Google Scholar : PubMed/NCBI

30 

Schaeffer D, Somarelli JA, Hanna G, Palmer GM and Garcia-Blanco MA: Cellular migration and invasion uncoupled: Increased migration is not an inexorable consequence of epithelial-to-mesenchymal transition. Mol Cell Biol. 34:3486–3499. 2014. View Article : Google Scholar : PubMed/NCBI

31 

Pinho SS and Reis CA: Glycosylation in cancer: Mechanisms and clinical implications. Nat Rev Cancer. 15:540–555. 2015. View Article : Google Scholar : PubMed/NCBI

32 

English NM, Lesley JF and Hyman R: Site-specific de-N-glycosylation of CD44 can activate hyaluronan binding, and CD44 activation states show distinct threshold densities for hyaluronan binding. Cancer Res. 58:3736–3742. 1998.PubMed/NCBI

33 

Guo HB, Lee I, Kamar M, Akiyama SK and Pierce M: Aberrant N-glycosylation of beta1 integrin causes reduced alpha5beta1 integrin clustering and stimulates cell migration. Cancer Res. 62:6837–6845. 2002.PubMed/NCBI

34 

Pinho SS, Seruca R, Gärtner F, Yamaguchi Y, Gu J, Taniguchi N and Reis CA: Modulation of E-cadherin function and dysfunction by N-glycosylation. Cell Mol Life Sci. 68:1011–1020. 2011. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

September 2018
Volume 40 Issue 3

Print ISSN: 1021-335X
Online ISSN:1791-2431

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
APA
Lee, J., Kim, M., Park, E., Shen, Y.N., Jeon, J., Cho, E. ... Lee, K. (2018). TMEM165, a Golgi transmembrane protein, is a novel marker for hepatocellular carcinoma and its depletion impairs invasion activity. Oncology Reports, 40, 1297-1306. https://doi.org/10.3892/or.2018.6565
MLA
Lee, J., Kim, M., Park, E., Shen, Y. N., Jeon, J., Cho, E., Park, S., Han, C. J., Choi, D. W., Jang, J. J., Suh, K., Hong, J., Kim, S. B., Lee, K."TMEM165, a Golgi transmembrane protein, is a novel marker for hepatocellular carcinoma and its depletion impairs invasion activity". Oncology Reports 40.3 (2018): 1297-1306.
Chicago
Lee, J., Kim, M., Park, E., Shen, Y. N., Jeon, J., Cho, E., Park, S., Han, C. J., Choi, D. W., Jang, J. J., Suh, K., Hong, J., Kim, S. B., Lee, K."TMEM165, a Golgi transmembrane protein, is a novel marker for hepatocellular carcinoma and its depletion impairs invasion activity". Oncology Reports 40, no. 3 (2018): 1297-1306. https://doi.org/10.3892/or.2018.6565