Background
Esophageal cancer is the eighth most common cancer worldwide and the sixth-leading cause of cancer-related deaths [
1]. The molecular mechanism underlying tumor formation and progression is still not fully illuminated. Over the last decade, E3 ubiquitin ligases, enzymes that confer specificity to the ubiquitination process, have been shown to play a significant role in carcinogenesis [
2‐
4].
Membrane-associated RING-CH (MARCH) protein family is comprised of 11 gene members among which MARCH8 was the first mammalian MARCH protein to be discovered. Overexpression of MARCH8 has been reported to downregulate immunomodulatory proteins viz. MHC II, B7-2, TfR (transferrin receptor), CD166, CD44, CD88, CD98, IL1RAP and syntaxin 4 [
5‐
11]. MARCH8 mediated downregulation of TNF-related apoptosis inducing ligand receptor 1 (TRAIL-R1) has been shown to prevent breast cancer cells from undergoing apoptosis suggesting it to be a potential target for knockdown studies which may provide therapeutic benefit to patients suffering from cancer [
12]. A recent study revealed the role of MARCH8 in embryogenesis wherein, overexpression of MARCH8 led to decreased surface expression of E-cadherin in zebrafish and
Xenopus leading to loss of cell adhesion and abnormal cell migration [
13,
14]. In addition to these reports, Kumar et al. identified MARCH8 as one of the differentially expressed gene in esophageal squamous cell carcinoma (ESCC) using 19.1K cDNA microarrays [
15]. However, its expression and clinical relevance in ESCCs has not yet been analysed.
In the present study, we have reported aberrant expression of MARCH8 gene in esophageal squamous cell carcinoma (ESCC). Moreover, we have analysed the role of MARCH8 gene in ESCC. We observed that silencing of MARCH8 affects proliferation, migration/invasion, colony formation potential and apoptosis of ESCC cells.
Methods
Study subjects
Thirty-five cancerous and distant matched non-malignant tissue (5 cm apart from tumor) biopsies were collected from patients with ESCC who underwent endoscopy at Department of Gastroenterology, AIIMS. One part of the tissue taken in 10% formalin and embedded in paraffin was used for hematoxylin/eosin staining and immunohistochemical analysis. The clinicopathological data were recorded in a predesigned performa that included site of lesion, histopathological differentiation, age, gender, nature of diet, tea, alcohol and tobacco consumption, and family history. The sites of esophageal squamous cell tumors included upper, mid and lower esophagus.
Cell culture and transfections
Human esophageal carcinoma cell line, KYSE-410 (ECACC 94072023), was obtained from Sigma-Aldrich (Bangalore, India). The cells were grown in RPMI-1640 media supplemented with 10% heat inactivated fetal bovine serum (FBS) and 1% antibiotics in a 5% carbon dioxide and 37 °C atmosphere. KYSE-410 cells were transfected with 50 nmol/l MARCH8 siRNA (5′-AAUGACUCAUGAAAUGUCC-3′, Ambion, CA, USA) or scrambled sequence siRNA (Ambion) using Lipofectamine 3000 (Invitrogen, CA, USA) as transfecting agent in a serum- and antibiotics-free medium.
Quantitative real time PCR (qRT-PCR)
Total RNA was extracted from cell line, ESCC and distant matched non-malignant tissues using RNAeasy mini kit (Qiagen, Copenhagen, Denmark) as per the manufacturer’s protocol. cDNA was synthesized from 1 µg of total RNA by reverse transcription PCR. To prevent genomic DNA amplification, primers were designed from exon–exon junction. The details of primer sequences are given in Table
1. A two-step real time PCR, for analysing the expression of MARCH8 mRNA, was performed as described before [
16].
Table 1
Primer sequences for qRT-PCR
1. | MARCH8 | TGCATCAGATCTCTGCCATT TGGACGTCATCTGCAACTTC | 56 | 435 |
2. | 5s rRNA | GTCTACGGCCATACCACCCTG AAAGCCTACAGCACCCGGTAT | 60 | 121 |
Immunohistochemistry
Paraffin-embedded sections (5 µm) of histologically confirmed human esophageal normal (n = 25) and ESCC (n = 35) tissues were obtained on poly-l-lysine coated slides. Briefly, the tissue sections were deparaffinized and rehydrated. Tris–EDTA buffer (10 mM Tris-base, 1 mM EDTA, pH 9.0) was used for carrying out antigen retrieval. To quench endogenous peroxidase activity, sections were incubated with 0.3% v/v hydrogen peroxide in methanol for 30 min, followed by blocking in 1% normal horse serum to prevent non-specific binding. After this, slides were incubated overnight with 1:50 diluted rabbit polyclonal anti-MARCH8 antibody of human origin (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4 °C. Next day, the slides were washed and coated with HRP conjugated anti-rabbit IgG [ImmPRESS anti-rabbit Ig (peroxidase) Polymer Detection kit, Vector Laboratories Inc, USA] for 30 min at RT. The colour was developed using diaminobenzidine as the chromogen. Haematoxylin was used for nuclear staining. ESCC tissue sections not treated with anti-MARCH8 antibody were used as negative controls. For MARCH8 protein expression, sections were counted as positive if epithelial cells showed immunopositivity in the nucleus/cytoplasm when observed independently by three of us (SS, RS and PD). The slides were scored based on the percentage of immunostained cells as ≤ 10% = 0; 11–20% = 1; 21–40% = 2; 41–60% = 3; 61–80% = 4 and > 81% = 5. Slides were also scored on the basis of staining intensity as faint = 1; moderate = 2 and strong = 3. Finally a total score was found by adding the scores of percentage positivity and intensity. If any disagreement in the grading between the 3 investigators occurred, those slides were reviewed by all three of us and unanimity was reached by discussion. Based on sensitivity and specificity, calculated by ROC curve analysis, a total score cut-off value of 4 was defined as MARCH8 immunopositivity.
In-silico prediction of MARCH8 protein subcellular localization
Subcellular localization of MARCH8 protein was predicted using CELLO v.2.5 database (
http://cello.life.nctu.edu.tw/) which is a multi-class SVM classification system. CELLO database uses four types of schemes: amino acid composition, dipeptide composition, partitioned amino acid composition and sequence composition based on physicochemical properties of amino acids [
17]. In addition to this, cNLS Mapper database (
http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi) was also used to predict nuclear localization signals (NLS) in MARCH8 protein sequence [
18].
Protein isolation and western blotting
For total protein isolation, the KYSE-410 cells were lysed in RIPA buffer (Sigma) and scraped off the dish. For nuclear and cytoplasmic protein extraction, cells were lysed using Cytoplasmic and Nuclear Protein Enrichment kit (Amresco, Ohio, USA) according to manufacturer’s protocol. The protein quantification was carried out by Bradford method (BioRad, USA) using BSA as a standard. Total cellular proteins as well as nuclear and cytoplasmic protein fractions (100 µg protein/lane) were resolved on 12% sodium dodecyl sulphate-poly-acrylamide gels, transferred to PVDF membranes and immunolabelled with rabbit polyclonal anti-GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase, 1:200 dilution, Santa Cruz Biotechnology), mouse monoclonal anti-proliferating cell nuclear antigen or PCNA (DAKO A/S Copenhagen, Denmark, 1:500 dilution) and anti-MARCH8 (1:100 dilution) antibodies in 1% bovine serum albumin for 1 h at room temperature. PVDF membranes were incubated with respective horseradish peroxidase (HRP)-tagged secondary antibodies. The HRP-tagged immunolabelled proteins were detected by Enhanced Chemiluminescence kit (Thermo Scientific, USA). The integrated density values (IDV) for each group were determined using Image J software (
https://imagej.nih.gov) and normalization was done by dividing the corrected IDV of MARCH8 protein in each group by corrected IDV of GAPDH protein in the corresponding group.
Confocal and immunofluorescence microscopy
To determine the localization of MARCH8 protein in cancer cells (KYSE-410), confocal and immunofluorescence microscopy were performed. Briefly, the cells were fixed in methanol at – 20 °C for 20 min and permeabilized with 0.5% Triton X-100 in 1X-PBS for 10 min. To prevent non-specific binding, the cells were blocked with 3% BSA and 0.05% Triton X-100 in PBS for 1 h. Then the cells were incubated with rabbit polyclonal anti-MARCH8 antibody of human origin (1:50 dilution) overnight at 4 °C, followed by incubation with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (Invitrogen) at room temperature for 30 min (1:1000 dilution). The cells were observed under a confocal microscope (Leica TCS SP2 AOBS), after counterstaining of the nuclei with DAPI for confocal microscopy and under inverted fluorescence microscope (Nikon), after nuclei counterstaining with propidium iodide (PI) for immunofluorescence microscopy.
MTT assay
Cell growth was analyzed by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. 1 × 104 KYSE-410 cells were seeded in a 96-well plate and transfected with siRNA after 24 h. Cell growth inhibition rate was determined at 24, 48 and 72 h. The cell growth inhibition rate was calculated with the equation: inhibition rate = (1 − ODtreatment/ODcontrol) × 100. The results were expressed as the percentage of inhibition relative to control cells.
1000 KYSE-410 cells transfected with MARCH8 siRNA/scrambled sequence siRNA and untransfected cells were cultured in 6-well plates for 1 week to check the number of colonies formed using 0.5% (w/v) crystal violet in distilled water.
Scratch assay
Scratch assay was performed to test the effect of MARCH8 silencing on cell migration. For Scratch assay, 4 × 105 cells were seeded on a 6-well plate overnight. After 24 h post-transfection, a scratch was made using a pipette tip. At 0, 24 and 48 h after scratching, cells were observed and images were taken at 4× magnification in inverted fluorescence microscope (Nikon). The migration area among different groups was measured as the average percent of wound closure as compared to that at 0 h.
Boyden chamber assay for cell migration and invasion
Boyden chamber assay was also performed to check the effect of MARCH8 knockdown on KYSE-410 cell migration and invasion. Briefly, 48 h post-transfection, 4 × 104 cells, re-suspended in 1× PBS, were seeded on the upper side of the Boyden chamber (SPL Lifesciences Co. Ltd., Korea) containing polycarbonate membrane filter (6.5 mm diameter; 8 µm pore size). Unlike migration, Matrigel basement membrane matrix (Corning, Massachusetts, USA) was used to coat the upper side of Boyden chamber to assess the effect of MARCH8 knockdown on invasive capacity of KYSE-410. Similar to migration assay, cells were added over the Matrigel on the upper side of Boyden chamber. The lower chamber was filled with a complete medium. Cells were incubated for 24 h, and those cells that did not migrate through the pores were detached using a cotton swab. Cells on the lower surface of the membrane were fixed in prechilled methanol, stained with 1 µg/µl of DAPI and counted by an inverted fluorescence microscope (magnification, 100×, Nikon). The mean number of cells migrated/invaded was calculated by dividing the average number of cells in each of the random fields within an insert by the area of the microscopic viewing field and then multiplied by the entire area of the Boyden chamber.
Cell cycle assay
1 × 105 cells, seeded in 6-well plates, were transfected with either MARCH8 siRNA or scrambled sequence siRNA. After 72 h, the cells were harvested and fixed in 70% ethanol overnight at − 20 °C. Next day, the cell pellet was resuspended in 10 µg/ml of PI and cell cycle distribution was analysed with LSR II flow cytometer (Becton–Dickinson) and the FACSDiva software-version 6.1.3 (Becton–Dickinson).
Apoptosis assay
To detect apoptosis, phycoerythrin (PE) Annexin V Apoptosis Detection kit I (BD Pharmingen, California, USA) was used. Fluorescence was detected using LSR II flow cytometer. Cells treated with 6 µg of camptothecin for 10 h and untreated cells, respectively, served as positive and negative controls for dual staining.
Statistical analysis
Data was statistically analyzed by using Statistical Program for Social Sciences (SPSS) software, version 17.0 (SPSS Inc., Chicago, IL, USA). Each experiment was performed in triplicates and the results were represented as mean ± SD using either Student’s t test or ANOVA. Mann–Whitney U test was applied to evaluate statistical differences in MARCH8 mRNA expression between ESCC and distant matched non-malignant tissue populations. The associations between clinicopathological parameters of ESCC patients and MARCH8 protein expression were examined by χ2 test. A p ≤ 0.05 was considered as a criterion for statistical significance.
Discussion
Initial studies of MARCH8 primarily focused on its immunomodulatory role. But, its relevance in cancers has not yet been fully elucidated. In this study, we showed overexpression of MARCH8 in esophageal cancer tissues and its silencing inhibited proliferation, migration, invasion and clonogenicity of EC cells. Additionally, MARCH8 silencing enhanced the apoptosis of EC cells.
Immunohistochemical analysis revealed an increased expression of MARCH8 in ESCC tissues as compared to distant matched non-malignant tissues. Moreover, all the dysplastic tissues showed increased MARCH8 protein expression suggesting its possible involvement in early stages of esophageal cancer. Interestingly, MARCH8 expression was found to be localized in nucleus of ESCC cells in addition to its cytoplasmic expression. Earlier, MARCH8 expression has been shown to be localized in early endosomes and late endosomes [
5,
8]. However, most of the MARCH8 protein subcellular localization information available till date relies on its ectopic expression in cells which may be one of the reasons that expression of MARCH8 in the nucleus was not reported earlier. In the present study, we have used antibodies to detect endogenous MARCH8 expression which offers more direct evidence of endogenous MARCH8 localization in the cell. Confocal microscopy and western blotting in ESCC cells further validated our immunohistochemical results. In addition to this, well-recognized nuclear localization signals were also found in MARCH8 protein sequence. However, further in-depth studies are necessary to examine the translocation of MARCH8 to the nucleus and its functional implication there.
Our results also demonstrated the effect of MARCH8 knockdown on cellular migration, invasion, growth inhibitory rate, and clonogenic potential of KYSE-410 cells. We found that inhibiting the MARCH8 expression resulted in higher growth inhibition of cells. To find out if this growth inhibition was related to cell cycle, DNA cell cycle analysis was carried out. It was observed that MARCH8 gene knockdown induces a significant increase in sub G0 and G2/M populations (cell death) and decreases the S-phase population suggestive of induced apoptosis due to MARCH8 silencing. To check this, apoptosis assay was performed which showed significantly more number of cells in early apoptotic phase after MARCH8 gene knockdown. This may be due to the fact that MARCH8 targets proteins that play significant role in apoptosis (Fas and TRAIL-R1) [
8,
12]. TRAIL receptor signaling has been associated with metastasis suppression [
19]. Van et al. [
12] demonstrated that a unique membrane-proximal lysine in the cytoplasmic tail of TRAIL-R1 interacts with MARCH8 which ubiquitinates TRAIL-R1 and thus diminishes its steady-state cell surface expression which suggests MARCH8 as a potential determinant for tumor cell sensitivity to TRAIL receptor-targeted therapy [
12].
Knockdown of MARCH8 was also found to suppress cancer cell properties like invasion, migration and colony formation ability of KYSE-410 cells. E-cadherin is a tumor-suppressor gene that prevents migration/invasion of the epithelial tumor cells [
20]. Moreover, change in the surface levels of E-cadherin due to E3 ubiquitin ligase activity of MARCH8 has been reported to reduce adherence of the cells in the animal cap during embryogenesis in zebrafish [
13]. In addition to this, MARCH7 which has recently been reported to be oncogenic was also found to regulate E-cadherin protein levels in ovarian cancer cells [
21]. This suggests that downregulation of E-cadherin by MARCH8 may also contribute to cancer development as loss of E-cadherin-mediated adhesion system has been well documented to be involved in malignant transformation of the cells [
22]. Moreover, most of the targets of MARCH8 have been found to be involved in immune modulation such as MHC II, B7.2, IL1RAP and TNF-α [
6,
8,
11,
23]. However, role of MARCH8 in immune surveillance is yet to be analysed. Besides this, clinical and functional implications of other MARCH proteins viz. MARCH7, 1 and 5 have also been reported in ovarian cancer [
19,
24,
25]. This suggests that MARCH gene silencing inhibits the aggressive behaviour of ESCC, indicating that this protein family may act as a potential therapeutic target for ESCC.
Authors’ contributions
SS has contributed to data acquisition, data analysis, data interpretation and in drafting of manuscript. AS is the clinician who provided the tissue samples and carried out endoscopic procedures and pathological examination of the tissues. PD is the pathologist who analysed all the tissue samples for immunohistochemical staining and scoring. RS has contributed to acquisition of funding, conception and design, data interpretation, and revised manuscript critically for intellectual content. All authors read and approved the final manuscript.