Association of MARCH7 with tumor progression and T-cell infiltration in esophageal cancer

MARCH7 is an E3 ubiquitin ligase known to regulate neuronal development,T-cell proliferation, and cell and tissue differentiation. But, the altered expression of MARCH7 has been observed in various malignancies. Herein, the cellular localization and role of MARCH7 have been elucidated in esophageal squamous cell carcinoma (ESCC), the information regarding which is currently limited. To check the expression of MARCH7 and its correlation with immune cells infiltration in ESCC, immunohistochemical analysis was performed. RNAi approach was used to investigate the role of MARCH7 in esophageal cancer cells. Interestingly, we found a significantly higher expression of MARCH7 protein in 84% of ESCC tissues than in distant matched non-malignant tissues (p ≤ 0.001). In addition to this, immunohistochemistry results have shown a negative correlation between MARCH7 protein expression and tumor-infiltrating immune cells such as CD8 + T cells (r = − 0.633, p = 0.001) and PD1 + T cells (r = − 0.560, p = 0.005). Furthermore, MARCH7 silencing inhibited the ESCC cell growth and reduced the clonogenic and invasion/migration potential of ESCC cells. MARCH7 silencing also significantly increased E-cadherin protein levels in ESCC cells relative to those in negative control cells (p < 0.05). Thus, MARCH7 is oncogenic and might have a possible role in esophageal carcinogenesis. Moreover, E-cadherin may be a downstream target of MARCH7 in ESCC.


Introduction
MARCH7 (Membrane Associated RING-CH 7), also known as axotrophin, belongs to the MARCH family of E3 ubiquitin ligases which add ubiquitin on target proteins, to signal their vesicular transfer amongst membrane compartments [1,2]. A report indicated that deficiency of MARCH7 potentiates proliferation and T cell-derived Leukemia inhibitory factor (LIH) secretion in T-cells upon mitogen stimulation [3], signifying that MARCH7 plays important roles in immune tolerance. MARCH7 was also reported to be involved in regulating systemic inflammation by ubiquitinating and degrading the NLRP3 (NLR family pyrin domain containing 3) inflammasome via cyclic adenosine monophosphate (cAMP) and dopamine D1 receptor (DRD1) signaling [4]. Several reports have also indicated that MARCH7 plays important roles in the cell proliferation, actin cytoskeleton remodelling, migration / invasion, neuronal development, and autophagy [5][6][7][8][9][10]. Recently, the role of MARCH7 role has been studied in cancer. Silencing of MARCH7 was found to decrease ovarian cancer cell proliferation, migration, and invasion and prevent ovarian cancer growth in mice [11].  observed the involvement of MARCH7 in VAV2-RAC1-CDC42 pathway which promoted the proliferation and invasion of cervical cancer cells [12]. MARCH7 was also found to promote the invasion and metastasis of endometrial cancer via the Snail-mediated pathway [13]. However, the role of MARCH7 in esophageal squamous cell carcinoma (ESCC) has not been studied yet.
Esophageal cancer has the seventh highest incidence and sixth highest mortality among all the cancers, worldwide Shivam Singh and Arjumand Bano shares equally as first authors. [14]. It is the seventh most common cancer among males and thirteenth most common cancer among females [14]. The molecular mechanisms underlying tumor formation and progression are not well understood. As MARCH7 has been shown to play roles in other cancers, we aimed here, to determine whether it is involved in the development and progression of ESCC. To this end, we evaluated the differential expression of MARCH7 gene in ESCC and distant matched non-malignant tissues by immunohistochemical analysis. We also assessed the correlation of MARCH7 expression with immune cell infiltration in ESCC. Moreover, RNA interference was used to further investigate the role of MARCH7 in esophageal cancer cells.

Sample collection
ESCC and distant matched non-malignant tissues were collected from patients undergoing endoscopy at the Department of Gastroenterology, All India Institute of Medical Sciences (AIIMS). The study has received the ethical approval from the Guru Gobind Singh Indraprastha University (GGSIPU) and AIIMS research committee prior to its instigation and all the procedures involving human participants were performed in accordance with the ethical standards of both of the institutes. The whole research was carried out in accordance with "The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans". All the individual participants included in the research plan were duly informed and their written informed consent was also obtained. For each patient, one pair of tissue samples was stored in 10% formalin vials to be embedded in paraffin and sectioned for immunohistochemical analysis. Information regarding various clinicopathological parameters was also obtained from each patient.

Transfections of cultured cells
Esophageal carcinoma cell line of human origin, KYSE-410 (ECACC 94,072,023), was attained from Sigma-Aldrich and was also checked for mycoplasma contamination (Bangalore, India). The cells were transfected with 50 nmol/l MARCH7 siRNA (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)
RNA was isolated from KYSE-410 cells using the Qiagen miRNeasy Mini Kit as per the manufacturer's protocol. The primers sequence details are given in Table 1. To analyse the MARCH7 mRNA expression, real time PCR was carried out as described before [16].

Colony formation assay
Thousand transfected and untransfectedKYSE-410 cells were cultured in 6-well plates for a week and the number of colonies formed was counted using 0.5% (w/v) crystal violet in distilled water.

Transwell assay
Transwell assay was also performed to check the effects of MARCH7 knockdown on KYSE-410 cell migration and invasion. The mean number of cells migrated/invaded was calculated as described before [15].

Statistical analysis
The statistical analysis was carried out using the Statistical Program for Social Sciences (SPSS) software, version 17.0 (SPSS Inc., Chicago, IL, USA). The correlation between clinic-pathological parameters of ESCC patients, such as age, gender, and stage and MARCH7/CD8/PD-1 protein expression were studied using the χ 2 test. A p-value ≤ 0.05 was considered as indicative of statistical significance. The reported p-values are 2 tailed. To describe the discrimination between distant matched non-malignant and cancerous conditions based on MARCH7 expression, Receiver Operating Characteristic (ROC) curves were constructed and area under curve (AUC), sensitivity and specificity were calculated.The correlation between the expression ofMARCH7 and CD8/PD-1 in tumor and distant matched non-malignant tissues was studied using Pearson correlation. Each experiment was performed in triplicate and the results obtained using either Student's t-test or ANOVA, are presented as mean ± SD (standard deviation).

Increased expression of MARCH7 protein in ESCC tissues
As MARCH7 mRNA expression was predicted to be differentially expressed in esophageal cancer tissues using GEPIA analysis tool (Please refer supplementary data; Fig. 1A and B), we next evaluated the protein expression profile of MARCH7 in endoscopic biopsy specimens collected from ESCC patients. Immunohistochemical analysis was carried out in ESCC (n = 25) and distant matched non-malignant tissues (n = 24).The results showed higher percentage positivity and amplified intensity of MARCH7 staining in 21/25 (84%) ESCC tissues than distant matched non-malignant tissues (Fig. 1C-G). Nuclear and cytoplasmic expression of MARCH7 was observed in ESCC tissues. Remarkably, MARCH7 overexpression was displayed by most preneoplastic tissues which is suggestive of its probable role in early stages of esophageal tumorigenesis (Fig. 1D).Chi square analysis showed a significant increase in MARCH7 expression in esophageal cancer (preneoplastic and neoplastic) tissues than distant matched non-malignant epithelia (p ≤ 0.001, Table 2, Fig. 1I). No staining was detected in the negative control samples, where primary antibody treatment was not given (Fig. 1H).
ROC curve analysis displayed an AUC of 0.983 (Fig. 1J).No correlation was observed between MARCH7 expression and clinic-pathological variables ( Table 2).

Inverse correlation of T-cell infiltration with MARCH7 protein expression in ESCC tissues
To confirm our in-silico results (supplementary data), indicating that MARCH7 expression affects tumor infiltration with t-lymphocytes in ESCC tissues, we analysed the expression of CD8 and PD-1 in 23 ESCC tissues ( Fig. 2B-E). Amongst these 23 esophageal tissues, 13 were moderately differentiated squamous cell carcinoma(MDSCC); 2 were poorly-differentiated squamous cell carcinoma (PDSCC), one was well-differentiated squamous cell carcinoma (WDSCC) and 7 were preneoplastic esophageal tissues. An inverse correlation between MARCH7 and PD-1 expression was observed in 18 out of 23 esophageal cancer tissues (r = − 0.560, p = 0.005, Fig. 2F). Similarly, an inverse correlation was observed between MARCH7 and CD8 expression in 18 out of 23 esophageal cancer tissues (r = − 0.633, p = 0.001, Fig. 2G). This suggests that there is less T-cell infiltration in MARCH7 positive tissues which might help this cancer in evading immune response. However, these results need to be further validated in a larger cohort.

Effect of MARCH7 silencing on mRNA and protein expression levels
Evaluation of MARCH7 mRNA levels at 24 and 48 h posttransfection showed a reduction of 62% (p < 0.05) and 65% (p > 0.05), respectively, in MARCH7 siRNA-treated group as compared to the scrambled sequence siRNA-treated group (Fig. 3A). Moreover, 80% and 66% decline was observed in the MARCH7 protein level at 24 and 48 h post-transfection in MARCH7 siRNA-treated group as compared to scrambled sequence siRNA-treated group as shown in Fig. 3B and C (p < 0.05).

Increase in cell growth inhibition upon silencing of MARCH7
To evaluate the effect of MARCH7 silencing on cell growth inhibition, MTT assay was performed using cells in the MARCH7 silencing group, negative control group, transfection reagent only control group and untransfected group. Data were obtained from day 1 to day 2 post-transfection. The inhibition in KYSE-410 cell growth was found to be highest in MARCH7 silencing group (27%) at 24 h posttransfection as compared to that in the negative control group (Fig. 3D, p < 0.05).

Reduction of clonogenic potential of KYSE-410 cells uponsilencing of MARCH7
The total number of colonies formed by MARCH7 siRNAtreated cells were 72 ± 11 as compared to 210 ± 21 in scrambled siRNA-treated or 243 ± 18 in untreated cells ( Fig. 3E and F, p < 0.05).

Silencing of MARCH7 suppresses invasion/migration of KYSE-410 cells
The influence of MARCH7 silencing upon migration and invasion of esophageal cancer cells was evaluated using the transwell assay. MARCH7 silencing caused decrease in migration and invasion of KYSE-410 cells at 24 h (p < 0.05). The number of KYSE-410 cells that had migrated through the Boyden chamber were 88 ± 24 and 631 ± 56 (24 h posttransfection) in the MARCH7 silencing and negative control groups, respectively ( Fig. 3G and H). Similarly, the numbers of KYSE-410 cells that invaded through the transwell insert were 29 ± 10 and 297 ± 16 (24 h post-transfection) in the MARCH7 silencing and negative control groups, respectively ( Fig. 3I and J).

Regulation of the E-cadherin protein levels by MARCH7 in KYSE-410 cells
E-cadherin and β-catenin complexes regulate the intercellular adhesion. E-cadherin negatively regulates the transport of β-catenin to the nucleus for activation and transcription of various genes [17,18]. In addition to this, in our previous study we reported the negative regulation of E-cadherin by MARCH8 which is another MARCH protein family member (15). Therefore, regulation of E-cadherin by MARCH7 was also explored in the present study. We found that the protein levels of E-cadherin were significantly increased in MARCH7 siRNA-treated groups compared to the scrambled sequence siRNA-treated group at 24 h post-transfection (p < 0.05, Fig. 4).

Discussion
Though MARCH protein family have been identified as important physiological regulators, certain MARCH proteins such as MARCH7 have recently been reported to be upregulated in a variety of cancers. However, its significance in esophageal cancer has not yet been explored.
The expression of MARCH7 was shown to be higher in ovarian cancer tissues than paired normal tissues [11]. Liu et al. 2019 also showed increased expression of MARCH7 in endometrial cancer tissues than that in normal endometrium tissues [13]. Furthermore, immunohistochemical studies conducted by Zhang et al. 2016 revealed that high MARCH7 and ubiquitin-specific protease 7 (USP7) expressions was correlated with lymph node metastasis in epithelial ovarian cancer patients [19]. In the present study, the immunohistochemical analysis revealed that MARCH7 protein expression is significantly upregulated in ESCC tissues as compared to that in distant matched non-malignant tissues. Additionally, most dysplastic tissues showed increased expression of MARCH7, suggesting its possible role in esophageal cancer development.We previously reported an increased expression of MARCH8 in ESCC tissues as compared to that in distant matched non-malignant tissues, which in combination with present findings, suggests that several MARCH proteins play important roles in regulating ESCC [15]. Infiltration of tumor with T-cells is linked with better prognosis and clinical outcomes in a majority of cancers, including ESCC [20]. Thus, we analyzed the expression of CD8 and PD-1 in TILs in MARCH7 immunopositive ESCC tissues using immunohistochemical analysis. The results revealed a negative correlation between MARCH7 and CD8/PD1 expression levels. Thus, MARCH7 might be promoting an immunosuppressive environment in ESCC tumors by decreasing CD8 + T cell infiltration. The negative correlation between the expression levels of MARCH7 and PD-1, which is expressed on T cells, suggests that PD-1-based immunotherapy might not work in MARCH7 immunopositive ESCC cases. Thus, MARCH7 might be involved in the progression of esophageal cancer through the modulation of immune response. However, in-depth validation of the link between MARCH7 and TILs in larger cohorts is required. MARCH7 was recently reported to be an oncogene and a potential oncotarget for cancer therapy [6,13,19,21].  reported that the silencing and overexpression of MARCH7 affected the SKOV3 cells proliferation, invasion and migration [11]. Additionally, Hu et al., 2018 mentioned that the interaction of the long noncoding RNA MALAT1 and Autophagy-Related Protein ATG7 with MARCH7 promoted invasion and autophagy in ovarian carcinoma [6]. The silencing of MARCH7 was also shown to repress the invasion and metastasis of endometrial cancer cells in vitro and in vivo [13]. Herein, we evaluated the possible involvement of MARCH7 in the proliferation and migration of esophageal cancer cells using the RNAi-mediated knockdown of MARCH7 followed by various cell assays,viz. MTT assay, colony formation assay and transwell assay. A decreased expression of MARCH7 mRNA in KYSE-410 cells treated with MARCH7 siRNA was observed. Maximum inhibition of cell viability was observed at 24 h post-transfection with 50 nM siRNA. MARCH7 gene knockdown led to a decrease in the proliferation, invasion/migration, and clonogenic potentials of ESCC cells. The E-cadherin gene is a tumor-suppressor gene that inhibits the metastasis of epithelial tumor cells [22]. Liu et al. 2019 revealed a positive Fig. 2 Correlation between MARCH7 expression and CD8 + and PD-1 + TILs in ESCC tissues. A TIMER generated image depicting correlation between MARCH7 expression and immune infiltration level in esophageal carcinoma. B-E PD-1 and CD8 expression in ESCC tissues wherein B and D represent no detectable staining of PD-1 and CD8, respectively and C and E represent positive staining (arrow) of PD-1 and CD8, respectively, in the stroma of ESCC tissues. F and G Correlations of IHC intensity scores of MARCH7 and PD-1/CD8. The correlation was calculated using Pearson correlation. Scale bars = 100 μm correlation of MARCH7 with Snail and Vimentin expression and a negative correlation with E-cadherin [13]. Moreover, MARCH7 was also found to negatively regulate E-cadherin protein levels in ovarian tumor cells [11]. This proposes that the negative regulation of E-cadherin by MARCH7 may add to cancer development, as the loss of E-cadherin-mediated intercellular adhesion system has been well recognised to be a contributing factor in the malignant transformation of cells [23]. However, this hypothesis needs to be validated through further study. Additionally, clinical and functional involvement of other MARCH proteins, viz. MARCH 1, 5 and 8 have also been described in cancers such as ovarian cancer, ESCC, gastric cancer and non-small cell lung carcinoma [15,24,25]. This, in combination with our findings, inhibition in MARCH7 siRNA-treated cells was greater than that in scrambled siRNA-treated cells at 24 h post-transfection. E and F Colony formation assay. The number of colonies formed by MARCH7 siRNA-treated cells was less than those of colonies formed by scrambled siRNA-treated or untreated cells. G-J Transwell assay. G and H The migration of MARCH7 siRNA-treated cells was lesser than that of scrambled siRNA-treated cells at 24 post-transfection (*p < 0.05). Matrigel invasion assay (I and J). Significantly lesser invasion potential was observed in MARCH7 siRNA-treated cells than in scrambled siRNA-treated cells (*p < 0.05). Scale bar = 100 µm suggests that MARCH gene silencing can inhibit the aggressive behaviour of ESCC, and MARCH family proteins may act as potential therapeutic targets in ESCC.
In conclusion, our study provides the first evidence indicating clinical and functional involvement of MARCH7 in ESCCs. Increased expression of MARCH7 in preneoplastic and neoplastic esophageal tissues and the effects of its knockdown on cancer cell properties revealed herein point towards the possible role of MARCH7 in esophageal tumorigenesis and its potential as a therapeutic target in ESCC.
Author Contribution SS: has contributed to data acquisition of MTT, colony formation, invasion/migration, western blotting; data analysis of all the experiments; data interpretation, in-silico analysis, statistical analysis and drafting and editing of manuscript. AB: has contributed in data acquisition of PD-1 and CD8 immunohistochemistry, carried out western blotting of MARCH7 and targets and performed in-silico data analysis. AS: is the clinician who provided the tissue samples and carried out endoscopic procedures for the same. PD: has contributed to histopathological analysis and grading of immuhistochemical slides. RS: has contributed to acquisition of funding, conception and design, data interpretation, revising manuscript critically for intellectual content and has given full approval of version to be published. All authors reviewed the manuscript.

Data availability
The PCR, western blotting, immunohistochemical staining and their supporting clinical data used to support the findings of this stud are available from the corresponding author upon request because the data also forms part of an on-going study. The bioinformatics data supporting this study are from previously reported studies and datasets, which have been cited. The processed data are available at Gene Expression Profiling Interactive Analysis (GEPIA) (http:// gepi.a. cancer-pku. cn/) and Tumor Immune Estimation Resource (TIMER) (http:// cistr ome. shiny apps. io/ timer/). Consent to participate A written informed consent was taken from the patients prior to sample collection.