Transcription Activation of Rab8A by PEA3 Augments Progression of Esophagus Cancer by Activating the Wnt/β-Catenin Signaling Pathway

Background Rab8A has been reported as an oncogenic gene in breast and cervical cancer. However, the function and molecular mechanism of Rab8A in esophagus cancer has not been reported. Methods Rab8A expression was detected by qPCR and western blotting assays, small interference RNA (siRNA) was applied to reduce Rab8A expression, and the biological behaviors of esophagus cancer cells were estimated by cell counting kit-8, colony formation, and transwell and western blotting assays. The transcriptional factor of Rab8A was verified by dual-luciferase assay and chromatin immunoprecipitation assay. The protein expression of key genes in the Wnt/β-catenin signaling pathway was determined by western blotting assay. M435-1279 was used to suppress the Wnt/β-catenin signaling pathway. Results A significant increase of Rab8A expression has been found in esophagus cancer cells. Knockdown of Rab8A suppressed the viability, colony formation, migration, and invasion abilities of esophagus cancer cells and induced apoptosis. PEA3 transcriptionally regulated Rab8A expression and promoted the viability, colony formation, migration, and invasion abilities of esophagus cancer cells and blocked apoptosis, which were diminished by si-Rab8A transfection. Additionally, the expression levels of key genes related to the Wnt/β-catenin signaling pathway were strengthened by PEA3 overexpression, which were reduced by si-Rab8A transfection. M435-1279 treatment significantly reduced the viability and colony formation of esophagus cancer cells. Conclusions The data showed that Rab8A was transcriptionally regulated by PEA3 and promoted the malignant behaviors of esophagus cancer cells by activating the Wnt/β-catenin pathway. The above results indicated that Rab8A may be considered as a promising biomarker for diagnosis and precision treatment in esophagus cancer.


Introduction
Esophagus cancer is still an integral cause of cancer-related death and has presented a drastic increase of greater than 6-fold in incidence rates worldwide [1]. In China, esophagus cancer is one of the highest morbidity and mortality cancers; nearly 258,000 new cases and 193,000 deaths of esophagus cancer were reported in 2017 [2]. The incidence rate of esophageal cancer varies considerably with location. Despite the great process in the diagnosis and treatment of esophagus cancer, effective biomarkers that contribute to precision diagnosis and therapy are still lacking [3]. Therefore, it is very important to seek for promising biomarker and therapeutic target for the patients with esophagus cancer.
Rab proteins belong to GTPases of the Ras superfamily, and functional damage of Rab8 proteins has been reported to be involved in tumorigenesis, such as Rab1, Rab2A, and Rab8 [4]. Rab8, as a small Ras-related GTPase, was implicated in protein trafficking and secretion [5]. Rab8A and Rab8B were two isoforms of Rab8 in humans, which has 80% homology. Rab8A has been reported to be involved in various cellular functions such as neuronal differentiation and membrane trafficking [6]. Additionally, the effect of Rab8A in cancer has been reported. For example, Rab8A has been found to promote breast cancer progression by elevating surface expression of tropomyosin-related kinase B [7]; Rab8A was reported to promote the growth and metastasis of cervical cancer [8]. However, the function and molecular mechanism of Rab8A in esophagus cancer has not been revealed.
The concept of transcriptional control was put forward half a century ago in bacterial systems [9]. Over the past decade, our knowledge of mammalian regulatory elements and the transcriptional and chromatin regulatory elements that function at these sites has grown considerably [10]. Many different cancers can be caused by mutations in regulatory sequences and transcription factors that interact with these regions [10]. We described the transcriptional regulation of Rab8A in esophagus cancer.
This study was performed to detect the function of Rab8A in esophagus cancer. We estimated the effect of Rab8A depletion on cell proliferation, apoptosis, invasion, and migration. It is of great significance to estimate the molecular pathology to clarify the pathogenesis and disease progression of esophagus cancer patients.

Tissue Collection.
A total of 7-paired esophagus cancer specimens and adjacent normal control tissues were collected from esophagus cancer patients undergoing surgery at the First Affiliated Hospital of Jinzhou Medical University. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation and with the Helsinki Declaration of 1964 and later versions. Informed consent or substitute for it was obtained from all patients for being included in the study.
2.6. Cell Cloning Ability Detection. KYSE150 and TE-1 cells were harvested, resuspended in DMEM including 10% FBS, and then implanted in 6-well plates (500 cells each well). After cultivation under standard condition for 2 weeks, the colonies were fixed and stained. Finally, cell colonies were counted and applied for comparisons of colony formation ability.
2.7. Transwell Assay. Cell suspension (100 μL) including 2 × 10 4 cells was added to the upper chamber of a 24-well transwell plate with 8 μm pore polycarbonate filters (Costar, Corning, NY, USA). In the lower chamber, 500 μL of DMEM including 10% FBS was added. After incubation for 24 h, the medium was removed from the upper chamber and cells in each group were erased by a cotton swab. The cells that had migrated to the lower chamber were fixed and stained. Migrating cells were captured under a microscope in five random fields of view. The steps of invasion assay were consistent with the migration assay, except that the upper 2 Disease Markers chamber was precoated with Matrigel. Three independent experiments were performed.

Chromatin Immunoprecipitation (ChIP) Assay.
To detect whether PEA3 could bind to the promoter of Rab8A, a ChIP assay was conducted according to the ChIP assay kit procedures (ab500, Abcam). Briefly, the treated cells were collected and lysed. Antibody against Rab8A and IgG was applied for immunoprecipitation. PCR amplification of the precipitated DNA was performed.
2.9. Dual-Luciferase Reporter Assay. The potential transcription factor binding sites between Rab8A and PEA3 were generated by online software UCSC (https://genome.ucsc .edu/) and PROMO (http://alggen.lsi.upc.es/). To determine the activity of the Rab8A promoter in the presence of PEA3 overexpression, luciferase assay was performed. First, based on the predicted transcription factor binding sites, wildtype Rab8A (WT-Rab8A), mutant-1 Rab8A (Mut1-Rab8A), and Mut1-Rab8A were amplified and transfected into PGL3 vector. Subsequently, cells were cotransfected with PGL3-Rab8A promoter and PEA3 overexpression vector or the corresponding control vector using Lipofectamine 2000. A total of 48 h following transfection, the relative luciferase activity was measured as normalized to Renilla luciferase activity using the Dual-Luciferase® Reporter Assay System (Promega Corporation).

Statistical Analysis.
At least three independent experiments were carried out in each group. All experimental data were presented as mean ± standard deviation. The differences were processed by Student's t-test (two groups) or one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test (three or more groups). P value less than 0.05 was deemed as a statistically significant difference. were established and cotransfected with corresponding control or PEA3 overexpression (OE) vector into cells. The data showed that PEA3-OE treatment increased luciferase activity in the WT group whereas the luciferase activity in the Mut1 or Mut2 group has no obvious change (Figure 3(a)). Data from ChIP assay showed that depletion of PEA3 reduced Rab8A enrichment compared with that in the si-NC group (Figures 3(b) and 3(c)). Additionally, we also discovered that depletion of PEA3 decreased Rab8A expression in KYSE150 and TE-1 cells, and Rab8A expression was significantly increased after PEA3-OE treatment (Figures 3(d)-3(g)). Moreover, we also detected the protein expression of Rab8A and PEA3 in esophageal cancer tissues. The results showed that Rab8A and PEA3 were both highly expressed in esophageal cancer samples (Figures 3(h) and 3(i)). The above findings revealed that PEA3 could bind to the promoter of Rab8A and regulate Rab8A expression.   [11][12][13]. Once the Wnt/β-catenin signaling pathway is overactivated, the patterns of target genes c-MYC and CyclinD1 related to cell proliferation and migration are increased, which promotes cell proliferation and migration, resulting in malignant transformation of cells [14]. In our study, we discovered that the protein levels of β-catenin, c-MYC, and CyclinD1 were significantly increased when PEA3 was overexpressed (Figures 5(a)-5(d)). Moreover, siRab8A transfection reduced the protein levels of β-catenin, c-MYC, and

Discussion
The tumor-promoting effect of Rab8A has been involved in various physiological activities and signaling pathways [7]. In this study, we discovered that Rab8A was highly expressed in esophagus cancer samples, and knockdown of Rab8A suppressed the growth, migration, and invasion as well as induced apoptosis. Additionally, PEA3 has been identified as an upstream transcriptional factor of Rab8A and positively regulated Rab8A expression. Moreover, the effect of siRab8A on esophagus cancer cells could be diminished by PEA3 upregulation.
The PEA3 subfamily, as a subgroup of the E26 transformation-specific family, includes ETV1, ETV4, and ETV5. Functionally, the PEA3 subfamily is related to motor coordination, axon guidance, metabolism, neuron development, hormonal regulation, fertility, and tumorigenesis [15]. PEA3, also named ETV4, has been found to be highly expressed in many tumors [15]. High level of PEA3 usually resulted in a more aggressive tumor phenotype and drug resistance [16]. As an oncogenic transcription factor, PEA3 was discovered to directly target to the 5 ′ and 3 ′ MYC enhancers, insinuating that PEA3 may modulate the expression of several important oncogenes [17]. The PEA3 transcription factor is usually activated in gastric cancer, hepatocellular carcinoma, colorectal cancer, and lung cancer [18][19][20][21]. In esophageal squamous cell carcinoma and esophageal adenocarcinoma, PEA3 overexpression significantly elevated MMP levels and activated metastatic progression [22,23]. In our study, PEA3 was identified as a transcriptional factor of Rab8A in esophagus cancer. Moreover, PEA3 was highly expressed in esophagus cancer samples, and overexpression of PEA3 promoted the proliferation, migration, and invasion of esophagus cancer cells as well        Figure 5: The Wnt/β-catenin signaling pathway was involved in the regulation of PEA3/Rab8A on KYSE150 and Eca109 cell behaviors. (a-d) Protein levels of c-MYC, β-catenin, and CyclinD1 in KYSE150 and Eca109 cells were detected by western blotting after control, PEA3-OE, or PEA3-OE+si-Rab8A treatment, respectively. * * P < 0:01 vs. control; ## P < 0:01 vs. PEA3-OE. (e-h) Protein levels of c-MYC, β-catenin, and CyclinD1 in KYSE150 and Eca109 cells were detected by western blotting after control, siRab8A, inhibitor, or siRab8A +inhibitor treatment, respectively. (i, j) The OD values in KYSE150 and Eca109 cells were detected by CCK-8 assay after control, siRab8A, inhibitor, or siRab8A+inhibitor treatment, respectively. (k, l) The cell clone number in KYSE150 and Eca109 cells was detected by colony formation assay after control, siRab8A, inhibitor, or siRab8A+inhibitor treatment, respectively. * * P < 0:01 vs. control; ## P < 0:01 vs. siRab8A. 12 Disease Markers as suppressed apoptosis. Additionally, the function of Rab8A depletion on esophagus cancer cells was reversed by PEA3 upregulation. The Wnt/β-catenin signaling pathway is a conserved signaling axis taking part in various physiological processes including proliferation, apoptosis, differentiation, invasion, migration, and tissue homeostasis [24,25]. Abnormal Wnt/β-catenin signaling pathway promotes tumor stem cell renewal, cell proliferation, and differentiation, thus performing an important role in tumorigenesis and therapeutic response [26]. In the Wnt/β-catenin signaling pathway, aberrant regulation of β-catenin, which is the crucial component of the Wnt/β-catenin signaling pathway, resulted in early events in carcinogenesis [27,28]. Additionally, the relationship between Rab8A and Wnt pathway has been reported. For example, Rab8A controls Wnt delivery in producing cells and is crucial for Paneth cell maturation [29]; Rab8A attenuates the Wnt signaling pathway and is required for mesenchymal differentiation into adipocytes [30]; Rab8A has been discovered to regulate the development of nonsmall-cell lung cancer by modulating the Wnt/β-catenin signaling pathway [31]. In our study, overexpression of PEA3 increased the protein levels of c-MYC, β-catenin, and CyclinD1, while siRab8A transfection diminished the effect of PEA3 overexpression and suppressed the protein levels of c-MYC, β-catenin, and CyclinD1. Additionally, the inhibitor of the Wnt/β-catenin signaling pathway was applied. We discovered that suppression of the Wnt/β-catenin signaling pathway could inhibit the growth ability of esophagus cancer cells.
Several deficiencies in our study should be pointed out. First, our results were achieved only based on cell experiments; animal experiments will be needed to validate the results. Additionally, more molecular mechanism about Rab8A in esophagus cancer should be revealed.

Conclusions
Taken together, our results showed that knockdown of Rab8A could diminish the growth, invasion, and migration abilities as well as induce the apoptotic ability. Moreover, Rab8A was transcriptionally regulated by PEA3 and interacted with PEA3 to regulate the malignant behavior of esophagus cancer cells. The Wnt/β-catenin signaling pathway was positively regulated by Rab8A/PEA3 in esophagus cancer cells. The above results indicated that Rab8A, transcriptionally modulated by PEA3, promoted the growth and metastasis abilities of esophagus cancer cells by activating Wnt/β-catenin signaling.

Data Availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.