RAB11A Promotes Cell Malignant Progression and Tumor Formation of Prostate Cancer via Activating FAK/AKT Signaling Pathway

Background RAB11A, a member of the GTPase family, acts as a regulator in diverse cancers development. The dysregulation of the FAK/AKT signaling pathway is mainly related to tumorigenesis. This study aimed to investigate the possible effect of RAB11A in prostate cancer and further explore the potential mechanisms. Results In this study, we illustrated the tumor-promoting effects of RAB11A based on in vivo and in vitro experiments. RAB11A expression was upregulated in prostate cancer cells. RAB11A knockdown decreased the prostate cancer cell proliferation, migration, and invasion. RAB11A also induced the epithelial-mesenchymal transition. PF562271 suppressed the malignant characteristics of prostate cancer cells caused by RAB11A knockdown. Furthermore, the interference of RAB11A reduced the tumor growth and the protein levels of p-FAK/FAK and p-AKT/AKT in vivo. Conclusion RAB11A promotes cell malignant progression and tumor formation in prostate cancer via activating FAK/AKT signaling pathway.


Introduction
Prostate cancer is one of the most commonly diagnosed cancers in men worldwide, with approximately 191,930 cases and 33,330 deaths each year [1]. Te incidence of prostate cancer is related to race and familial inheritance, especially in Africa and the USA [2]. Prostate cancer is usually diagnosed in the elderly (age >65 years) and ranks as the second cancer-related death in men [1]. Malignant transformation in the prostate is a multistage process that is initiated by localized prostate cancer, followed by adenocarcinoma with local invasion, and ends with metastatic prostate cancer [3]. Terapies for prostate cancer are mainly based on the pathologic evaluation of a prostate biopsy [4]. Surgery and radiotherapy are the main therapeutic approaches for early prostate cancer; however, they cannot efectively prevent tumor metastasis. Currently, the understanding of tumorigenesis mechanism of prostate cancer remains limited, which is an obstacle in the development of cancer therapies.
RAB11A is the frst identifed member of the small GTPase family, playing a crucial role in polarization during collective cell migration [5]. Previous studies found that RAB11A plays a critical role in cancer malignant progression through regulating the growth factor signaling [6,7]. By activating of WNT signaling, RAB11A enhanced the proliferative capability and motility of esophageal cancer cells [8]. RAB11A is overexpressed in colorectal carcinoma and suppresses the expression of E-cadherin, which induces epithelial-mesenchymal transition (EMT) [9]. Besides, RAB11A is up-regulated in human gastric cancer cells and facilitates the proliferation and invasion of cancer cells through the FAK/AKT pathway [10]. Tese studies suggest RAB11A as an oncoprotein during cancer progression. However, the potential efects and mechanisms of RAB11A in prostate cancer remain unknown.
Te focal adhesion kinase (FAK) signaling pathway is related to cell motility, which positively regulates tumorigenesis and metastasis [11]. FAK is upregulated in multiple cancers, such as gastric and prostate cancers, suggesting the deactivation of FAK signaling might be a possible approach for the treatment of cancer [10,12]. FAK is an upstream target of ATP-dependent tyrosine kinase (AKT), and AKT acts as a migratory regulator in cancer cells [13]. Meanwhile, the activation of AKT could enhance the migration and invasion capabilities of cancer cells [14,15]. Previous studies showed the FAK/AKT signaling pathway is activated in prostate cancer [12,16]. Terefore, the FAK/AKT signaling pathway might be an efective target for cancer treatment, but its role in prostate cancer remains majorly elusive.
Terefore, the aim of the present study was to investigate the potential role of RAB11A in the development of prostate cancer. Meanwhile, the potential mechanism of RAB11A against prostate cancer was explored through in vitro and in vivo experiments to identify a novel potential target for the treatment of prostate cancer.
When the confuency of PC-3 cells was up to 70-90%, they were transfected with si-RAB11A, overexpressing (oe)-RAB11A, and empty vector (si-NC and oe-NC) plasmids (40 μL, 1 × 10 8 TU/mL). After transfection for 48 h, the medium was discarded, and complete medium with 2.5 μg/ mL puromycin was added to flter out the stable transfected cell line. Te stable transfected cells were conducted for subsequent experiment. To analyze the efect of RAB11A on FAK/AKT signaling pathways, PC-3 cells were pretreated with 0.5 μM PF562271 (a FAK inhibitor) for 24 h before the transfection.

Animal Model.
Animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Xiamen University (XMULAC20220034-1). A total of 10 male BALA/c nude mice, obtained from Gem-Pharmatech Co. Ltd. (Nanjing, China), were cultured for 5-6 weeks, weighed 18−21 g, and were raised under specifc nonpathogen conditions at 24−26°C and 40%-60% humidity. Mice were put into two groups at random (n � 5 each group): si-NC and si-RAB11A groups. After one week of adaptation, mice were subcutaneously injected with 3 × 10 6 PC-3 cells transfected with si-NC or si-RAB11A. Ten days after transplantation, tumor sizes were detected every fve days, and the volume was calculated: tumor volume (v) � 1/ 2 × length × (width 2 ). After 30 days, mice were sacrifced via intraperitoneal injection with 160 mg/kg pentobarbital sodium, and death was considered as the asystole. Tumor tissues in each mouse were collected, weighed, and imaged.

Reverse Transcription-Quantitative PCR (RT-qPCR)
Analysis. Total RNAs were extracted from cells and tumor tissues by Trizol (Invitrogen). cDNA was reversely transcribed using FastKing gDNA Dispelling RT Supermix (TIANGEN, KR118-02) and stored at −20°C. Te SYBR Green PCR Master Mix (Lifeint, Guangzhou, China) was used to amplify cDNA in a cycler apparatus of the real-time system (MX3000P, Agilent Stratagene, California, USA). Te thermocycling conditions were as follows: 95°C for 3 min, 40 cycles of annealing at 95°C for 12 s, and extension at 62°C for 40 s. Te 2 −∆∆Ct method was employed to analyze the relative expression of genes. Te composition of the RT-qPCR reaction mixture was listed at Table 1. Primer sequences used for RT-qPCR were presented in Table S2.

Western Blot Analysis.
Te transfected cell and tumor tissues lysing were accomplished with the radio immunoprecipitation assay bufer (RIPA, Beyotime, Shanghai, China) for total protein extraction. Protein concentration was determined using a bicinchoninic acid assay kit (BCA, Beyotime). Proteins were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) by electrophoresis and transferred to polyvinylidene fuoride (PVDF) membrane. Te membrane was blocked with 5% fat-free milk at room temperature for 1 h. Ten, the membrane was incubated overnight at 4°C with primary antibodies, as listed at Table S3. Membranes were washed with TBST (a bufer combined with Tris bufered saline and Tween, Beyotime; 1 : 10) for 10 min thrice and incubated with secondary antibodies for 1 h. Finally, protein bands were visualized by the ECL reagents (Applygen, Beijing, China) and were quantifed by Image J software.

Cell Counting Kit (CCK)-8 Assay.
Te cell viability assay was implemented with a CCK-8 assay kit (Beyotime). Briefy, transfected cells were digested by trypsin and then centrifuged at 1,000 g for 5 min to discard the supernatant. Te fresh complete medium was used to suspend the cells. After suspension, cells (1 × 10 5 /mL) were seeded into 96-well plates with 40 μL/well and incubated at 37°C with 5% CO 2 . After 24 h, each well was added with 10 μL CCK-8 solution and incubated for 2 h. Te absorbance value at 450 nm was measured by a microplate reader (DR-3518G, Wuxi Hiwell Diatek, Wuxi, China).

Colony Formation
Assay. Cells (200 cells/well) were seeded into 6-well plates and cultured in 2 mL complete medium for 2 weeks. After 2 weeks of cell culture, the medium was discarded, and cells were rinsed with PBS twice. Following the fxation with 1 mL methanol for 15 min, cells were stained with crystal violet. Subsequently, the plate was reversely put on a white paper and then captured for further colony cell number counting by Image J software.

Wound Healing Assay.
Te cells in the logarithmic period were digested by trypsin for about 1 min, which was ended by the addition of complete medium. Ten, the cells were centrifuged at 1,000 g for 5 min to discard the supernatant, and adjusted density with fresh complete medium. Cells (1 × 10 8 cells/well) were seeded in 6-well plates with complete medium. After 24 h incubation, cells were scratched with pipette tip, washed with PBS three times, and then put into a chamber at 37°C with 5% CO 2 . Cell migration was observed between 0 and 24 h. Te cell migration at 0 h was captured as a control for further comparison with the migration condition at 24 h.

Transwell Invasion Assay.
Te Transwell chamber was pretreated with 50 μL Matrigel (50 mg/L, dilution: 1 : 4) and incubated at 37°C for 2 h. After digestion by trypsin, cells were washed and resuspended by PBS to a cell density of 1 × 10 6 /mL. Te cells were suspended in serum-free medium. Upper and lower chambers of Transwell were added with 200 μL cell suspension and 600 μL medium with 10% FBS, respectively. Following incubation at 37°C with 5% CO 2 for 24 h, the medium was discarded, and cells were fxed with formaldehyde for 30 min. After fxation, cells were stained with 0.1% crystal violet for 20 min, and imaged three random regions under a microscope (SC180, Olympus, Japan).

Statistical
Analysis. Data are presented as mean-± standard deviation. GraphPad Prism 7.0 software (GraphPad software, USA) was employed to analyze all experimental data. A one-way ANOVA was applied to compare data obtained from multiple groups. Statistical signifcance was regarded as P < 0.05.

RAB11A Expression is Upregulated in Prostate Cancer
Cells. RAB11A is a crucial regulator in multiple cancers. We frst used RT-qPCR and western blot assays to measure the expression level of RAB11A in prostate cancer cells. Results showed both the mRNA and protein expression of RAB11A were remarkably overexpressed in human prostate cancer cell lines (DU145, VCaP, and PC-3) compared with those in normal RWPE1 cells (P < 0.05). Notably, the expression level of in PC-3 cells was relatively highest (P < 0.01) (Figure 1).

RAB11A Promotes Prostate Cancer Cell Proliferation.
Since RAB11A expression was obviously higher in PC-3 cells relative to normal cells, we examined the efects of RAB11A on PC-3 by transfecting PC-3 cell lines with siRNA-RAB11A (si-RAB11A), overexpress RAB11A (oe-RAB11A), and a negative control (NC). Te mRNA and protein levels of RAB11A were reduced by si-RAB11A and enhanced by oe-RAB11A (P < 0.01) (Figure 2(a)). CCK-8 and colony formation assays showed that, compared to the NC groups, the interference of RAB11A remarkably reduced the proliferation of PC-3 cells, and RAB11A overexpression enhanced cell proliferation (P < 0.01) (Figures 2(b) and 2(c)).

RAB11A Facilitates the Migration, Invasion, and Epithelial-Mesenchymal Transition (EMT) of Prostate Cancer
Cells. Next, we determined the infuence of RAB11A on the invasion and migration of cancer cells. Wound healing assay demonstrated the decreased migratory potential of PC-3 cells with RAB11A knockdown and the increased migration with RAB11A overexpression (P < 0.01) (Figure 3(a)). Transwell assay suggested that RAB11A overexpression markedly enhanced cell invasion, while RAB11A knockdown presented the opposite efect (P < 0.01) (Figure 3(b)). Subsequently, we used western blot assay to detect the levels of E-cadherin, N-cadherin, and vimentin. Results revealed  Evidence-Based Complementary and Alternative Medicine that the knockdown of RAB11A, compared with the NC group, enhanced the level of the epithelial marker Ecadherin but suppressed the expression of mesenchymal markers N-cadherin and vimentin; meanwhile, RAB11A overexpression presented opposite efects (P < 0.01) (Figure 3(c)).

RAB11A Promotes Proliferation, Migration, Invasion, and EMT of Prostate Cancer Cells via Activating FAK/AKT
Signaling Pathway. Activation of FAK/AKT promotes prostate cancer cell aggression. Tus, we hypothesized that the potential mechanism of RAB11A in prostate cancer might be associated with the FAK/AKT signaling pathway.
To verify this, we pretreated PC-3 cells with the FAK inhibitor PF562271 before transfection with si-RAB11A. Results showed that PF562271 did not afect the RAB11A expression level in transfected PC-3 cells (Figures 4(a)   Evidence-Based Complementary and Alternative Medicine mice and the sizes of collected tumors are presented in Figure 6(a), showing the absence of RAB11A reduced tumor sizes. Te weight and volume of tumor tissues in the si-RAB11A group were signifcantly lower than si-NC group (P < 0.01) (Figure 6(b)). RT-qPCR showed that RAB11A expression, compared with the si-NC group, was remarkably decreased by si-RAB11A (P < 0.01) (Figure 6(c)). On the other hand, the protein levels of FAK, AKT, p-FAK, and p-AKT were detected by western blot assay. Results showed the p-FAK/FAK and p-AKT/AKT protein levels are obviously decreased in si-RAB11A group (P < 0.01) (Figure 6(d)).

Discussion
Prostate cancer is the secondary diagnosed malignancy in men worldwide, with high mortality. Te etiology of prostate cancer remains elusive because of the lacked evidence of genetic and pathology [17]. Previous studies showed there are several signaling pathways were mainly involved in prostate cancer, including MEK/ERK, FAK/AKT, and p75NTR signaling pathway [12,18,19]. Risk factors regulating these signaling pathways could be regarded as a potential target in the treatment of prostate cancer. RAB11A is overexpressed in diverse types of human cancers, such as lung and gastric cancer [6,10]. In this work, we found that the RAB11A is upregulated in prostate cancer cells and promotes the progression of prostate cancer in vitro and in vivo through activating FAK/AKT signaling. RAB11A is a major GTPase of vesicular trafcking and membrane dynamics, the alterations of which promote tumorigenesis [20]. It has been reported that RAB11A facilitates gastric cancer progression and metastasis [10]. Our data showed a similar efect of RAB11A overexpression in prostate cancer, promoting the proliferation, invasion, and Evidence-Based Complementary and Alternative Medicine migration of cancer cells. Meanwhile, the RAB11A expression was positively related with mesenchymal markers (N-cadherin and vimentin) but negatively with epithelial marker E-cadherin. Tese results demonstrate that RAB11A plays an oncological role in prostate cancer. FAK is an important regulator of cell migration and invasion [21,22]. FAK signaling pathway was activated in multiple human cancers including prostate cancer [16]. AKT is activated by FAK stimulation, regulating cell migration and invasion [23]. Accumulating evidence suggests that the activation of FAK/AKT signaling promotes tumorigenesis. CCK3 contributes to the EMT process in prostate cancer by activating FAK/AKT signaling [12]. Knockdown of RABL3, which belongs to the Rab subfamily, inhibits the proliferation and invasion of oral squamous cell carcinoma through deactivating the FAK/AKT pathway [24]. As another member of the Rab subfamily, RAB11A in the present study showed similar functions, facilitating the proliferation, migration, and invasion of prostate cancer. Terefore, we speculated that RAB11A promotes prostate cancer progression via the FAK/AKT pathway. Our data showed that si-RAB11A reduced the expression of p-FAK/FAK and p-AKT/AKT. PF562271 (a FAK inhibitor) enhanced the inhibitory efect of RAB11A on the FAK/AKT signaling pathway and on the malignant progression of prostate cancer. Tese results demonstrate that RAB11A could potentially promote the malignant progression of prostate cancer by activating the FAK/AKT signaling pathway. Meanwhile, in vivo experiment showed that the interference of RAB11A reduced the tumor growth and downregulated the protein levels of p-FAK/FAK and p-AKT/AKT in tumor tissues of prostate cancer. Taken together, our data suggest that RAB11A, as an oncogenous protein, promotes prostate cancer malignant progression and tumorigenesis through activating FAK/AKT signaling.
In conclusion, the current study identifed the role of RAB11A as a tumor promoter overexpressed in human prostate cancer. Te possible mechanism of RAB11A promoting prostate cancer is associated with the activation of the FAK/AKT pathway. Meanwhile, this study suggests the FAK/AKT signaling pathway as a therapeutic target to regulate the progression and development of prostate cancer. However, the understanding of the underlying intermediate mechanism by which RAB11A regulates the FAK/AKT signaling pathway remains unrevealed.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Ethical Approval
Tis study was approved by the Ethic Committee of Afliated Hospital of Xiamen University (XMULAC20220034-1).