Lysosomal-Associated Transmembrane Protein 5 Promotes Proliferation, Migration, and Invasion of Clear Cell Renal Cell Carcinoma

Clear cell renal cell carcinoma (ccRCC) is the most aggressive and deadly cancer of the urinary system and is regulated by multiple signaling pathways. However, the specific molecular mechanisms underlying ccRCC have not been fully studied or demonstrated. This study aimed to elucidate the function of lysosomal-associated transmembrane protein 5 (LAPTM5) in ccRCC cell lines and animal models and determine the potential underlying mechanisms. Our results demonstrated that LAPTM5 expression in patients with ccRCC was significantly higher in the tumor group than that in the adjacent nontumor group. Moreover, LAPTM5 promoted proliferation, migration, and invasion of ccRCC cells through the gain and loss of the function of LAPTM5 in 786-0 and Caki-1 cell lines. Similar results regarding LAPTM5 overexpression were obtained in BALB/c nude mice. In addition, LAPTM5 activated the Jun N-terminal kinase (JNK)/p38 signaling cascade by interacting with Ras-related C3 botulinum toxin substrate 1 (RAC1). Treatment with an RAC1 inhibitor eliminated the effects of LAPTM5 in ccRCC. In conclusion, these results indicate that LAPTM5 may be a new therapeutic target for ccRCC via activation of the RAC1-JNK/p38 axis.


Introduction
Renal cell carcinoma (RCC) is among the ten most common tumors in the world [1]. Te incidence rate of RCC is third among all cancers worldwide, and its associated mortality rate ranks frst [2]. Clear cell renal cell carcinoma (ccRCC) is the most common type of RCC, accounting for approximately 70% of all cases. Notably, the ccRCC incident rate demonstrates an increasing trend [3]. RCC treatment, including that for ccRCC, has transitioned from nonspecifc immunotherapy to the targeted therapy and immunotherapeutic agents [3]. Te development of novel treatment options have led to alleviated symptoms in some patients; however, the ccRCC mortality rate is high and the prognosis remains poor, particularly in patients with high pathological ccRCC grades and stages [4]. Tus, illustrating the underlying molecular mechanisms and identifying new therapeutic targets for ccRCC treatments is crucial.
Lysosomal-associated transmembrane protein 5 (LAPTM5), also known as E3 and CD40-ligand-activated specifc transcript, is a member of the LAPTM family. LAPTM5 is transferred from the Golgi apparatus to lysosomes through interactions with NEDD4, an E3 ubiquitin-protein ligase [5][6][7]. In contrast to the two widely expressed family members LAPTM4a and 4b, LAPTM5 is primarily located in late endosomes and lysosomes [7]. LAPTM5 contains a ubiquitin interaction region, which interacts with E3 ubiquitin ligases and plays important roles in ubiquitination [7]. Functionally, LAPTM5 participates in the progression of cellular immunity, protein transport, apoptosis, and tumorigenesis [8][9][10]. A recent study showed that LAPTM5 may serve as a biomarker related to testicular germ cell tumor prognosis and diagnosis, and the survival rate was higher in the low LAPTM5 expression group, what's more, the author considered that LAPTM5 may afect the immune function [8]. In addition, previous studies suggest that LAPTM5 is related to the activation of mitogen-activated protein kinase (MAPK), NF-κB, PI3K/Akt, and other pathways [8,11]. Tese signaling pathways play key roles in the progression of numerous tumors, including ccRCC [12][13][14]. However, the role of LAPTM5 in ccRCC and its potential molecular mechanisms are yet to be fully elucidated.
Results obtained from Te Cancer Genome Atlas (TCGA) database demonstrated that LAPTM5 expression levels were higher in ccRCC tissues than that in paracancerous tissues. Western blotting and reverse transcription quantitative (RT-q) polymerase chain reaction (PCR) revealed that LAPTM5 expression was higher in the human ccRCC tumor group than that in the corresponding nontumor group. Moreover, LAPTM5 overexpression promoted tumor proliferation, migration, and invasion in 786-0 and Caki-1cell lines, whereas LAPTM5 knockdown exerted the opposite efects. In addition, LAPTM5-overexpressed 786-0 cells were injected into nude mice and the same results were obtained. Te weight, volume, and protein expression levels of migration-, invasion-, and proliferation-associated markers in LAPTM5-overexpressed mice were higher than those in the control group. Moreover, our results demonstrate that LAPTM5 activates the Jun N-terminal kinase (JNK)/p38 axis by binding to Ras-related C3 botulinum toxin substrate 1 (RAC1), and the efects of LAPTM5 on tumor growth were attenuated following treatment with an RAC1 inhibitor. In conclusion, the results of our study demonstrate that LAPTM5 regulates ccRCC development by regulating the RAC1-JNK/p38 signaling cascade.

Human ccRCC Samples.
Te present study included patients who were pathologically diagnosed with ccRCC postoperatively at the First Afliated Hospital of Gannan Medical University. Tis study was approved by the Ethics Review Committee of the First Afliated Hospital of Gannan Medical University, and informed consent was obtained from all patients prior to the treatment (approval no. CCSC-2021101201). Tis study was conducted in accordance with the principles of the Declaration of Helsinki.

Animal Models.
All animal experiments were approved by the First Afliated Hospital of Gannan Medical University and carried out in accordance with the guidelines for the care and use of experimental animals. Te 786-0 cells stably transfected with LAPTM5 (2 × 10 6 cells in 200 μl phosphate bufered saline (PBS)) and the corresponding negative controls (200 μl PBS) were injected subcutaneously into 6-week-old BALB/c nude mice (n � 6 per group). After 28 days, the mice were sacrifced by intraperitoneal injection of sodium pentobarbital (100 mg/kg), and death was verifed by respiratory and cardiac arrest and pupil dilation. Te masses and volumes of the obtained tumors were detected and subsequently photographed.

Bioinformatics
Analysis. RNA sequencing data were downloaded from TCGA database (https://portal.gdc. cancer.gov/). Diferential gene analysis was performed using the R package "DESeq2" (R Foundation for Statistical Computing, Vienna, Austria). Moreover, the R package "ggplot2" was used to visualize the data. Te "survminer" package was used to visualize the survival probability, and the survival package was used for statistical analysis of survival data. A t-test was used to analyze tumor stage correction between the LAPTM5 and control groups.

Plasmid Construction.
For the LAPTM5-overexpressed plasmids, the full-length LAPTM5 cDNA was cloned into the lentiviral vector of pHAGE using cytomegalovirus promoter and FLAG antibodies. For LAPTM5 knockdown plasmids, three short hairpin (sh) RNAs were selected (Sigma-Aldrich, St. Louis, MO, USA; Merck KGaA, Darmstadt, Germany) for cloning into the lentiviral vector pLKO.1; expression was detected using Western blot and RT-qPCR. To determine the molecular mechanism, RAC1 was cloned into the pcDNA5 vector using HA antibody. All primers used in this study are listed in Table S1 in the Supplementary Material.
2.6. Cell Transfection. pMD2.G, psPAX2, and the corresponding recombinant plasmids (including the empty vector) were cotransfected into 293T cells, and the supernatant was collected after 24 and 48 h using polyethylenimine linear. Te 786-0 and CAKI-1 cells transfected with supernatant and polybrene (10 μl/ml) were added. After 48 h, puromycin (4 μl/ml) was used to select and obtain target cells.

5-Ethynyl-2′-Deoxyuridine Labeling
Assay. Approximately 5,000 786-0 or Caki-1 cells were inoculated per well in 48-well plates and subsequently cultured in 5% CO 2 at 37°C for 48 h. Ten, 5-ethynyl-2′-deoxyuridine (EdU) reagent was added to the cell culture medium and incubated for 2 h. Te plates were then fxed and stained according to the instructions of the EdU kit (cat. no. C0071S; Beyotime Institute of Biotechnology, Shanghai, China). Fluorescent images were captured to compare the proliferation of each group.

Colony Formation Assay.
A total of 1,000-1,500 cells were inoculated per well into 6-well plates at 37°C and 5% CO 2 . Following 10-15 days of culture, the cells were fxed with 4% paraformaldehyde at room temperature for 20 min and subsequently stained with 0.1% crystal violet for 15 min prior to imaging.

Wound-Healing Assay.
Multiple horizontal lines were drawn on the back of 6-well plates with a marker. A total of 5-10 × 10 5 cells were inoculated into each well. After overnight incubation, the cells reached full confuence. Subsequently, a 20-μl pipette tip was used to scratch along the previously marked line on the back of the plate. Te cells were then treated with PBS 2-3 times and the medium was replaced with fresh DMEM with 1% FBS. Cells were cultured in a 37°C, 5% CO 2 incubator and images were captured under the microscope after 0 and 24 h. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to calculate wound healing in each group.

Transwell Assay.
Te migration and invasion ability of ccRCC cells were assessed using a transwell assay (cat. no. 3421; Corning Inc., Corning, NY, USA). Te 786-0 cells were inoculated into the upper chamber of a 24-well plate. Following incubation at 37°C and 5% CO 2 for 6 h, the migrating cells were fxed using 4% paraformaldehyde and stained with 0.1% crystal violet. Te cells in the upper chamber were gently removed and imaged, and the number of migrating cells was counted. Te upper chamber was coated with Matrigel and the cells were inoculated into the upper chamber. Te aforementioned protocols were used for invasion. Caki-1 cells were incubated at 37°C and 5% CO 2 for 24 h, and the aforementioned procedures were performed to determine the migration and invasion of Caki-1-1 cells.
2.11. RT-qPCR. Total RNA was extracted using 1 ml Trizol reagent (cat. no. 15596-026; Termo Fisher Scientifc), then total RNA was reverse transcribed into cDNA using a cDNA synthesis kit (cat. no. 04896866001; Roche Diagnostics GmbH, Basel, Switzerland). RT-qPCR was performed using SYBR green PCR master mix. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. All primer sequences are listed in Table S2 in the Supplementary Material.

Western Blot Analysis.
Tissue blocks and cells were added to 1 ml lysate (radioimmunoprecipitation assay (RIPA), phenylmethylsulfonyl fuoride complete, Phos-STOP, NaF, and Na3VO4), and tissue blocks were crushed using a grinder (30 Hz/s for 90 sec). Both the cells and tissues were centrifuged at 4°C and 12,000 rpm for 30 min and the supernatant was collected. Te protein concentration was determined using a bicinchoninic acid protein assay kit. For sample preparation, 10X dithiothreitol, 10X loading bufer, and RIPA bufer were added to the cells. Te samples were subjected to 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis and subsequently transferred onto polyvinylidene difuoride membranes. Primary antibodies were added, and the membranes were incubated at 4°C overnight. Following the primary incubation, secondary antibodies were added for 1 h at room temperature (25°C). GAPDH was used as an internal control. Te antibodies used in this study are listed in Table S3 in the Supplementary Material.

Coimmunoprecipitation
Assay. LAPTM5-FLAG and RAC1-HA were constructed and cotransfected into 293T cells. After 24 h, samples were collected, and FLAG antibody was added and shaken at 4°C overnight. Te subsequent immunoprecipitate was treated with precooled immunoprecipitation bufer 5-6 times and 2× loading bufer was added; the interaction between LAPTM5 and RAC1 was detected using HA and FLAG antibodies. Te aforementioned protocols were repeated, and the target proteins were immunoprecipitated using the HA antibody; the interaction between LAPTM5 and RAC1 was detected using Western blot analysis.

Statistical Analysis.
All data are presented as means± standard deviations and were statistically analyzed using SPSS software (version 23.0; IBM Corp., Armonk, NY, USA). For normally distributed data, a twotailed Student's t-test was used for comparisons between the two groups; one-way analysis of variance was used for comparisons among multiple groups, followed by Bonferroni's post hoc analysis (data meeting the homogeneity of variance) or Tamhane's T2 analysis (missing variance). For data with skewed distributions, nonparametric statistical analysis was performed using the Mann-Whitney U test for the two groups and Kruskal-Wallis test for multiple groups. P < 0.05 was considered statistically signifcant.   Journal of Oncology

LAPTM5 is Highly Expressed and Related to Poor Prognosis in ccRCC.
To explore the role of LAPTM5 in ccRCC, the LAPTM5 expression levels were determined in tumor and adjacent healthy tissues of patients with ccRCC obtained from TCGA database and GEO database (GSE 6344 and GSE 781). Te results suggested that LAPTM5 expression was higher in patients with ccRCC than that of patients in the control group (Figures 1(a)-1(e)). Moreover, the results of TCGA database showed that LAPTM5 is signifcantly associated with tumor stage. Te LAPTM5 expression levels in stages III and IV were signifcantly higher than those in stages I and II (Figure 1(f )). Survival analysis was subsequently conducted, and the results suggest that increased LAPTM5 expression is closely related to poor prognosis of ccRCC ( Figure 1(g)). To further verify these results, RT-qPCR and Western blot analyses were performed in human ccRCC tissues. Te results obtained from the RT-qPCR and Western blot analyses support the previously obtained results (Figures 1(h)-1(i)). We selected 786-0, Caki-1, and 769-P cell lines to explore the LAPTM5 expression in ccRCC cell lines. Compared with the renal epithelial cell lines HK-2, the results showed that LAPTM5 was upregulated in three ccRCC cell lines (Figure 1(j)) and we chose 786-0 and Caki-1 for the further experiment. In conclusion, LAPTM5 may be involved in ccRCC progression.

LAPTM5 Promotes ccRCC Proliferation In Vitro.
LAPTM5 overexpression (Figure 2(a)) and knockdown were performed in 786-0 and Caki-1 cell lines. Following LAPTM5 knockdown using three diferent plasmids, shLAPTM5-3 was selected for follow-up experiments (Figure 2(e)). Te efects of LAPTM5 on cell proliferation were determined using a clone formation assay, EdU staining, and proliferation marker levels. We found that the number of cell clones and the cell proliferation rate (Figures 2(b) and 2(c)) in the LAPTM5-overexpression group were higher than that in controls, indicating that LAPTM5 promoted cell proliferation in ccRCC. In addition, the expression of the molecular markers of proliferation, namely, proliferating cell nuclear antigen (PCNA) and cyclin-D1, were determined in both cell lines, suggesting that both markers were higher in the LAPTM5-overexpression group, compared with that in the control group (Figure 2(d)). Moreover, the above results showed that LAPTM5 knockdown decreased the number of cell clones, cell proliferation rate, and molecular marker expression (PCNA and cyclin-D1) in 786-0 and Caki-1 cells (Figures 2(f )-2(h)). Tus, these results demonstrate that LAPTM5 promotes cell proliferation in vitro.

LAPTM5 Promotes Migration and Invasion of ccRCC In
Vitro. To verify the role of LAPTM5 in 786-0 cell migration, a wound healing assay was performed, which suggested that overexpressed LAPTM5 promoted cell migration, whereas knockdown inhibited cell migration (Figure 3(a)). Tese results were validated in Caki-1 cells (Figure 3(b)). Te function of LAPMT5 in cell invasion and migration was determined using a transwell assay, which revealed that LAPTM5 overexpression promoted 786-0 and Caki-1 cell migration (Figure 3(c)) and invasion (Figure 3(d)). Te expression of migration-and invasion-associated molecular markers was also detected. Te results demonstrated that Ncadherin expression was increased and E-cadherin expression was decreased in the LAPTM5-overexpression group (Figure 3(e)). In addition, the transwell assay revealed that LAPTM5 knockdown inhibited 786-0 and Caki-1 cell migration and invasion (Figures 3(f ) and 3(g)). Moreover, the N-cadherin level was reduced and E-cadherin was promoted in the LAPTM5-knockdown group compared with that in the control group (Figure 3(h)). In conclusion, these fndings suggest that LAPTM5 promotes the migration and invasion of ccRCC cells in vitro.

LAPTM5 Promotes ccRCC Development and Growth In
Vivo. Te efects of LAPTM5 were evaluated in vivo using subcutaneous tumorigenesis experiments in BALB/c mice. Te 786-0 cell line with LAPTM5 overexpression was injected into mice, and the time until tumor visualization was observed. Notably, the tumor could be visualized after 18 days. Weight changes in the BALB/c mice were also observed from days 18 to 28, and the tumor volume was recorded on day 28. No signifcant changes in the weight of the mice were observed (Figure 4(a)), and the tumor volume in the LAMPT5overexpression group was larger than that in the control group (Figure 4(b)). In addition, the tumor mass in the LAPTM5 overexpression group was signifcantly larger than that in the control group (Figures 4(c) and 4(d)). Molecular markers of proliferation, migration, and invasion were detected in ccRCC mice using Western blotting. Te results demonstrated that PCNA, cyclin-D1, and N-cadherin levels increased, while E-cadherin levels decreased (Figure 4(e)). Overall, these results indicate that LAPTM5 promotes tumor growth in vivo.

LAPTM5 Activates JNK/p38 Pathway by Binding to RAC1.
Te potential mechanisms underlying ccRCC were explored in the present study. Notably, the MAPK pathway is widely involved in numerous cellular activities, such as proliferation, migration, and apoptosis and is closely associated with ccRCC. Te results demonstrated that LAPTM5 overexpression activated p38 and JNK phosphorylation, whereas the expression levels of total and phosphorylated extracellular signal-regulated kinase (ERK) did not change ( Figure 5(a)). LAPTM5 knockdown inhibited p38 and JNK phosphorylation in 786-0 cells, although no change was observed in ERK expression levels ( Figure 5(b)). We also found that LAPTM5 overexpressed activated the phosphorylation of JNK and p8 in Caki-1 cell lines ( Figure 5(c)). In addition, results consistent with those previously described were obtained in vivo following subcutaneous tumorigenesis in BALB/c nude mice ( Figure 5(d)). Tus, these results suggest that LAPTM5 regulates ccRCC growth through MAPK pathways. Moreover, our results demonstrate that RAC1 upstream from MAPK interacts with LAPTM5 ( Figures 5(e) and 5(f )). We also conducted GSTpulldown assays to verify the direct interaction between LAPTM5 and RAC1 ( Figure 5(g) and 5(h)). Tus, LAPTM5 might regulate MAPK activation by interacting with RAC1.
3.6. RAC1 Knockdown Regulates ccRCC Phenotype through LAPTM5. Treatment with an RAC1 inhibitor (NSC 23766) was administered to determine whether RAC1 is crucial to LAPTM5 for growth regulation in ccRCC. Te wound healing and EdU assays demonstrate that the promoting efects of LAPTM5 on tumor proliferation were signifcantly weakened following RAC1 inhibitor treatment (Figures 6(a) and 6(b)). Moreover, the efects of LAPTM5 on tumor migration and invasion were eliminated following RAC1 inhibitor treatment (Figures 6(c) and 6(d)). Tese fndings support the notion that LAPTM5 regulates ccRCC growth through the RAC1 signaling pathway.

Discussion
ccRCC poses a severe threat to the lives of patients and involves numerous signaling pathways. Exploring and clarifying the potential mechanisms of the pathways involved in ccRCC is crucial for the development of novel ccRCC treatment options. We focused on the function of LAPTM5 as a marker of ccRCC, and the results demonstrate that LAPTM5 regulates JNK and p38 phosphorylation by directly interacting with RAC1 to promote proliferation, migration, and invasion of ccRCC. When RAC1 is inhibited, the ccRCC phenotype is no longer afected by LAPTM5. Based on our results, we suggest that LAPTM5 can serve as an oncogene in the development of ccRCC.
As a lysosomal transmembrane protein, LAPTM5 is widely involved in various cell functions, including protein   transport, immune cell activation, signal pathway activation, autophagy, and oxidative stress [9,[15][16][17][18][19][20]. LAPTM5 is also widely involved in the progression of various cellular activities and signaling pathways. LAPTM5 plays a negative regulatory role in T and B cell activation [21] and is widely involved in tumor growth. In addition, LAPTM5 inhibits tumor invasion, clonogenicity, and tumorigenicity by inhibiting NF-κB activation, which may act as a therapeutic target for CD40+gliomas [22]. LAPTM5 knockdown leads to apoptosis in lung cancer cells; thus, inhibiting LAPTM5 expression has potential for the treatment of cancer [23]. LAPTM5 also mediates cell death in neuroblastoma. Notably, the E3 ubiquitin ligase, ITCH, promotes degradation by ubiquitination to prevent LAPTM5-mediated programmed death [24]. In bladder cancer, LAPTM5 knockdown inhibited cell proliferation and the cell cycle [25].
ccRCC is a urinary system tumor, and the potential function of LAPTM5 in ccRCC remains to be fully explored and elucidated.
A previous study found that LAPTM5 was downregulated in most human tumors, including RCC. Notably, the authors compared 15 RCC cell lines with nontumor tissues and found that the mRNA expression of LAPTM5 was downregulated [10]. Tis was an extensive screening through RT-PCR. However, in our study, we found that LAPTM5 was upregulated in ccRCC tumor tissues compared with that in the adjacent healthy tissues using Western blot and RT-PCR, which is consistent with previous bioinformatics analyses of kidney renal clear cell carcinoma (Figure 1(a)). Proliferation, migration, and invasion are key factors in tumor development [26,27]. In our study, we found that LAPTM5 promoted proliferation, migration, and invasion of ccRCC cells in vitro and promoted tumor growth and survival in vivo; however, the underlying mechanisms remain unknown. Numerous pathways are involved in the progression of ccRCC, including MAPK, wingless/integrated, and PI3K/Akt pathways [28][29][30]. JNK, p38, and ERK are important components of the MAPK pathway [31]. In LAPTM5 knockdown macrophages, the activation of tumor necrosis factor receptor-mediated MAPK and NF-κB pathways was reduced, indicating that LAPTM5 is a positive regulator of the infammatory signaling axis in macrophages [21]. In addition, LAPTM5 regulates protein stability. Notably, A20 is a powerful inhibitor of the NF-κB and MAPK signaling cascade, and LAPTM5 directly promotes A20 degradation by binding with it in HeLa cells in the endolysosomal system [21]. Tus, we hypothesized that LAPTM5 regulates ccRCC via MAPK pathways. Our fndings further demonstrate that LAPTM5 promotes p38 and JNK phosphorylation in ccRCC cells and in animal models by directly binding to RAC1. Numerous previous studies have reported that p38 and JNK are involved in ccRCC survival and growth and play a signifcant regulatory role in ccRCC tumorigenesis, metastasis, and angiogenesis [32][33][34], which is consistent with our fndings. Notably, ERK is a key downstream factor of MAPK and is involved in ccRCC progression [35,36]. However, no signifcant changes in ERK expression were observed in the present study. ERK may be regulated by other important molecules, and further investigations are therefore required.   Journal of Oncology 13 RAC1 belongs to the Rho guanosine triphosphatase family and acts as a "molecular switch" in various cell signal transduction processes. In addition, RAC1 is an upstream molecule of MAPK located in lysosomes [37]. When RAC1 is activated, it plays a crucial role in tumorigenesis, growth, and death [38][39][40]. In gliomas, high RAC1 expression promotes tumor migration and invasion by inducing epithelial-mesenchymal transition and increasing matrix metalloproteinase expression [41]. Zhu et al. demonstrated that GINS complex subunit 4 promotes gastric cancer development by regulating RAC1 [39]. RAC1 is overexpressed in ccRCC cells, thereby promoting proliferation, migration, metastasis, and angiogenesis and is required for MAPK pathway activation [42]. In the present study, we hypothesized that LAPTM5 regulates the MAPK pathway by interacting with RAC1. Te results of our study demonstrate an interaction between LAPTM5 and RAC1. Moreover, these results demonstrate that treatment with an RAC1 inhibitor rescued the ccRCC phenotype caused by LAPTM5. Collectively, these fndings indicate that LAPTM5 regulates ccRCC growth by activating the RAC1-JNK/p38 axis. Tis study had some limitations. Notably, LAPTM5 knockdown was not performed in vivo, and the interaction between LAPTM5, RAC1, and MAPK requires further investigation.

Conclusions
In conclusion, the function and molecular mechanisms of LAPMT5 were investigated in ccRCC. Our results demonstrate that LAPTM5 regulates ccRCC cell proliferation, migration, and invasion by interacting with RAC1 and activating downstream JNK/p38 pathways.