TBC1 domain family member 23 interacts with Ras‐related protein Rab‐11A to promote poor prognosis of non‐small‐cell lung cancer via β1‐integrin

Abstract Non‐small‐cell lung cancer (NSCLC) accounts for approximately 80% of lung cancer cases. TBC1D23, a member of the TBC/RABGAP family, is widely expressed in human tissues; however, its role in NSCLC is currently unknown. Immunohistochemical analysis was conducted on 173 paraffin‐embedded lung tissue sections from patients with NSCLC from 2014 to 2018 at the First Affiliated Hospital of China Medical University. MTT, colony formation assay, cell cycle assay, scratch assay, transwell assay, Western blotting and real‐time PCR were employed on multiple NSCLC cell lines modified to knock down or overexpress TBC1D23/RAB11A. Immunoprecipitation, immunoprecipitation‐mass spectrometry, immunofluorescence and flow cytometry were performed to explore the interaction between TBC1D23 and RAB11A and TBC1D23 involvement in the interaction between RAB11A and β1 integrin in the para‐nucleus. TBC1D23 was correlated with tumour size, differentiation degree, metastasis, TNM stage and poor prognosis. TBC1D23 was involved in the interaction between RAB11A and β1 integrin in the para‐nucleus, thus activating the β1 integrin/FAK/ERK signalling pathway to promote NSCLC. Furthermore, TBC1D23 promoted NSCLC progression by inducing cell proliferation, migration and invasion. This study indicated the relationship between TBC1D23 expression and the adverse clinicopathological characteristics of patients with NSCLC, suggesting that TBC1D23 may be an important target for NSCLC treatment.


| INTRODUC TI ON
Lung cancer is a malignant tumour with the highest morbidity and mortality in the world, posing a serious threat to human health and demonstrating an annually increasing trend. 1 According to the 2018 global cancer statistics, lung cancer patients accounted for 11.6% of the total number of malignant tumours and 18.4% of all malignant tumour-related deaths. Among these, NSCLC accounts for approximately 80% of cases. [2][3][4] The five-year survival rate for NSCLC is no more than 15%. 5,6 TBC1D23, a member of the TBC/RABGAP family, is widely expressed in human tissues and cell lines. The TBC/RABGAP family of proteins has a TBC (Tre2-Bub2-Cdc16) domain 7 that regulates RAB GTPase activation (RABGAP). 8 Previous research has demonstrated the important role played by TBC1D23 in intracellular substance transport. 9,10 TBC1D23 also affects neuron growth, brain development and autoimmunity. 11,12 However, unlike other members of the TBC family, the TBC domain of TBC1D23 is catalytically inactive and lacks the catalytic Arg and Gln residues needed for RABGAP function. 13 Marin-Valencia et al.
reported that TBC1D23 had no RABGAP function, but this did not preclude interactions between TBC1D23 and the RAB family. 14 RAB11A, a member of the RAB small GTPase family, is widely expressed in mammals. 15 RAB11A is located in recycling endosomes, the trans-Golgi network and the plasma membrane. 16 It plays an important role in ligand and receptor recycling, cytokinesis, cell secretion and intracellular signal transduction. [17][18][19] Recent studies have demonstrated that RAB11A can affect tumour development through regulation of β1-integrin circulation. 20,21 β1-integrin, a crucial transmembrane receptor, 22 promotes disease development via multiple signalling pathways. 23 The FAK pathway is one of the most important pathways in cancer development. 24 The MAPK pathway is a common downstream signalling pathway of FAK, which is activated in turn by a tertiary kinase cascade. 25,26 The β1-integrin/FAK/ MAPK signalling is an essential mechanism of tumorigenesis and development, involved in inducing epithelial-mesenchymal transition, promoting tumour cell proliferation, inhibiting tumour cell apoptosis and promoting tumour angiogenesis. Therefore, many anti-tumour drugs have been developed to inhibit this pathway, with the goal of delaying tumour progression. 27,28 The current study aimed to identify the relationship between TBC1D23 expression and NSCLC and to determine whether TBC1D23 promotes the proliferation, migration and invasion of NSCLC cells. Furthermore, the interaction between TBC1D23 and RAB11A was investigated to determine its influence on the β1 integrin/FAK/ERK signalling pathway.

| Western blotting
We used lysis buffer and phenylmethylsulfonyl fluoride (PMSF) to lyse cells and tissues and extract total proteins. The total protein was separated by 10% gel SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes

| Immunofluorescence
Cells were cultured in a 24-well plate for 24 h. Cells were then immobilized with 2% paraformaldehyde for 20 min, followed by

| Cell culture and treatment
Human bronchial epithelial (HBE) cells were obtained from ATCC and

| Cell migration and invasion
Scratch assays were performed with cells 48 h after transfection.
Cells were cultured in serum-free medium for 2 h, then a channel was marked in the cells, and the width of the channel was recorded.
The width of the channel was measured after incubation for 24 h and the difference between the widths of the two channels was calculated. Each experiment was repeated in triplicate.

| Co-Immunoprecipitation (Co-IP) and IP-mass spectrometry
Immunoprecipitation was conducted in the same manner as de- Protein Technology for mass spectrometry analysis. Each experiment was repeated in triplicate.

| Statistical Analysis
Chi-square tests were used to compare immunohistochemistry scoring results using SPSS v17.0 software (SPSS Inc.,) for statistical analysis. Two-tailed t tests were used to compare differences between experimental and control groups using ImageJ software (National Institutes of Health, Bethesda) for statistical analysis. p values <0.05 were considered statistically significant.

| TBC1D23 is highly expressed in NSCLC and correlated with poor prognosis
IHC revealed positive staining of tumour nests and negative staining of alveolar and bronchial epithelial cells ( Figure 1A). High TBC1D23 expression was correlated with tumour size (p < 0.001), differentiation degree (p < 0.001), metastasis (p = 0.027) and TNM stage (p < 0.001).
TBC1D23 expression was not correlated with sex (p = 0.237), age (p = 0.920) or tumour type (p = 0.416) (Table S1). According to survival curve analysis of 494 cases with NSCLC in the Human Protein Atlas database, high TBC1D23 expression was correlated with poor prognosis for NSCLC ( Figure 1B), which concurred with our findings.
Next, we analysed 15 pairs of tissue samples (tumour and normal) using Western blotting, revealing higher TBC1D23 expression in tumour tissues than in normal tissues for 12/15 pairs (p = 0.0147) ( Figure 1C). Real-time PCR analysis demonstrated higher TBC1D23 RNA expression in tumour tissues than in normal tissues for 10/15 pairs (p = 0.0195), which suggested that TBC1D23 had a higher transcription level in NSCLC ( Figure 1D). Previous studies reported that TBC1D23 was located in the cytoplasm, especially in organelles such as the Golgi apparatus. 7,9,13 We also detected the localization of TBC1D23. IHC staining indicated that TBC1D23 was located in the cytoplasm ( Figure 1A).
Immunofluorescence analysis of the NSCLC cell lines A549, H1299, SK-MES-1, H460 and H292 confirmed that TBC1D23 was located in the cytoplasm ( Figure 1E).  Figure 2D, Figure S3A). Conversely, the cell proliferation and colony formation abilities of the TBC1D23 overexpression groups were significantly higher than those of the control groups ( Figure 2B,2C). The results of the cell cycle assays revealed that high TBC1D23 expression level promoted the G1/S transition ( Figure 2D). 29,30 All assays were repeated with transfected A549 cells, and the same results were obtained ( Figure 2B-D). Western blot analysis indicated that cyclin B1, cyclin D1, CDK2 and CDK6 were decreased when TBC1D23 was knocked down and increased when TBC1D23 was overexpressed ( Figure 2E). Taken together, the results indicated that TBC1D23 promoted NSCLC proliferation.  Figure 3C, Figure S2). Taken together, the results indicated that TBC1D23 promoted the migration and invasion of NSCLC.

| TBC1D23 promotes the biological behaviour of NSCLC by interacting with RAB11A
Next, we explored the mechanism responsible for NSCLC promotion by TBC1D23. Firstly, we selected the A549 cell line for immunoprecipitation-mass spectrometry analysis (IP-MS) of TBC1D23. We found that TBC1D23 may interact with RAB11A ( Figure 4A, Figure S3B,C). Immunoprecipitation in A549 cells revealed that TBC1D23 interacted with RAB11A ( Figure 4B). Next, immunofluorescence of A549 cells indicated that TBC1D23 and RAB11A co-located in the para-nucleus ( Figure 4C), suggesting that TBC1D23 can interact with RAB11A. H1299 and SK-MES-1 cells were also analysed by IP and immunofluorescence, and the same results were obtained ( Figure 4B,4C).
Does TBC1D23 affect RAB11A expression? RAB11A expression did not change whether TBC1D23 was knocked down or overexpressed in the transfected cells ( Figure 4D), indicating that TBC1D23 did not influence RAB11A expression.
We then explored whether TBC1D23 promoted NSCLC through its interaction with RAB11A. We employed si-RAB11A to knock down RAB11A in TBC1D23-overexpressing A549 cells. The enhancing effect of the TBC1D23 plasmid was restored after RAB11A was These results suggested that TBC1D23 promoted NSCLC by interacting with RAB11A.

| TBC1D23 interacts with RAB11A to activate the β1 integrin/FAK/ERK pathway
Next, we explored how TBC1D23 interacts with RAB11A to promote NSCLC. Western blot analysis revealed that the expression of β1 integrin, P-FAK, P-MEK, and P-ERK was downregulated in TBC1D23 knockdown SK-MES-1 cells compared with the control SK-MES-1 cells. However, expression of P-JNK and P-p38 was unaltered ( Figure 6A). Expression of β1 integrin, P-FAK, P-MEK and P-ERK was upregulated in TBC1D23-overexpressing H1299 cells compared with control H1299 cells. Again, expression of P-JNK and P-p38 was unaltered ( Figure 6A). Corresponding results were obtained with A549 cells ( Figure 6A). Taken together, the results suggested that TBC1D23 promoted NSCLC by activating the β1 integrin/FAK/ERK pathway.
Next, we knocked down RAB11A in TBC1D23-overexpressing A549 cells. Expression of β1 integrin, P-FAK, P-MEK and P-ERK was restored. We obtained the same result in TBC1D23-overexpressing H1299 cells ( Figure 6B). These results indicated that TBC1D23 interacted with RAB11A to activate the β1 integrin/FAK/ERK pathway, which promoted the biological behaviour of NSCLC.
Finally, we constructed a TBC1 domain-deleted splice variant (△TBC1) ( Figure S1A). Firstly, we transfected the wild plasmid of TBC1D23 (WT) and △TBC1 into A549 cells for immunoprecipitation analysis and found that without the △TBC1 domain, TBC1D23 could not interact with Rab11A ( Figure S1B). Then, we transfected

| TBC1D23 is involved in the interaction between RAB11A and β1 integrin in the para-nucleus
We further explored the mechanism underlying promotion of NSCLC by TBC1D23. Results of real-time PCR indicated that β1 integrin mRNA expression was unaltered whether TBC1D23 was knocked down or overexpressed ( Figure 6C), indicating that TBC1D23 affected β1 integrin at the post-transcriptional stage.
According to Nader et al., the microtubule regrowth system after NZ washout can be used to synchronize disassembly of focal adhesions, thus synchronizing β1 integrin to initiate internal circulation. 22 β1-Integrin reportedly combines with RAB11A in the para-nucleus endocytic recycling compartment (PERC) 60 min after NZ washout. 21 In the current study, IP demonstrated that RAB11A could interact with β1 integrin ( Figure S2A).
We validated the method by Nader et al. in A549 cells without transfection and found that maximum β1 integrin content was recovered from the cell membrane 90 min after NZ washout ( Figure 6D).
Flow cytometry analysis revealed that the β1 integrin content recycled to the cell membrane was significantly lower in the knockdown groups than in the control groups 90 min after NZ washout ( Figure 6E).
Immunofluorescence analysis then revealed that β1 integrin and RAB11A did not co-localize in the para-nucleus in A549 cells with TBC1D23 knockdown 60 min after NZ washout. The same results were obtained for TBC1D23 knockdown SK-MES-1 cells ( Figure 7A).
Next, we overexpressed TBC1D23 in cells with TBC1D23 knockdown and found that RAB11A and β1 integrin were co-localized in the para-nucleus at 60 min ( Figure 7B). This indicated that TBC1D23 was involved in the interaction between RAB11A and β1 integrin in the para-nucleus.
Finally, IHC staining of 28 tissue pairs from NSCLC patients indicated that high β1 integrin expression was observed in tissues with high TBC1D23 expression, and the reverse was also true ( Figure 7C).

| DISCUSS ION
The current study was based on lung tissue samples from 173 patients with NSCLC. TBC1D23 expression in carcinoma nests was significantly higher than that in normal tissues and was associated with poor prognosis. TBC1D23 was shown to promote NSCLC pro- Moreover, β1-integrin binds to ligands on the cell membrane and enters cells through endocytosis. 21 After screening early endosomes, some β1-integrin undergoes lysosomal degradation by late endosomes. 31,32 When separated from the ligand, β1-integrin can be recycled in two ways. One is a short circulation pathway mediated by RAB4, in which β1 integrin returns directly from the early endosome to the plasma membrane, and the other is a long recycling pathway mediated by RAB11A, in which β1 integrin is returned to the cell membrane through PERC with the assistance of ARF6. 33 When more β1 integrin is recycled to the cell membrane through PERC, less β1-integrin is degraded and the overall amount of β1 integrin increases. Therefore, TBC1D23 may form a complex with RAB11A in the PERC. After dissociation from the ligand, β1-integrin is transported to the PERC. Under joint action of the TBC1D23-RAB11A complex, β1-integrin returns to the cell membrane through the intracellular long circulation pathway to complete the recycling process. In turn, more β1-integrin activates the FAK/ERK pathway to promote the progression of NSCLC. In this process, both TBC1D23 and RAB11A are indispensable.
Moreover, β1 integrin was also present in our IP-MS results ( Figure S2B), suggesting that TBC1D23 may also interact with β1 integrin. If this is the case, TBC1D23 most likely recruits β1 integrin to the RAB11A-mediated PERC, connecting RAB11A to β1 integrin and thus mediating circulation of β1 integrin. The decreased amount of β1 integrin that eventually returns to the cell membrane is due to decreased β1 integrin content in the PERC. Therefore, we plan to use isotopes to label TBC1D23, RAB11A and β1-integrin in further experimental studies to observe their real-time intracellular localization.
Our study had some limitations. We did not construct a mutation in TBC1D23 nor detect which fragment of TBC1D23 interacted with RAB11A. These provide avenues for future studies. And our study provides insight into the relationship between TBC1D23 expression and adverse clinicopathological characteristics of patients with NSCLC, suggesting that TBC1D23 is an important target for treatment.

ACK N OWLED G EM ENTS
This work was supported by the Liaoning Province Colleges and Universities Innovation Team (LC2015029), Liaoning Provincial Education Department. We would like to thank Editage for Englishlanguage editing.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interests. Funding acquisition (lead); Project administration (lead).

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this published article. Further data are available from the corresponding author upon request.