Rab25 promotes erlotinib resistance by activating the β1 integrin/AKT/β‐catenin pathway in NSCLC

Abstract Objectives Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR‐TKI) has significant therapeutic efficacy in non‐small‐cell lung cancer (NSCLC) patients. However, acquired resistance is inevitable and limits the long‐term efficacy of EGFR‐TKI. Our study aimed to investigate the role of ras‐associated binding protein 25 (Rab25) in mediating EGFR‐TKI resistance in NSCLC. Materials and Methods Rab25 expression in NSCLC patients was measured by immunohistochemical staining. Western blotting was used to analyse the expression of molecules in the Rab25, EGFR and Wnt signalling pathways. Lentiviral vectors were constructed to knock in and knock out Rab25. The biological function of Rab25 was demonstrated by cell‐counting kit‐8 and flow cytometry. The interaction between Rab25 and β1 integrin was confirmed by co‐immunoprecipitation. Results Rab25 overexpression induced erlotinib resistance, whereas Rab25 knockdown reversed this refractoriness in vitro and in vivo. Moreover, Rab25 interacts with β1 integrin and promotes its trafficking to the cytoplasmic membrane. The membrane‐β1 integrin induced protein kinase B (AKT) phosphorylation and subsequently activated the Wnt/β‐catenin signalling pathway, promoting cell proliferation. Furthermore, high Rab25 expression was associated with poor response to EGFR‐TKI treatment in NSCLC patients. Conclusions Rab25 mediates erlotinib resistance by activating the β1 integrin/AKT/β‐catenin signalling pathway. Rab25 may be a predictive biomarker and has potential therapeutic value in NSCLC patients with acquired resistance to EGFR‐TKI.

phase 3 trial, the median progression-free survival (PFS) of advanced NSCLC patients was 11 months in the erlotinib group vs 5.5 months in the chemotherapy (gemcitabine/cisplatin) group. 1 However, almost all patients develop acquired resistance to erlotinib after 6-12 months of treatment. [2][3][4] Although a substantial number acquired resistance mechanisms have been identified, such as the T790M mutation and c-MET amplification, almost 30% of the acquired resistance cases remain unexplainable. 5 Therefore, appropriate biomarkers that correlate with EGFR-TKI acquired resistance are urgently needed for better prognostic and therapeutic strategies.
Ras-associated binding protein 25 (Rab25) is a member of the Rab small GTPase family that regulates intracellular vesicle trafficking. 6 Membrane trafficking is involved in many cellular pathways related to carcinogenesis, such as cell proliferation, invasion, metastasis and polarity loss. 7 Jeong 8 noted that Rab25 upregulates β1 integrin levels and induces snail and fascin expression, leading to epithelial-mesenchymal transition (EMT) in breast and ovarian cancer cells. Furthermore, Rab25 interacted with α5β1 integrin and regulated the trafficking of this molecule, which promoted tumour progression in ovarian cancer cells. 9 In our previous study, Rab25 was a candidate oncogene in NSCLC, and its driving role in EGFR-TKI resistance was investigated.
In our laboratory, we have established erlotinib-resistant cell lines by exposing cells to increasing doses of erlotinib and did not detect the T790M mutation in the clones. 10 The Rab25 protein levels were increased in the erlotinib-resistant cell lines. Furthermore, we observed that Rab25 induced β1 integrin trafficking to the cytoplasmic membrane and stabilized β1 integrin protein to activate the Wnt signalling pathway, ultimately leading to cell survival. Therefore, our study elucidated the mechanism by which Rab25 mediates acquired EGFR-TKI resistance without the T790M mutation in NSCLC.

| Patients and samples
Lung cancer specimens were obtained from Xinqiao Hospital, and the protocol was approved by the Medical Ethics Committee of Xinqiao Hospital. Between May 2015 and March 2017, 37 lung adenocarcinoma patients who had received EGFR-TKI treatment (erlotinib or gefitinib) were enrolled in this retrospective study, and tumour samples were collected. In addition, four tumour samples after acquired resistance to EGFR-TKI were collected. The clinical response was evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria. Patients who had progressive disease (PD) or stable disease (SD) for 9 months or less were regarded as EGFR-TKI poor responders (n = 11). Individuals with a status of complete response (CR), partial response (PR) or SD for more than 9 months were classified as EGFR-TKI good responders (n = 26). Rab25 protein expression was detected by immunochemistry. In addition, a survival analysis assessing the period from diagnosis to death or study completion (May 2018) was performed. Details of immunohistochemical staining of tumour samples are provided in supporting information.

| Western blot and co-immunoprecipitation analyses
Cells were washed twice with phosphate-buffered saline (PBS),

| Statistical analysis
All quantitative data are presented as the means ± SEM, and all data test was used to evaluate the significance of the difference.

| Rab25 is involved in erlotinib resistance
We previously observed that Rab25 expression was higher in NSCLC tissue than in adjacent normal tissue, and high Rab25 expression was significantly correlated to worse prognosis. 11 To further explore with the corresponding parental cells ( Figure 1H). Moreover, Rab25knockdown PC9/ER and HCC827/ER cells ( Figure 1D,E) exhibited increased sensitivity to erlotinib ( Figure 1G) and greater numbers of apoptotic cells ( Figure 1I).

| Wnt signalling is correlated with Rab25mediated erlotinib resistance
In our previous study, the Wnt signalling pathway was observed to be closely associated with lung adenocarcinoma (data not shown).
Rab25 has been reported to activate the Wnt pathway and promote cell proliferation. 12,13 GSK-3β, a crucial molecule in the Wnt pathway, can be phosphorylated by AKT. 14 Interestingly, erlotinib was observed to inhibit p-EGFR and p-ERK expression but not p-AKT or Rab25 expression in the PC9/ER and HCC827/ER cells ( Figure 1A).
Therefore, we speculated that Rab25 was associated with the observed AKT activation and the stimulation of the Wnt pathway.
We observed that Rab25 overexpression induced the expression of crucial molecules in the Wnt pathway (p-GSK-3β and β-catenin) and facilitated erlotinib resistance in the PC9 and HCC827 cells ( Figure 2A). In addition, Rab25 knockdown led to the inhibition of these proteins and erlotinib re-sensitization in the PC9/ER and HCC827/ER cells ( Figure 2B)

| β1 integrin-mediated activation of the Wnt signalling pathway
Rab25 was reported to regulate integrin-recycling vesicle trafficking to the cytoplasmic membrane. As an important member of the integrin family, β1 integrin activates PI3K/AKT and regulatory proteins. 15 Rab25 overexpression induced β1 integrin and p-AKT expression in the PC9 and HCC827 cells ( Figure 3A). In contrast, Rab25 knockdown led to a decrease in these proteins, and the effects were enhanced by erlotinib treatment in the PC9/ ER and HCC827/ER cells ( Figure 3B). In addition, silencing β1 integrin expression reduced Rab25-mediated p-AKT, p-GSK-3β and β-catenin expression, suggesting that β1 integrin is important for Rab25-induced Wnt signalling activation ( Figure 3C). As shown in Figure 3D,E, the results of a co-IP assay confirmed the direct interaction between Rab25 and β1 integrin. Upon examination by confocal microscopy, we observed that Rab25 colocalized with β1 integrin in the cytoplasm of the PC9 and HCC827 parental cells in the presence of erlotinib ( Figure 3F). However, the increased recycling of β1 integrin from the cytosol to the cell surface was observed in the PC9/ER and HCC827/ER cells ( Figure 3G). In combination, these results suggest that the Rab25-mediated regulation of β1 integrin recycling was responsible for activating the Wnt pathway and inducing erlotinib resistance.

| Rab25 induces erlotinib resistance in vivo
To further assess the ability of Rab25 to affect the erlotinib response in vivo, stable Rab25-overexpressing cells (PC9/Rab25 and HCC827/Rab25) were subcutaneously injected into nude mice followed by erlotinib treatment. Compared to the tumours in the control group, those in the PC9/Rab25 and HCC827/Rab25 groups exhibited significantly larger tumour volumes after erlotinib treatment for 14 days (Figure 4A,B). Moreover, mice injected with stable Rab25-depleted cells (PC9/ER/shRab25 and HCC827/ER/shRab25) showed smaller tumour volumes than did the PC9/ER/shctrl-and HCC827/ER/shctrl-injected mice ( Figure 4A,B). Furthermore, the immunohistochemical analysis results showed higher Rab25, β1 integrin, β-catenin and ki-67 expression in tumour tissues from the PC9/Rab25 and HCC827/Rab25 groups than those observed in the PC9/vector and HCC827/vector groups ( Figure 4C,D). Consistent with the tumour volume data, the tumour tissue samples from the PC9/ER/shRab25 and HCC827/ER/shRab25 groups expressed lower Rab25, β1 integrin, β-catenin and ki-67 protein levels than those from the PC9/ER/shctrl and HCC827/ER/shctrl groups ( Figure 4E,F). These results demonstrate that Rab25/β1 integrin/βcatenin signalling is crucial for erlotinib resistance in vitro and in vivo.

| Elevated Rab25 expression is associated with EGFR-TKI resistance
Finally, we examined whether Rab25 expression may be correlated with EGFR-TKI resistance in NSCLC. We retrospectively collected specimens from 37 NSCLC patients who had received EGFR-TKI therapy and detected Rab25 expression by IHC staining. The clinicopathologic features of these patients are summarized in Table 1. Most patients who had a poor response to EGFR-TKI treatment exhibited high Rab25 protein levels ( Figure 5A,B), and this group of patients had shorter PFS and OS than their counterparts with low levels of Rab25 (PFS of 10 months vs 16 months, OS of 28 months vs 49 months, respectively) ( Figure 5D-F). In addition, Rab25 expression was increased in the acquired resistance to erlotinib or gefitinib tumour samples compared to the initial specimens ( Figure 5C). These findings indicate that Rab25 may be involved in EGFR-TKI in NSCLC.

| D ISCUSS I ON
In the last decade, EGFR-TKI has been a milestone therapy in NSCLC patients harbouring EGFR-sensitive mutations. However, acquired resistance over a short or long period of time is inevitable. Although the T790M mutation is the most common cause of resistance (40%-55%), 16 the remaining large proportion of relapsed patients exhibit a variety of other triggers, that is, mutations in MET, KRAS and PIK3CA. 17 Moreover, some unelucidated mechanisms of EGFR-TKI resistance remain unknown.
Rab25, together with Rab11a and Rab11b, belongs to the Rab11 family. Rab25 participates in apical recycling and transcytosis to sustain polarity in epithelial cells. 6 Since the loss of cell polarity is an essential characteristic of cancer, Rab25 has been well studied in cancers. As an oncogene or tumour suppressor, the reported roles of Rab25 are diverse in existing publications. In ovarian cancer, 12 breast cancer 18 and lung cancer, 19 Rab25 is reported to enhance tumour progression. However, Rab25 is regarded as a tumour suppressor in colon cancer. 20 In our previous study, Rab25 protein was high in lung cancer tissue, and it was significantly correlated with clinical progression. 6 Thus, we considered that Rab25 promotes tumour growth in NSCLC. In this study, we treatment. However, β1 integrin silencing reduced the AKT phosphorylation. Even so, the mechanism of β1 integrin-mediated phosphorylation of some kinases is still not fully understood.
The results of a previous study revealed that Rab25 recycles β1 integrin from lysosomal degradation to the plasma membrane, extending the biological role of the recycled protein. 25  In summary, the results of our study indicate that Rab25 facilitates erlotinib resistance by mediating β1 integrin trafficking to the cytoplasmic membrane, inducing AKT phosphorylation and activating the Wnt/β-catenin signalling pathway, resulting in cell F I G U R E 4 Rab25 mediates erlotinib resistance in lung cancer in vivo. (A) Mice were implanted subcutaneously with the indicated cell lines (5 × 10 6 ), and tumours were grown to ~100 mm 3 and then treated with erlotinib for 14 d. Subsequently, the mice were euthanized, and the tumours were dissected. (B) Tumour growth curve representing the mean ± SEM of the tumour volumes of five mice in the indicated xenograft groups. **P < 0.01, one-way ANOVA. (C-F) Immunohistochemical staining assay to confirm the expression of Rab25, β1 integrin and β-catenin in the indicated group of tumour samples. Tumour cell proliferation was measured by Ki-67 staining. Scale bars, 50 μm proliferation. Thus, Rab25 may be an independent predictive biomarker for EGFR-TKI treatment, and the combination of Rab25 and EGFR inhibitors may be a potential therapeutic mode to reverse resistance to EGFR-TKI therapy.

ACK N OWLED G EM ENTS
This work was supported by the National Natural Science Foundation of China (No. 81672841). The authors thank the patients for providing their clinical data.

CO N FLI C T O F I NTE R E S T
All authors declared no conflicts of interest.