PSCC is more common in developing countries, such as China, than in the United States and Europe. Chinese PSCC patients account for 1/3 of new cases worldwide and PSCC is substantial health concern due to its mental and physical effects [29, 30]. Although the TNM staging system, especially the pT stage, is a convenient and effective clinical prognostic tool for evaluating survival, the ability to perform precise and individualized assessment with this approach is limited by tumor heterogeneity [7, 8, 31]. Recently, several sequencing studies in Europe and the Americas have revealed the clinically relevant genomic alterations in PSCC and provided a better understanding of tumor progression and targets for therapy [7–11]. However, potential prognostic biomarkers and specific mechanisms of action remain unknown due to the lack of experimental data and large-cohort clinical validation; in particular, analysis of the genomic expression pattern in Chinese PSCC patients had not been previously reported [7–11]. Therefore, for the first time, we performed whole-transcriptome microarray profiling of paired PSCC tissues to explore the genomic landscape in Chinese patients and to investigate the potential biomarkers and mechanisms of PSCC.
In this study, we found that the expression of RAB20, a small GTPase family member located on chromosome 13q34 , was upregulated in PSCC matched tumor tissues, especially in metastatic lymph nodes. qPCR, WB and IHC further confirmed that RAB20 was overexpressed in the cytoplasm of five PSCC cell lines and 78 PSCC tumor tissues compared with that in the corresponding normal controls. The results highlight that RAB20 might be a crucial oncogene participating in the development and progression of PSCC, although the roles of RAB20 in PSCC have not yet been reported.
Recent studies have revealed that RAB20, which plays a role in the control of endocytotic vesicle transport, is involved in the progression of multiple cancers [15–17, 33]. Amillet et al.  first identified that the overexpression of RAB20 in pancreatic intraductal neoplasia lesions is an early event in the course of pancreatic cancer progression . Habermann et al.  demonstrated that RAB20 overexpression and amplification indicated genomic instability in colorectal adenomas and was correlated with high-grade histopathological features and tumor recurrence . In addition, the overexpression of RAB20 in triple-negative breast cancer has been associated with advanced disease stage and poor patient prognosis .
To further explore the clinical significance of RAB20 expression in PSCC, correlation and survival analyses were conducted on 259 PSCC patients, the largest cohort reported to date, with a median follow-up time of over six years. The overexpression of RAB20 in PSCC was positively associated with advanced clinicopathological features and a shorter 5-year CSS time. Moreover, RAB20 was found to be a strong independent prognostic indicator of poor clinical outcomes, identifying PSCC patients with an increased risk for tumor progression and a shorter survival time. These findings underscore the clinical significance of RAB20 in PSCC and imply that RAB20 plays oncogenic roles in tumor progression.
A recent study on neuronal network formation showed that RAB20, as a novel regulator, participated in neurite outgrowth and cell proliferation . Liu et al.  found that the restoration of RAB20 expression in hepatocellular carcinoma cells inhibited cell growth, motility and metastasis. To further investigate the oncogenic functions of RAB20 in regulating the malignant phenotype, validation experiments were conducted in vitro and in vivo. We observed that knockdown of RAB20 in our newly established PSCC cell lines repressed colony formation, cell proliferation, and migration along with repressing tumor growth in xenografted nude mice. Interestingly, GSEA showed that RAB20-silenced Penl2 cells were not only enriched in cell proliferation pathways but also in cell cycle checkpoint pathways. Habermann et al.  reported that RAB20 amplification triggered EGFR recycling and promoted cell proliferation by increasing the formation of cyclin A-CDK2 complexes in the S/G2 cell cycle phases . Therefore, we explored the cell cycle distribution by flow cytometry, and the results indicated that RAB20-silenced PSCC cells exhibited G2/M phase cell cycle arrest but unaffected G1/S transition. Similarly, knockdown of RAB20 did not inhibit the expression of the CDK2-cyclinE1 complex, the key effector in the G1/S phase, but significantly suppressed cdc2-cyclinB1 levels at the G2/M transition . Overall, we found that RAB20-mediated cancer progression could be tumor-type specific and promotes PSCC proliferation by regulating G2/M cell cycle checkpoints.
Cdc25c phosphatase promotes the mitotic cell G2/M transition by triggering cdc2 dephosphorylation to activate the cdc2-cyclinB1 complex [26, 28]. The activation of cdc25c requires phosphorylation within the N-terminal domain at Thr48, Thr67, Ser122, Thr130 or Ser216 sites, which is regulated by the checkpoint protein kinases Chk1 and Chk2 and p53 pathways . We found that knockdown of RAB20 did not alter the phosphorylation of p53 or the transcriptional activation of p21 (a downstream protein in the p53 pathway) . However, in shRAB20 PSCC cells, the Chk1-mediated phosphorylation of cdc25c at Ser216 was increased and the cdc2-cyclinB1 complex was inhibited. Subsequently, we measured the cell cycle stage distribution and protein expression when Chk1 was repressed in shRAB20 PSCC cells. We found a remarkable decrease in the proportion of cells in G2, and the expression levels of the cdc2-cyclinB1 complex and p-cdc25c (Ser216) were restored. The results further demonstrate that RAB20 induces G2/M phase cell arrest via the Chk1/cdc25c pathway in PSCC.
Our study also has some limitations. We focused on the oncogenes that were upregulated in the comprehensive genomic sequencing and did not study the potential tumor suppressor genes that might promote the progression of PSCC. Second, the detailed mechanisms by which RAB20 regulates Chk1 in G2/M cell cycle arrest should be further investigated. Third, more efforts at multiple centers are required to determine the potential value of RAB20 as a biomarker for PSCC.