Expression of Rab1A is upregulated in human lung cancer and associated with tumor size and T stage

Rab1A is highly expressed in lung cancer tissues

Immunohistochemical staining was performed to evaluate Rab1A expression in 60 lung cancer tissues and paired adjacent noncancerous tissues. Positive staining appeared brown. Rab1A was predominantly expressed in the cytoplasm, and the percentage of nuclear expression is very low (less than 5%) in both noncancerous tissues and cancerous tissues. In contrast to the noncancerous tissues, Rab1A was significantly highly expressed in tumor cells (Figure 1A), while faint staining was present in stromal and lymphoid cells in paired noncancerous tissues. When using the median H score of 67.5 as a cutoff value, the overall rate of positive staining in lung cancer tissues (95%) was significantly higher than in paired noncancerous tissues (5%; P = 0.000; Table 1). Compared with matched noncancerous tissues (median H score = 15), Rab1A staining was much stronger in lung cancer tissues (median H score = 189; Figure 1B). On an individual basis, Rab1A scored higher in approximately 96.67% (58/60) of tumors than in the corresponding noncancerous tissues, although Rab1A staining was remarkably variable, differing by as much as 100-fold between different tumor samples (Figure 1C). For different histological subtypes of lung cancer, Rab1A showed similar expression patterns (Figure 1D) with a 100% positive staining rate in adenocarcinoma, adenosquamous carcinoma and large cell lung cancer tissues (Table 2). The H score in the adenocarcinoma group (median, 211.5) was higher than others histological types while the small cell lung cancer cohort showed the lowest H score (median, 121.5; Figure 1E).

Rab1A expression is correlated with clinical parameters

To further delineate the clinical significance of Rab1A expression, we examined the relationship between Rab1A staining and widely recognized clinicopathologic factors. Cases were divided into high and low Rab1A staining groups based on the median H score of 180 in cancer tissues. The Kruskal-Wallis test showed that Rab1A staining is significantly correlated with tumor size (P = 0.002), and T stage (P = 0.039), as shown in Table 3. Large tumors and late T stage tumors consistently displayed higher Rab1A expression when compared with small tumor size and early T stage, indicating that Rab1A may promote tumor growth and progression. However, there were no significant associations observed between Rab1A expression and age, gender, lymph node status and AJCC stage.

Rab1A is highly expressed in lung cancer cell lines but not correlated with expression of key signaling pathways

We further examined Rab1A expression in a panel of lung cancer cell lines. Compared with the immortalized lung/bronchus epithelial cell line BEAS-2B, Rab1A is highly expressed in all lung cancer cell lines with great variability, ranging from a 6-fold increase in the A549 and H1650 cell lines to a 1.5-fold increase in H460 and SK-LU-1 cells lines (Figure 2A and B). These findings are consistent with Rab1A expression levels in lung cancer tissues. Because Rab1A has been reported to be correlated with hyperactive mTORC1 signaling in human colorectal cancer [8], we examined whether Rab1A expression was correlated with mTORC1 signaling in lung cancer. However, no significant correlation between Rab1A and P-S6K1 (T389) was observed in lung cancer cell line panels. We further examined substrates of other key cancer cell signaling pathways, including P-AKT (S473), P-ERK (T202/Y204), P-C-JUN (S63) and P-MK2 (T334), but no correlation between expression of these proteins and Rab1A expression was observed (Figure 2C-G), indicating that, under physiological conditions, Rab1A expression is not correlated with the basal expression levels of the mTORC1, mTORC2, MEK, JNK and p38MAPK signaling pathways in lung cancer.

Rab1A knockdown has no effect on mTOR signaling or lung cancer cell growth

It has been reported that Rab1A knockdown dramatically attenuated mTORC1 signaling and cell growth in CRC and HCC. Therefore, we examined whether Rab1A knockdown had similar effects in human lung cancers. Although Rab1A was successfully knocked down in a panel of lung cancer cell lines, no changes were observed with regards to mTORC1 (P-S6K1(T389)) or mTORC2 (P-AKT (S473)) signaling (Figure 3A). Consistent with these findings, cell growth was not affected by Rab1A knockdown in an MTT assay (Figure 3B).

Tissue array and immunohistochemistry

Sixty de-identified lung cancer tissues with paired adjacent noncancerous tissues were collected from the hospital between January 2007 and March 2008 and developed into tissue arrays by Shanghai OUTDO Biotech (Shanghai, China). Twenty adenocarcinomas, 10 squamous carcinomas, 10 adenosquamous carcinomas, 10 large cell carcinomas and 10 small cell carcinomas were included. The study protocols were approved by the SJTUSM (Shanghai Jiao Tong University School of Medicine) Ethics Committee. All procedures adhere to the BRISQ Guidelines for reporting research on human biospecimens.

Immunohistochemical detection of Rab1A was performed using a streptavidin-biotin complex method as described previously [8]. The primary antibody against Rab1A (Proteintech Group) was used at a concentration of 1:100. To score a tumor cell as positive, cytoplasmic or nuclear staining was performed. For quantitative analysis, a Histo (H) score was calculated based on the staining intensity and percentage of stained cells using Aperio ScanScope systems (Vista, CA, USA). The intensity score was defined as follows: 0, no appreciable staining in cells; 1, weak staining in cells comparable with stromal cells; 2, intermediate staining; 3, strong staining. The fraction of positive cells was scored as 0%-100%. The H score was calculated as previously described, by multiplying the intensity score and the fraction score [8], producing a total range of 0-300. Based on the distribution of the data, a cutoff of 67.5 was used for Rab1A positivity. Tissue sections were examined and scored separately by two independent investigators blinded to the clinicopathologic data.

Cell lines and culture conditions

Immortalized bronchial epithelial cells (BEAS-2B) and nine human lung cancer cell lines (A549, H1299, H1650, H1975, H2030, H292, H460, H838, SK-LU-1) were purchased from ATCC (American type culture collection). All cell lines were confirmed to be free of mycoplasma contamination. BEAS-2B and H292 cells were cultured in Dulbecco’s Modified Eagle Medium with 10% fetal bovine serum (FBS). A549, H1299, H1650, H1975, H2030, H460, H838 and SK-LU-1 cells were cultured in RPMI 1640 medium with 10% FBS. All cell lines were maintained at 37°С in a 5% CO₂ incubator. Cell lines were immediately expanded upon receipt and aliquots frozen to allow the cell lines to be restarted every three to four months from the same batch of cells. Cell phenotypes were verified in every experiment.

siRNA transfection

Small interfering RNAs (siRNAs) targeting human Rab1A was synthesized by GenePharma (Shanghai, China) and have been validated in our previous studies [8]. Sequences: Rab1A siRNA, AAU AAC UGG AGG UGA UUG UUC, Negative control siRNA: UUC UCC GAA CGU GUC ACG UTT. Transfections were performed on a panel of lung cancer cell lines. Cells were incubated with siRNA oligonucleotides and Lipofectamine 3000 (ThermoFisher, Pittsburgh, PA) transfection reagent for 12 h. The cell culture medium was then replenished with fresh culture medium and the cells were analyzed.

MTT assay

To determine the effects of Rab1A knockdown, cell growth was analyzed using an MTT assay as previously described [20]. Cells were harvested 12 h post-transfection and re-seeded in 96-well plates at an initial density of 3 × 10³ cells/well. Assays were performed at 48 h post-seeding according to the manufacturer’s protocols. Data represent the mean ± SD (absorbance value at 490 nm) from three independent triplicate experiments.

Antibodies and immunoblotting

Cells were harvested 48 h post-transfection and subjected to immunoblotting as described previously [8]. Reagents were obtained from the following sources: antibody for Rab1A, Proteintech Group (Rosemont, IL); antibodies for P-S6K1(T398), S6K1, P-AKT(S473), AKT, P-ERK(T202/Y204), ERK, P-MK2(T334), MK2 and Tubulin, Cell Signaling Technology (Danvers, MA). HRP-labeled GAPDH, anti-mouse and anti-rabbit antibodies were purchased from Santa-Cruz Biotechnology (Dallas, Texas). The EDTA-free Complete Protease Inhibitor Cocktail and PhosSTOP were obtained from Roche (Basel, Switzerland). All primary antibodies were confirmed to be reactive only to the targets by the manufacturer and used at 1:1000, and secondary antibodies were used at 1:10000. The data represent at least three independent experiments.

Statistical analysis

Statistical analyses were carried out using SPSS 20.0 software. The statistical analysis of numeration data was performed using Pearson’s chi-square test or Fisher’s exact chi-square test. The comparisons of continuous data between groups were performed using the Kruskal-Wallis test. A nonparametric Spearman’s correlation test was performed to analyze the correlation between Rab1A and P-S6K1, P-AKT1, P-ERK, P-MK2 and P-c-Jun expression levels. All statistical tests were conducted at a two-sided significance level of 0.05.