Stable silencing of ROR1 regulates cell cycle, apoptosis, and autophagy in a lung adenocarcinoma cell line

Abstract

Lung cancer has the highest mortality and recurrence rate among cancers in the world. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) has been widely recognized for its role in promoting the growth and metastasis of lung cancer, but its comprehensive role and molecular mechanisms in regulating cell cycle, apoptosis, and autophagy remain unclear. In this study, a series of ROR1-stably silenced monoclonal clones from lung adenocarcinoma cell lines PC9, PC9erlo, and NCI-H1975 were successfully selected and confirmed by qRT-PCR, western blot, and flow cytometry, and used as cell models in the following assays. Our study clearly shows that blocking ROR1 significantly downregulates cell cycle-inducing molecules such as CDK4 and Cyclin E1, and anti-apoptotic molecules such as Bcl-XL and Bcl-2, while it markedly upregulates pro-apoptotic molecules such as Bak, Caspase-3, and Caspase-7, which extends our previous observation on the molecular mechanism of ROR1-mediated tumor growth in lung adenocarcinoma. Our data also show that silencing ROR1 promotes autophagy since the key molecules involved in autophagy including ATG7, ATG12, BNIP3L, LC3A, LC3B, and NBS1 were up-regulated. We further screened key phosphokinase signaling pathways downstream of ROR1 in lung adenocarcinoma by a human phospho-kinase array. Our data indicate that blocking ROR1 could deactivate Akt, then activate GSK-3α/β by de-phosphorylation, and finally deactivate mTOR. In this way blocking ROR1 could effectively regulate the cell cycle, apoptosis, and autophagy in lung cancer.

Keywords: ROR1; apoptosis; autophagy; cell cycle; lung adenocarcinoma.

Figure 1

Silencing of ROR1 with lentivirus-mediated shRNA in lung adenocarcinoma cell lines. The infection efficiency was demonstrated by GFP fluorescence intensityunder fluorescence microscope (A) and the relative expression levels of ROR1 were analyzed by flow cytometry (B). Mock, cells cultured with RPMI-1640 culture medium with 10% FBS alone; Lv-shCon, cells infected with lentivirus containing non-related shRNA; Lv-shROR1, cells infected with lentivirus containing ROR1-specific shRNA.

Figure 2

Selection of ROR1 stably-silenced clones from lung adenocarcinoma cell lines by western blot. Clones (From left to right: R1, R2, R3, R4 and R5) were screened from cell lines PC9, PC9erlo, and NCI-H1975 which have been infected with Lv-shROR1 by finite dilution method (A). The integrated density analysis showed the changes in expression (B). Mock, cells cultured with RPMI-1640 culture medium with 10% FBS alone; MN, clones selected from cells infected with Lv-shCon; R, clones selected from cells infected with Lv-shROR1.

Figure 3

Identification of clones with ROR1 stably-silenced by RT-PCR and flow cytometry. The infection efficiency of Lv-shROR1 in selected clones (R3 and R4 from PC9, R1 and R2 from PC9erlo, R3 and R5 from NCI-H1975) was demonstrated by GFP fluorescence intensity under fluorescence microscope (A) and the relative expression levels of ROR1 were analyzed by qRT-PCR (B) and flow cytometry (C), respectively. Asterisks indicate statistically significant differences compared with the non-related shRNA group (*P<0.05). Statistical analyses was performed using one-way ANOVA (LSD method) (R3 and R4 from PC9, R1 and R2 from PC9erlo, R3 and R5 from NCI-H1975 compared with MN). Mock, cells cultured with RPMI-1640 culture medium with 10% FBS alone; MN, clones selected from cells infected with Lv-shCon; R, clones selected from cells infected with Lv-shROR1.

Figure 4

ROR1 regulates cell cycle- and apoptosis-related molecules. The expression levels of ROR1 and cell cycle-related molecules (A) and apoptosis-related molecules (C) and β-actin were detected by western blot. The integrated density analysis showed the changes of expression (B, D). Asterisks indicate statistically significant differences compared with the non-related shRNA group (*P<0.05). Statistical analyses were performed using Student’s T test (R1 compared with MN). Mock, cells cultured with RPMI-1640 culture medium with 10% FBS alone; MN, clone selected from PC9erlo cell lines infected with Lv-shCon; R1, clone 1 selected from PC9erlo cell lines infected with Lv-shROR1.

Figure 5

ROR1-silenced induces autophagy. Twenty autophagy-related proteins were screened in clone R1 from PC9erlo by a commercially available human autophagy array kit (A). The relative expression levels of autophagy-related proteins were graphed (B). MN, clone selected from PC9erlo cell lines infected with Lv-shCon; R1, clone 1 selected from PC9erlo cell lines infected with Lv-shROR1.

Figure 6

Screening ROR1-mediated phospho-kinase signaling pathways. Detecting the relative phosphorylation levels of 43 human protein kinases in clone R1 from PC9erlo cell lines by a human phospho-kinase assay kit (A). The relative expression levels of Akt and GSK-3α/β were graphed (B). The phosphorylation levels of Akt, GSK-3α/β, mTOR and β-actin were detected by western blot (C). MN, clone selected from PC9erlo cell lines infected with Lv-shCon; R1, clone 1 selected from PC9erlo cell lines infected with Lv-shROR1.

Figure 7

Schematic representation of the proposed mechanism of ROR1 in lung adenocarcinoma cell lines.