Down-regulation of RCC1 sensitizes immunotherapy by up-regulating PD-L1 via p27kip1 /CDK4 axis in non-small cell lung cancer

Abstract

In recent years, although Immune Checkpoint Inhibitors (ICIs) significantly improves survival both in local advanced stage and advanced stage of non-small cell lung cancer (NSCLC), the objective response rate of ICI monotherapy is still only about 20%. Thus, to identify the mechanisms of ICI resistance is critical to increase the efficacy of ICI treatments. By bioinformatics analysis, we found that the expression of regulator of chromosome condensation 1 (RCC1) in lung adenocarcinoma was significantly higher than that in normal lung tissue in TCGA and Oncomine databases. The survival analysis showed that high expression RCC1 was associated with the poor prognosis of NSCLC. And the expression of RCC1 was inversely related to the number of immune cell infiltration. In vitro, knockdown of RCC1 not only significantly inhibited the proliferation of lung adenocarcinoma cells but also increased the expression levels of p27^kip1 and PD-L1, and decreased the expression level of CDK4 and p-Rb. In vivo, knockdown of RCC1 significantly slowed down the growth rate of tumour, and further reduced the volume and weight of tumour model after treated by PD-L1 monoclonal antibody. Therefore, RCC1 could up-regulate the expression level of PD-L1 by regulating p27^kip1 /CDK4 pathway and decrease the resistance to ICIs. And this study might provide a new way to increase the efficacy of PD-L1 monoclonal antibody by inhibiting RCC1.

Keywords: ICI; PD-L1; RCC1; non-small cell lung cancer; p27KIP1/CDK4.

FIGURE 1

The expression level of RCC1 in lung cancer and normal tissues. (A) The expression level of RCC1 was found in TCGA database. (B) The expression of RCC1 in lung cancer and normal lung tissues was analyzed by Oncomine database. (C) The Human Protein Atlas database was used to detect the immunohistochemical staining of RCC1 in lung cancer tissues and normal lung tissues. Data are shown as the mean ± SD. *P < .05, **P < .01 and ***P < .001

FIGURE 2

The comparision of the prognostic information between high and low expression of RCC1 from three survival databases. Figure A‐F were from the KM‐plotter database. Figure G‐J were from the Prognoscan database. Figure K was from the TISIDB database.

FIGURE 3

Effect of RCC1 expression on the function of lung adenocarcinoma cells. (A and B) Knockdown of RCC1 expression hindered A549 and H1299 cells growth. (C and D) A549 and H1299 cells were used for flow cytometry analysis assay to measure cell apoptosis. (E and F) A549 and H1299 cells were used for flow cytometry analysis assay to measure cell cycle. Data are shown as the mean ± SD. *P < .05, **P < .01 and ***P < .001

FIGURE 4

The high expression level of RCC1 was related to the decrease of immune cell infiltration in TISIDB

FIGURE 5

The expression levels of p27^kip1/CDK4/p‐Rb/PD‐L1 downstreams of RCC1. (A, C and E) The mRNA expression levels of p27^kip1/CDK4/PD‐L1 in A549 cells detected by qPCR. (B, D and F) The mRNA expression levels of p27^kip1/CDK4/PD‐L1 in H1299 cells detected by qPCR. (G and H) The protein expression levels of p27^kip1/CDK4/p‐Rb/PD‐L1 in A549 cells detected by Western blot. (I and J) The protein expression levels of p27^kip1/CDK4/p‐Rb/PD‐L1 in H1299 cells detected by Western blot. Data are shown as the mean ± SD. *P < .05, **P < .01 and ***P < .001

FIGURE 6

RCC1‐shRNA plus PD‐L1 monoclonal antibody suppressed tumor growth in vivo. (A, B and C) Established a stable RCC1‐shRNA knockdown transfectant‐ RCC1‐shRNA‐Lewis and the negative control line detected by qPCR, Western blot and Fluorescence. (D and E) Image of the tumors before and after we resected. (F and G) Growth curve of tumors volume and tumors weight in 4 groups. (H) Representative images of immunohistochemistry (IHC) staining and stained for RCC1, p27^kip1, CDK4 and PD‐L1. Data are shown as the mean ± SD. *P < .05, **P < .01 and ***P < .001

FIGURE 7

A summary which indicated by in vitro and in vivo results in this study