The pathological significance of abnormal RON and PD-L1 expression in colorectal cancer


 Background: PD-L1 immunotherapy remains poorly efficacious in colorectal cancer. The RON receptor tyrosine kinase plays an important role in regulating tumor immunity. Here, we identify patterns of RON and PD-L1 expression and explore the clinical significance of these patterns in colorectal cancer.
Methods: Gene expression data were obtained from the Gene Expression Omnibus database (GEO; n = 290) and patients at the First Affiliated Hospital, Zhejiang University School of Medicine (FAHZUSM; n = 381) and were analyzed to determine the prognostic value of RON and PD-L1 expression within the tumor cells and the tumor microenvironment of colorectal cancer. Human colorectal cancer cell lines were treated with BMS-777607 to explore the relationship between RON activity and PD-L1 protein expression. Signaling pathways and protein expression perturbed by RON inhibition were evaluated by cellular immunofluorescence and western blot.
Results: In the GEO cohort, cut-off values for RON and PD-L1 expression of 7.70 and 4.30, respectively, were determined. Stratification of patients based on these cutoffs demonstrated that high expression of RON and PD-L1 associated with poor prognosis. In the FAHZUSM cohort, rates of high expression of RON in tumor cells, high PD-L1 expression in tumor cells and in tumor infiltrating monocytes, and both high RON and high PD-L1 expression in the tumor microenvironment were 121 (32%), 43 (11%), 91 (24%), and 51 (13.4%), respectively. High expression of RON and high expression of PD-L1 in the tumor cell compartment was significantly correlated (p < 0.001). High expression of RON and PD-L1 were independent prognostic factors for poorer overall survival. Concurrent high expression of both RON and PD-L1 in the tumor microenvironment was significantly associated with poor prognosis. In vitro , BMS-777607 inhibited the phosphorylation of RON, inhibited PD-L1 expression, and attenuated activation of the ERK1/2 and AKT signaling pathways in colorectal cancer cells.
Conclusions: RON, PD-L1 and the crosstalk between these proteins plays an important role in predicting the prognostic value of colorectal cancer. Moreover, phosphorylation of RON upregulates the expression of PD-L1, which provides a novel approach to immunotherapy in colorectal cancer.

Overall survival (OS) was evaluated using the Kaplan-Meier method, and survival 251 differences between groups were assessed using the log-rank test. Univariate and 252 multivariate Cox regression analysis was used to calculate the relationships between 253 clinical statistics and the overall survival (OS) of patients. A p-value of p < 0.05 was 254 considered to be statistically significant. All confidence intervals (CIs) are stated at the 255 95 % confidence level. 256 257

Patient Demographics 259
In the GEO cohort, the median age of all 287 CRC patients was 70 years, ranging from 260 24 to 97 years. There were 158 male patients (55.1%) and 129 female patients (44.9%).

Expression patterns of RON and PD-L1 in the GEO cohort 269
In the GEO cohort, the cut-off values for RON and PD-L1 (TC) mRNA in the tumor tissue 270 were determined by the X-tile program to be 7.70 and 4.30, respectively (Figure 1a-b), 271 and the χ2 log-rank value for RON and PD-L1 were 4.544 and 4.078, respectively. 272 Patients in the GEO cohort were divided into two groups according to RON and PD-L1 status: RON ≤ 7.70 and RON >7.70, and PD-L1 ≤4.30 and PD-L1 >4.30 (Figure 1a-b). 274

Expression of RON and PD-L1 in CRC tumor tissue 275
In colorectal cancer tissue samples, multiple immunofluorescence staining was used to

FAHZUSM cohort 291
In the FAHZUSM cohort, CRC patients were divided into two groups based on high or 292 low expression of RON and PD-L1 based on IHC staining. In 381 CRC patients, high and 293 low RON expression in tumor cells (TC) were 121 (31.8%) and 260 (68.2%), respectively. 294 High and low expression of PD-L1 in TC and TIMC were 43 (11.3%) and 91 (Table 2A). High expression of RON and PD-L1 (TIMC) was associated with T 309 stage, principal diagnosis, histological type, and treatment (p < 0.05; Table 2B). 310

Prognostic significance of RON and PD-L1 expression in CRC 311
In the GEO cohort, Univariate Cox regression modeling showed that age, T stage, M 312 stage, disease stage, RON, and PD-L1 (TC) expression were significantly correlated 313 with poor prognosis of CRC patients (p < 0.05). Multivariate analysis after adjustment 314 showed that only age, T stage, and M stage were independent prognostic factors for OS 315 in CRC patients (p < 0.05); disease stage, RON expression, and PD-L1 expression were 316 not significant factors in the multivariate analysis (p > 0.05; Table 3). Kaplan-Meier analysis showed that high expression of RON, PD-L1 (TC), and both high RON and 318 PD-L1 were all associated with poor OS (P < 0.05; Figure 1a-c). High expression of RON 319 was associated with patient age and treatment type, and high expression of PD-L1 (TC) 320 was associated with the primary site of tumor (P < 0.05; Table 1). 321 In the FAHZUSM cohort, Kaplan-Meier analysis showed that high expression of RON in 322 TC, high PD-L1 in TC, or high PD-L1 in TIMC in colorectal cancer tissue samples was 323 correlated with lower overall survival (OS) (p < 0.05) (Figure 4a- Table 3). We further analyzed the relationship between OS and 330 RON and PD-L1 expression in the tumor microenvironment. High expression of both 331 RON and PD-L1 in TC predicted a significantly worse overall survival, and high RON in 332 TC and high PD-L1 in TIMC was associated with significantly worse prognosis (p < 0.001; 333

Effects of regulating RON phosphorylation on PD-L1 expression 335
In order to study the relationship between RON and PD-L1 in CRC cells, HT29 cells 336