MUC1-C promotes the suppressive immune microenvironment in non-small cell lung cancer

Abstract

The cancer immune microenvironment is of importance for the effectiveness of immunotherapy; however, its dysregulation is poorly understood. The MUC1-C oncoprotein is aberrantly overexpressed in non-small cell lung cancer (NSCLC) and has been linked to the induction of PD-L1. The present work investigated the effects of targeting MUC1-C in an immuno-competent MUC1 transgenic (MUC1.Tg) mouse model. We show that Lewis Lung Carcinoma cells expressing MUC1-C (LLC/MUC1) exhibit upregulation of PD-L1 and suppression of interferon-γ (IFN-γ). In studies of LLC/MUC1 cells growing in vitro and as tumors in MUC1.Tg mice, treatment with the MUC1-C inhibitor, GO-203, was associated with the downregulation of PD-L1 and induction of IFN-γ. The results further demonstrate that targeting MUC1-C results in enhanced effector function of CD8+ tumor-infiltrating lymphocytes (TILs) as evidenced by increased expression of the activation marker CD69, the degranulation marker CD107α, and granzyme B. Notably, targeting MUC1-C was also associated with marked increases in TIL-mediated killing of LLC/MUC1 cells. Analysis of gene expression data sets further showed that overexpression of MUC1 in NSCLCs correlates negatively with CD8, IFNG and GZMB, and that decreases in CD8 and IFNG are associated with poor clinical outcomes. These findings in LLC/MUC1 tumors and in NSCLCs indicate that MUC1-C→PD-L1 signaling promotes the suppression of CD8+ T-cell activation and that MUC1-C is a potential target for reprogramming of the tumor microenvironment.

Keywords: CD8+ T-cells; IFN-γ; MUC1-C; NSCLC; PD-L1.

Figure 1.

MUC1-C regulates PD-L1 and IFN-γ expression in LLC NSCLC cells. (A) Lewis Lung Carcinoma (LLC) NSCLC cells stably expressing a control empty vector (LLC/Vector) or full length MUC1 (LLC/MUC1) were analyzed for MUC1, PD-L1 and IFN-γ mRNA levels by qRT-PCR. The results (mean ± SEM of 3 biologic replicates each performed in triplicate) are expressed as relative mRNA levels as compared with that obtained for the vector cells (assigned a value of 1). (B) Lysates from LLC/Vector and LLC/MUC1 cells were immunoblotted with the indicated antibodies. (C) LLC cells expressing a control empty vector (LLC/Vector) or MUC1-C (LLC/MUC1-C) were analyzed for MUC1, PD-L1 and IFN-γ mRNA levels by qRT-PCR. The results (mean ± SEM of 3 biologic replicates each performed in triplicate) are expressed as relative mRNA levels as compared with that obtained for the vector cells (assigned a value of 1). (D) LLC/MUC1 cells were treated with empty NPs or 2.5 µM GO-203/NPs for 72 h. Cells were analyzed for MUC1, PD-L1 and IFN-γ mRNA levels by qRT-PCR. The results (mean ± SEM of 3 biologic replicates each performed in triplicate) are expressed as relative mRNA levels as compared with that obtained for the NP-treated cells (assigned a value of 1). (E) Lysates from the designated LLC/MUC1 cells were immunoblotted with the indicated antibodies.

Figure 2.

Targeting MUC1-C activates the LLC tumor immune microenvironment in a MUC1.Tg mouse model. (A) Mice bearing established LLC/MUC1 tumor xenografts (∼150 mm³) were treated weekly with intraperitoneal injections of empty NPs (squares) or 15 mg/kg GO-203/NPs (triangles). The results are expressed as tumor volume (mean ± SEM, 6 mice per group). * denotes p < 0.05. (B) Tumors harvested from empty NP- and GO-203/NP-treated mice (day 10) were analyzed for MUC1, PD-L1 and IFN-γ mRNA levels by qRT-PCR. The results (mean ± SEM of 3 biologic replicates each performed in triplicate) are expressed as relative mRNA levels as compared with that obtained for the control NP-treated mice (assigned a value of 1). (C) Tumors obtained on day 10 were immunoblotted with the indicated antibodies. (D-F) Single cell suspensions were generated from the LLC/MUC1 tumor tissues and subjected to FACS analysis. (D) In a representative histogram, tumor cells from NP-treated (profile #1) and GO-203/NP-treated (profile #2) mice were analyzed for PD-L1 expression (left). An isotype identical antibody was used as an internal control (profile #3) (left). The percentage of PD-L1-positive tumor cells is expressed as the mean ± SEM for 5 tumors per group(right). (E) Expression levels of Ki67 on T-cells relative to PD-L1 on tumor cells. (F) Tumor-infiltrating CD8+ cells were analyzed for CD69 expression. The results are expressed as the percentage (mean ± SEM for 5 tumors per group) of CD69 positive cells.

Figure 3.

Functional evaluation of TILs from LLC/MUC1 tumors. (A-E) Immune cells were isolated from LLC/MUC1 tumors and then stimulated ex vivo for 6 h. (A) The CD45+CD3+ tumor-infiltating population was analyzed for CD8+ T-cells and CD4+Foxp3+ Tregs. The results (mean ± SD for 4 tumors per group) are expressed as the percentage CD4+Foxp3+ cells (left) and the CD8+/CD4+Foxp3+ ratio (right). (B) Representative histogram depicting IFN-γ production by CD8+ T-cells from NP-treated (profile #3) and GO-203/NP-treated (profile #2) LLC/MUC1 tumors (left). An isotype identical antibody was used as an internal control (profile #1) (left). The results are expressed as the percentage (mean ± SEM for 5 tumors per group) of IFN-γ+ cells (right). (C) Representative histogram showing CD107α expression by CD8+ T-cells from NP-treated (profile #3) and GO-203/NP-treated (profile #2) LLC/MUC1 tumors (left). The results are expressed as the mean fluorescent intensity (MFI; mean ± SEM of 5 tumors per group)(right). (D) CD8+ T-cells were analyzed for granzyme B secretion. The results are expressed as the percentage (mean ± SEM for 5 tumors per group) of granzyme B positive cells. (E) Lymph nodes obtained from NP- and GO-203/NP-treated mice were incubated with LLC/MUC1 target cells at the indicated ratios. The results are expressed as percentage cytotoxicity (mean ± SEM for 5 mice per group) comparing NP-treated mice (open bars) with GO-203/NP-treated mice (solid bars).

Figure 4.

Overexpression of MUC1 in NSCLC negatively correlates with CD8, IFNG and granzyme (B)(GZMB). (A) Microarray data from Oncomine database are expressed as box plots (25th-75th percentiles) for MUC1 expression in normal lung tissues (n = 20) and lung adenocarcinoma (n = 226). The data were log2 transformed and median centered. (B-D) RNA sequencing data of lung cancer patients was obtained from cBioPortal TCGA data set. Correlations between MUC1 expression and that for CD8 (B), IFNG (C) and GZMB (D) were assessed using Spearman's rank correlation coefficient, where p < 0.05 was considered as statistically significant. (E-F). Expression of CD8 and IFNG in NSCLC correlates with survival. Kaplan–Meier plots comparing the overall survival of patients with NSCLC in the TCGA data set. Patients were stratified with the high (red) or low (blue) expression of CD8 (E) and IFNG (F) against the median average. The survival curves were compared using log-rank (Mantel–Cox) test. HR, hazard ratio.