TMEM160 promotes tumor immune evasion and radiotherapy resistance via PD-L1 binding in colorectal cancer

Abstract

Background: The effectiveness of anti-programmed cell death protein 1(PD-1)/programmed cell death 1 ligand 1(PD-L1) therapy in treating certain types of cancer is associated with the level of PD-L1. However, this relationship has not been observed in colorectal cancer (CRC), and the underlying regulatory mechanism of PD-L1 in CRC remains unclear.

Methods: Binding of TMEM160 to PD-L1 was determined by co-immunoprecipitation (Co-IP) and GST pull-down assay.The ubiquitination levels of PD-L1 were verified using the ubiquitination assay. Phenotypic experiments were conducted to assess the role of TMEM160 in CRC cells. Animal models were employed to investigate how TMEM160 contributes to tumor growth.The expression and clinical significance of TMEM160 and PD-L1 in CRC tissues were evaluated by immunohistochemistry(IHC).

Results: In our study, we made a discovery that TMEM160 interacts with PD-L1 and plays a role in stabilizing its expression within a CRC model. Furthermore, we demonstrated that TMEM160 hinders the ubiquitination-dependent degradation of PD-L1 by competing with SPOP for binding to PD-L1 in CRC cells. Regarding functionality, the absence of TMEM160 significantly inhibited the proliferation, invasion, metastasis, clonogenicity, and radioresistance of CRC cells, while simultaneously enhancing the cytotoxic effect of CD8 + T cells on tumor cells. Conversely, the upregulation of TMEM160 substantially increased these capabilities. In severely immunodeficient mice, tumor growth derived from lentiviral vector shTMEM160 cells was lower compared with that derived from shNC control cells. Furthermore, the downregulation of TMEM160 significantly restricted tumor growth in immune-competent BALB/c mice. In clinical samples from patients with CRC, we observed a strong positive correlation between TMEM160 expression and PD-L1 expression, as well as a negative correlation with CD8A expression. Importantly, patients with high TMEM160 expression exhibited a worse prognosis compared with those with low or no TMEM160 expression.

Conclusions: Our study reveals that TMEM160 inhibits the ubiquitination-dependent degradation of PD-L1 that is mediated by SPOP, thereby stabilizing PD-L1 expression to foster the malignant progress, radioresistance, and immune evasion of CRC cells. These findings suggest that TMEM160 holds potential as a target for the treatment of patients with CRC.

Keywords: Colorectal cancer; PD-L1; SPOP; TMEM160.

Fig. 1

TMEM160 interacts with PD-L1. A The BioGRID database was used to search for the interacting proteins of CD274. B HCT116 cells were transfected using Flag-PD-L1 and Myc-TMEM160 for immunostaining with Flag antibodies (green), Myc antibodies (red), and DAPI antibodies (blue). Scale bar, 20 µm. C A prediction model for TMEM160 was obtained from the UniProt database (left), while a prediction model for PD-L1 was obtained from the Protein Data Bank (PDB) database (right). D The predicted interface of one of the complexes formed by TMEM160 and PD-L1. E, F Results of Co-IP of exogenous PD-L1 and TMEM160 in HEK-293 T and SW480 cells. G, H Endogenous Co-IP assays were performed in DLD1 and HCT116 cells to assess the interaction of PD-L1 with TMEM160. I The GST pull-down assay was conducted to confirm the direct binding between the His-PD-L1 and the GST-TMEM160 fusion protein

Fig. 2

TMEM160 stabilizes PD-L1 protein expression by inhibition of ubiquitination-dependent degradation of PD-L1. A, B Western blotting assays and C RT-qPCR were performed to test the expression of TMEM160 and PD-L1 at the protein and mRNA levels, respectively. The expression analysis was performed following interference with TMEM160 expression using two different siRNAs (#1 and #2) in HCT116 and DLD1 cells, as well as TMEM160 overexpression in SW480 and RKO cells. D IF experiments of HCT116 cells transfected with siScr or siTMEM160#1 plasmid were assessed with PD-L1 expression by the fluorescence intensity of PD-L1. E Flow cytometric analysis of DLD1 cells transfected with siScr or siTMEM160#1 plasmid were assessed with surface PD-L1 expression. F, G To assess the effect of TMEM160 expression on PD-L1 stability, the CHX assay was performed in DLD1 cells transfected with siTMEM160#1 and SW480 cells with Myc-TMEM160. H Western blotting assay detected that MG132 can reverse the reduced expression of PD-L1 due to TMEM160 downregulation. I The ubiquitination assay was conducted in HCT116 cells to analyze the impact of TMEM160 overexpression on PD-L1 ubiquitination

Fig. 3

TMEM160 competitively binds to PD-L1 with SPOP, inhibiting its ubiquitination degradation. A The BioGRID database was used to search for the interacting proteins of TMEM160. B, C Endogenous Co-IP assays were performed in DLD1 and HCT116 cells to assess the interaction of TMEM160 with SPOP. D, E DLD1 and RKO cells were collected for western blotting analysis to investigate the effects of SPOP and TMEM160 overexpression on the expression PD-L1 in these cells. The whole-cell lysates were collected 48 h after transfection. F, G The Co-IP assay demonstrated that TMEM160 competes with SPOP for binding to PD-L1 in DLD1 and SW480 cells. H The results of the ubiquitination assay showed that competitive binding of TMEM160 to PD-L1 with SPOP reduces the ubiquitination degradation of PD-L1

Fig. 4

TMEM160 promotes malignant biological behavior in CRC cells. After the knockdown of TMEM160 in HCT116 and DLD1 cells using siTMEM160#1 and siTMEM160#2. A, C The CCK-8 assay demonstrated that cell proliferation was inhibited when TMEM160 was reduced. B, D The results of the transwell assay indicated a significant reduction in both cell migration and invasion. E The colony formation ability of CRC cells was significantly impaired when TMEM160 expression was downregulated. F-J SW480 and RKO cells that were stably transfected with Myc-TMEM160 plasmids exhibited significant enhancements in CRC cell proliferation (F, H), migration, invasion (G, I), and colony formation abilities (J). Independent biological experiments were repeated three times each, helping to ensure the reliability and reproducibility of the results. Statistical significance is denoted by *p < 0.05 and **p < 0.01. Scale bar, 100 μm

Fig. 5

Deletion of TMEM160 impairs the role of PD-L1 in T cell-mediated tumor cytotoxicity and reduces radiotherapy resistance in CRC cells. A The association of TMEM160 with immunity was predicted using the TIMER database. B Schematic representation of TMEM160 correlation with immune infiltration. C FACS analysis of Jurkat cell-mediated killing of tumor cells using Annexin V and propidium iodide (PI) double staining. D The quantitative measurement of apoptotic cells in the Q2 and Q3 quadrants of each group, with more than 10⁴ cells counted in each group. E CCK8 analysis was performed to detect tumor cell viability after incubation with activated Jurkat cells. F, G To investigate the protein levels of TMEM160 and PD-L1 after radiotherapy (RT), measurements were performed in two experimental groups: siTMEM160#1 and TMEM160 overexpression. Both groups were subjected to 8 Gy of radiation. H, I The colony formation assays were visually represented through representative photographs, depicting the surviving proportions of DLD1 and RKO cells following irradiation with 0, 2, 4, and 8 Gy. J, K The proliferation of DLD1 and RKO cells was assessed using CCK8 assays. The independent biological experiments were repeated three times. Statistical significance is denoted *p < 0.05, **p < 0.01 and ***p < 0.001

Fig. 6

TMEM160 plays a crucial role in tumor immune evasion. A, B To establish CDX models, nude mice were subcutaneously injected with HCT116 cells that had been stably transfected with LV-ShTMEM160#1 or LV-ShScr. C, D In the LV-TMEM160#1 group, the tumor volume and weight were lower than those in the control group (mean ± SEM, n = 6/group). E, F To establish CDX models, BALB/c mice were subcutaneously injected with CT26 cells that had been stably transfected with LV-ShTMEM160#1 or LV-ShScr. G, H In the LV-TMEM160#1 group, the tumor volume and weight were markedly lower than those in the control group (mean ± SEM, n = 6/group). I, J Western blotting and IHC were conducted to detect the protein levels of TMEM160 and PD-L1 in tumor tissues obtained from subcutaneous CDX models constructed by HCT116 cells. K, L The protein levels of TMEM160 and PD-L1 and the infiltration of CD8 + T lymphocytes in subcutaneous CDX models constructed from CT26 cells were determined by western blotting and IHC

Fig. 7

TMEM160 is associated with PD-L1 expression in human CRC and predicts poor patient prognosis. A Western blotting assays were employed to detect the protein levels of TMEM160 in eight pairs of CRC tissue samples. B IHC analysis was conducted to detect the protein levels of TMEM160 in tumor tissues (n = 125) and adjacent normal tissues (n = 125) obtained from patients clinically diagnosed with CRC. C The patients with CRC were categorized into groups based on the IHC results of TMEM160 protein levels. Kaplan–Meier analysis was then conducted to assess the survival outcomes in each group. D IHC analysis was performed on serial sections of both tumor tissues and paired normal tissues. The purpose of the analysis was to evaluate the expression levels of TMEM160, PD-L1, and CD8A proteins. To assess the correlation between TMEM160 expression and the expression of PD-L1 and CD8A, a chi-square test was employed. E The correlation between TMEM160 protein expression and the protein expression of PD-L1 and CD8A was analyzed using IHC assays performed on CRC tissues that were obtained from clinical patients. F Kaplan–Meier survival analysis was performed on a cohort of 125 patients, who were classified into four groups based on the protein levels of TMEM160 and PD-L1

Fig. 8

TMEM160 promotes immune escape and radiotherapy resistance to colorectal cancer cells by binding to PD-L1. In the context of CRC, the interaction between membrane-localized PD-L1 and PD-1 on T cells plays a critical role in inhibiting T cell activation. When the expression of TMEM160 is low, PD-L1 can bind to SPOP, which triggers the proteasomal degradation of PD-L1. As a result, the expression of PD-L1 localized as the cellular membrane decreased, ultimately promoting T cell activation. Conversely, when TMEM160 expression was increased in vivo, the binding of TMEM160 to PD-L1 increased, so that the binding of PD-L1 to SPOP decreased, resulting in a decrease in PD-L1 ubiquitination and stabilizing its expression, leading to immune escape and radiotherapy resistance in CRC cells. This suggests that TMEM160 expression levels can impact the stability and localization of PD-L1, ultimately influencing immune regulation and the response to radiotherapy in CRC. The figure was drawn using Figdraw