Cancer Res Treat.  2023 Jan;55(1):231-244. 10.4143/crt.2021.1526.

PD-L1 Upregulation by the mTOR Pathway in VEGFR-TKI–Resistant Metastatic Clear Cell Renal Cell Carcinoma

• Jeong SU
• Hwang HS
• Park JM
• Yoon SY
• Shin SJ
• Go H
• Lee JL
• Jeong G
• Cho YM

• KMID: 2538007
• DOI: http://doi.org/10.4143/crt.2021.1526

Abstract

Purpose
Tyrosine kinase inhibitors (TKI) targeting vascular endothelial growth factor receptor (VEGFR) signaling pathways have been used for metastatic clear cell renal cell carcinoma (mCCRCC), but resistance to the drug develops in most patients. We aimed to explore the underlying mechanism of the TKI resistance with regard to programmed death-ligand 1 (PD-L1) and to investigate signaling pathway associated with the resistant mechanism.
Materials and Methods
To determine the mechanism of resistance, 10 mCCRCC patients from whom tumor tissues were harvested at both the pretreatment and the TKI-resistant post-treatment period were included as the discovery cohort, and their global gene expression profiles were compared. A TKI-resistant renal cancer cell line was established by long-term treatment with sunitinib.
Results
Among differentially expressed genes in the discovery cohort, increased PD-L1 expression in post-treatment tissues was noted in four patients. Pathway analysis showed that PD-L1 expression was positively correlated with the mammalian target of rapamycin (mTOR) signaling pathway. The TKI-resistant renal cancer cells showed increased expression of PD-L1 and mTOR signaling proteins and demonstrated aggressive tumoral behaviour. Treatment with mTOR inhibitors down-regulated PD-L1 expression and suppressed aggressive tumoral behaviour, which was reversed with stimulation of the mTOR pathway.
Conclusion
These results showed that PD-L1 expression may be increased in a subset of VEGFR-TKI–resistant mCCRCC patients via the mTOR pathway.

Keyword

Renal cell carcinoma; Receptor protein-tyrosine kinases; B7-H1 antigen; TOR serine-threonine kinases

Figure

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  Fig. 1 Upregulation of CD274 gene (programmed death-ligand 1 [PD-L1]) expression in a subset of the discovery cohort. (A) Normalized CD274 (PD-L1) mRNA expression was increased in post-treatment tissues of three patients (patients 3, 4, and 10) compared with matched pretreatment tissues by microarray analysis. (B) Representative images of PD-L1 immunohistochemical staining performed on pre- and post–tyrosine kinase inhibitor treatment clear cell renal cell carcinoma tissues of patient 3 (×200). (C) While post-treatment tissues of the three patients (patients 3, 4, and 10) showed positive immunoreactivity on PD-L1 staining in tissue microarray, the PD-L1 expression of patient 5 was decreased from 50% to negative immunoreactivity. Pretreatment tissue of patients 6 and 10 and post-treatment tissue of patient 8 were needle biopsy specimens and were not available for PD-L1 immunohistochemical staining after use in the microarray experiment.

• 

  Fig. 2 Differential gene expression associated with programmed death-ligand 1 (PD-L1) upregulation in tyrosine kinase inhibitor–resistant post-treatment clear cell renal cell carcinoma of the discovery cohort. (A) Heat map overview of differential gene expression between three patients showing PD-L1 upregulation and the remaining seven patients. The levels of mammalian target of rapamycin complex 1 (mTORC1)–associated genes, including PLOD2, SYTL2, NAMPT, SLC7A5, HSPA9, BHLHE40, ATP2A2, LDHA, ACLY, EIF2S2, CALR, CDKN1A, HSPA5, and SLC2A, were increased in patients with increased PD-L1 expression (individual gene names do not appear). Shades of red represent elevated levels and green and blue represent reduced levels of gene expression (see colour scale). (B) Enrichment result for the “hallmark mTORC1 signaling” gene set from the gene set enrichment analysis of microarray data from the above two groups with PD-L1 upregulation noted or not. The enrichment score is calculated by walking down a list of genes ranked by their correlation with up-regulated PD-L1 expression, increasing a running-sum statistic when a gene in that gene set is encountered (each black vertical line underneath the enrichment plot) and decreasing it when a gene that is not in the gene set is encountered. The enrichment score is the maximum deviation from zero encountered in the walk. FDR, false discovery rate.

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  Fig. 3 Development of 769-P/suR cells with programmed death-ligand 1 (PD-L1) expression and increased aggressive tumoral behavior. (A) Expression of PD-L1 at the protein level in each of the five sunitinib-sensitive and sunitinib-resistant cell lines. 769-P successfully established sunitinib resistance with increased PD-L1 expression. (B) 769-P/suS cells were treated with sunitinib at the indicated concentrations, and relative cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. 769-P cells treated with 5 μM sunitinib were chosen for further studies. (C) The proliferative capacities of 769-P/suS and 769-P/suR cells were assessed by colony formation assay. Representative photographs of crystal-violet-stained cell colonies showed that colony numbers and sizes were increased more in 769-P/suR cells than in 769-P/suS cells (survival fraction, 35). The invasion (D) and migration (E) abilities of the 769-P/suR cells were determined by the Matrigel invasion assay and the scratch test assay, respectively. The numbers of 769-P/suR cells that invaded the underside of the Matrigel membrane and migrated into the wound area were greater than the number of 769-P/suS cells. (F) Expression of PD-L1, phospho–mammalian target of rapamycin (p-mTOR) and phospho-S6 ribosomal protein (p-S6RP) was greater in 769-P/suR cells than in 769-P/suS cells on Western blot analysis.

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  Fig. 4 Down-regulated programmed death-ligand 1 (PD-L1) expression and decreased aggressive tumoral behavior in 769-P/suR cells treated with dactolisib and everolimus. (A) Inhibition of the mammalian target of rapamycin (mTOR) signaling pathway decreases PD-L1 expression by 769-P/suR cells in a time-dependent manner. (B) Dactolisib and everolimus, which inhibit mTOR signaling pathway-related proteins, reduced PD-L1 expression by 769-P/suR cells after 72 hours. (C) 769-P/suR cells treated with dactolisib and everolimus showed decreased colony numbers and sizes compared with 769-P/suR cells treated with dimethyl sulfoxide (DMSO) in the colony formation assay (survival fraction, 0.01 and 0.4, respectively). (D) The migration ability of 769-P/suR cells treated with dactolisib and everolimus was determined by the scratch test assay. The number of 769-P/suR cells treated with dactolisib and everolimus that migrated into the wound area was less than the number of 769-P/suR cells treated with DMSO. (E) The invasion ability of 769-P/suR cells treated with dactolisib and everolimus was determined by the Matrigel invasion assay. The number of 769-P/suR cells treated with dactolisib and everolimus that invaded the underside of the Matrigel membrane was less than the number of 769-P/suR cells treated with DMSO.

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  Fig. 5 Increased programmed death-ligand 1 (PD-L1) expression via activation of the mammalian target of rapamycin (mTOR) pathway in 769-P/suS cells by MHY1485. (A) Western blot analysis was performed to detect the change in total protein level of mTOR pathway-related protein and PD-L1 in 769-P/suS cells treated with MHY1485 as an mTOR activator for 24, 48, or 72 hours. (B) 769-P/suS cells treated with MHY1485 showed increased colony numbers and sizes compared with 769-P/suS cells treated with dimethyl sulfoxide (DMSO) in the colony formation assay (survival fraction, 27). The number of 769-P/suS cells treated with MHY1485 that migrated into the wound area and invaded the underside of the Matrigel membrane was greater than the number of 769-P/suS cells treated with DMSO in the scratch test assay (C) and the Matrigel invasion assay (D), respectively.

Reference

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