Data supporting the roles of BAP1, STING, and IFN-β in ISGF3 activation in ccRCC

Abstract

The data presented in this article are companion materials to our manuscript titled "BAP1 maintains HIF-dependent interferon beta induction to suppress tumor growth in clear cell renal cell carcinoma" (Langbein et al., 2022), where we investigated the downstream effects of BAP1 (BRCA1-associated protein 1) expression in clear cell renal cell carcinoma (ccRCC) cell lines and mouse xenograft models. In the manuscript, we showed that BAP1 upregulates STING (stimulator of interferon genes) expression and activity in ccRCC cells, leading to IFN-β transcription and activation of interferon stimulated gene factor 3 (ISGF3), the transcription factor that mediates the effects of type I interferons (IFNs). Here, we suppressed additional components of the type I IFN pathway, including IRF9 (a component of ISGF3), IFNAR1 (the type I IFN receptor), and STING (a stimulator of IFN production) by shRNA to investigate their involvement in BAP1-mediated upregulation of ISGF3 activity. We also inhibited extracellular IFN-β via neutralizing antibody treatment in BAP1-expressing cells to ascertain the role of the secreted cytokine in this pathway. ISGF3 activity was assessed by western blot analysis and qPCR measurement of its transcriptional targets. To examine the relevance of our observations in another model system, we characterized primary kidney cells from WT and Bap1 ^fl/fl mice by cytokeratin 8 immunohistochemistry and examined the effect of Bap1 knockout on Sting protein expression. Finally, we treated mice bearing BAP1 knockdown xenografted tumors with diABZI, a STING agonist, and measured immune cell recruitment via CD45 immunohistochemistry. These data can serve as a starting point for further investigation on the roles of BAP1 and other tumor suppressor genes in interferon pathway regulation.

Keywords: BAP1; Kidney cancer; Therapeutic; Tumor suppressor genes; Type I interferon.

Fig. 1

ISGF3 activity is elevated in BAP1-expressing cells. (A) SDS-solubilized whole cell lysates of UMRC6 cells expressing GFP or a BAP1 construct and control (SCR) or IRF9 shRNAs and blotted with the indicated antibodies (n=3). (B) SDS-solubilized whole cell lysates of UMRC6 cells expressing GFP or a BAP1 construct, treated with the indicated concentrations of ruxolitinib or IFN-⍺2 (2000 IU/ml) for 24 h, and blotted with the indicated antibodies (n=3). For ruxolitinib: +, 0.1 µM; ++, 0.3 µM; +++, 1 µM.

Fig. 2

IFN-β treatment increases ISGF3 target levels in IFNB1 knockdown cells. (A) RT-qPCR measurement of the indicated transcripts in Ren-02 shSCR and shIFNB1-01 cells treated with CTRL (PBS) or IFN-β for 24 h (n=2). (B) RT-qPCR measurement of the indicated transcripts in UMRC6 GFP shSCR, BAP1 shSCR, and BAP1 shIFNB1-01 cells treated with CTRL (PBS) or IFN-β for 24 h (n=2). (C) RT-qPCR measurement of the indicated transcripts in UMRC6 GFP shSCR, BAP1 shSCR, and BAP1 shIFNB1-01 cells treated with CTRL (PBS) or IFN-β for 24 h (n=2). In all panels, t-tests were performed comparing the control to the IFN-treated within each cell line, and only comparisons with p<0.1 are shown. IFN +, 25 pg/ml; IFN ++, 100 pg/ml.

Fig. 3

IFNAR1 knockdown reduces ISGF3 target expression. (A) SDS-solubilized whole cell lysates of UMRC6 cells expressing control (GFP) or BAP1 constructs and control (SCR) or IFNAR1 shRNAs and blotted with the indicated antibodies (n=2). (B) SDS-solubilized whole cell lysates of UMRC6 cells expressing the indicated constructs and blotted with the indicated antibodies. (C, D) RT-qPCR measurement of the indicated transcripts in UMRC6 cells expressing control (GFP) or BAP1 constructs and control (SCR) or IFNAR1 shRNAs (n=3, n=4 for OAS1, IFI44L, IFIT1).

Fig. 4

An IFN-β neutralizing antibody decreases ISGF3 target expression in BAP1-expressing cells. (A) UMRC6 BAP1-expressing cells were treated with CTRL (PBS), anti-goat IgG (mock antibody treatment), or increasing doses of anti-IFN-β antibody for 5 days, and qPCR was performed on the indicated transcripts (n=3). (B) Ren-02 cells were treated with CTRL, anti-goat IgG, or increasing doses of anti-IFN-β antibody for 3 days, and qPCR was performed on the indicated transcripts (n=3). +, 0.1 µg/ml; ++, 0.2 µg/ml.

Fig. 5

Bap1 knockout reduces STING protein levels in primary kidney cells from Bap1^fl/fl mice. (A) Phase contrast images of WT and Bap1^fl/fl mouse primary kidney cells. (B) Cytokeratin 8 immunohistochemistry performed on normal mouse kidney, normal human kidney, ccRCC and WT mouse primary kidney cells. (C) EBC-solubilized lysates of primary kidney cells from WT or Bap1^fl/fl mice treated with control or Cre adenovirus and blotted with the indicated antibodies.

Fig. 6

STING knockdown reduces ISGF3 target expression. (A) EBC-solubilized lysates from Ren-02 cells expressing control or STING shRNA and blotted with the indicated antibodies (n=3). (B) EBC-solubilized lysates from UMRC6 cells expressing GFP or BAP1 and control or STING shRNA and blotted with the indicated antibodies (n=2).

Fig. 7

A STING agonist increases CD45⁺ immune cell presence in BAP1-deficient xenografted tumors. H&E and IHC staining of tumor tissue from representative mice bearing Ren-02 shBAP1-74 xenografted tumors and treated with vehicle (control) or diABZI.