Investigating the effects of PTEN mutations on cGAS-STING pathway in glioblastoma tumours

Abstract

Background: PTEN is a tumour suppressor gene and well-known for being frequently mutated in several cancer types. Loss of immunogenicity can also be attributed to PTEN loss, because of its role in establishing the tumour microenvironment. Therefore, this study aimed to represent the link between PTEN and cGAS-STING activity, a key mediator of inflammation, in tumour samples of glioblastoma patients.

Methods: Tumour samples of 36 glioblastoma patients were collected. After DNA isolation, all coding regions of PTEN were sequenced and analysed. PTEN expression status was also evaluated by qRT-PCR, western blot, and immunohistochemical methods. Interferon-stimulated gene expressions, cGAMP activity, CD8 infiltration, and Granzyme B expression levels were determined especially for the evaluation of cGAS-STING activity and immunogenicity.

Results: Mutant PTEN patients had significantly lower PTEN expression, both at mRNA and protein levels. Decreased STING, IRF3, NF-KB1, and RELA mRNA expressions were also found in patients with mutant PTEN. Immunohistochemistry staining of PTEN displayed expressional loss in 38.1% of the patients. Besides, patients with PTEN loss had considerably lower amounts of IFNB and IFIT2 mRNA expressions. Furthermore, CD8 infiltration, cGAMP, and Granzyme B levels were reduced in the PTEN loss group.

Conclusion: This study reveals the immunosuppressive effects of PTEN loss in glioblastoma tumours via the cGAS-STING pathway. Therefore, determining the PTEN status in tumours is of great importance, like in situations when considering the treatment of glioblastoma patients with immunotherapeutic agents.

Keywords: Glioblastoma; Immunogenicity; Interferon response; PTEN; cGAS-STING pathway.

Fig. 1

PTEN expression status in wild-type (WT) and mutant (M) genotypes. (A) qRT-PCR results of PTEN mRNA expression normalised to GAPDH (*p = 0.008); (B) Western Blot analysis of PTEN protein expression normalised to B-actin (**p = 0.006); (C) Western Blot images of PTEN in the GBM tissues. PTEN genotypes are indicated as WT and M at the top of the gel; (D) Immunohistochemistry analysis of PTEN showing intact expression; (E) Weaker staining of PTEN with focal loss. Cases were clustered as PTEN low; (F) PTEN immunohistochemistry image demonstrates complete PTEN loss, images at 100x magnification; (G) Comparison of PTEN IHC expression in relation to PTEN genotypes

Fig. 2

Effects of PTEN genotypes on cGAS-STING pathway. (A) mRNA expression graphs of STING, IRF3, NF-KB1, and RELA genes in wild-type (WT) and mutant (M) patients; (B) Protein expressions of STING, IRF3, NF-KB, and Phospho-NF-KB normalized to B-actin; (C) Western Blot images of protein expressions. PTEN genotypes are indicated as WT and M at the top of the gel

Fig. 3

Effects of PTEN expression to tumour immunogenicity. (A) Transcription levels of interferon-stimulated genes according to immunohistochemically expression status of PTEN (*: p = 0.033; **: p = 0.015); (B) Normalised mRNA expression of GZMB (*: p = 0.045); (C) cGAMP production measured by ELISA in tumour lysates; (D) Immunohistochemistry analysis of CD8 showing 3 grades of infiltration. E: Comparison of CD8 infiltration in relation to PTEN status, images at 40x magnification

Fig. 4

TCGA database analysis. Median PTEN expression was used to discriminate low (n = 81) and high (n = 81) expression groups. (A) mRNA expression levels of interferon-stimulated genes; (B) CD4+; and (C) CD8 + cell infiltration in relation to PTEN expression