Early phase trials targeting the T-cell inhibitory molecule PD-L1 have shown clinical efficacy in cancer. This study was undertaken to determine whether PD-L1 is overexpressed in triple-negative breast cancer (TNBC) and to investigate the loss of the phosphatase and tensin homolog (PTEN) as a mechanism of PD-L1 regulation. The Cancer Genome Atlas (TCGA) RNA sequencing data showed significantly greater expression of the PD-L1 gene in TNBC (n=120) compared to non-TNBC (n=716) (P<0.001). Breast tumor tissue microarrays were evaluated for PD-L1 expression which was present in 19% (20 of 105) TNBC specimens. PD-L1+ tumors had greater CD8+ T-cell infiltrate than PD-L1− tumors (688 cells/mm versus 263 cells/mm; P<0.0001). To determine the effect of PTEN loss on PD-L1 expression, stable cell lines were generated using PTEN shRNA. PTEN knockdown led to significantly higher cell-surface PD-L1 expression and PD-L1 transcripts, suggesting transcriptional regulation. Moreover, PI3K pathway inhibition using the AKT inhibitor MK-2206 or rapamycin resulted in decreased PD-L1 expression, further linking PTEN and PI3K signaling to PD-L1 regulation. Co-culture experiments were performed to determine the functional effect of altered PD-L1 expression. Increased PD-L1 cell surface expression by tumor cells induced by PTEN loss led to decreased T cell proliferation and increased apoptosis. PD-L1 is expressed in 20% of TNBC, suggesting PD-L1 as a therapeutic target in TNBC. Since PTEN loss is one mechanism regulating PD-L1 expression, agents targeting the PI3K pathway may increase the antitumor adaptive immune responses.

Cushing's disease, also known as adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas (PAs) that cause excess cortisol production, accounts for up to 85% of corticotrophin-dependent Cushing's syndrome cases. However, the genetic alterations in this disease are unclear. Here, we performed whole-exome sequencing of DNA derived from 12 ACTH-secreting PAs and matched blood samples, which revealed three types of somatic mutations in a candidate gene, USP8 (encoding ubiquitin-specific protease 8), exclusively in exon 14 in 8 of 12 ACTH-secreting PAs. We further evaluated somatic USP8 mutations in additional 258 PAs by Sanger sequencing. Targeted sequencing further identified a total of 17 types of USP8 variants in 67 of 108 ACTH-secreting PAs (62.04%). However, none of these mutations was detected in other types of PAs (n = 150). These mutations aggregate within the 14-3-3 binding motif of USP8 and disrupt the interaction between USP8 and 14-3-3 protein, resulting in an elevated capacity to protect EGFR from lysosomal degradation. Accordingly, PAs with mutated USP8 display a higher incidence of EGFR expression, elevated EGFR protein abundance and mRNA expression levels of POMC, which encodes the precursor of ACTH. PAs with mutated USP8 are significantly smaller in size and have higher ACTH production than wild-type PAs. In surgically resected primary USP8-mutated tumor cells, USP8 knockdown or blocking EGFR effectively attenuates ACTH secretion. Taken together, somatic gain-of-function USP8 mutations are common and contribute to ACTH overproduction in Cushing's disease. Inhibition of USP8 or EGFR is promising for treating USP8-mutated corticotrophin adenoma. Our study highlights the potentially functional mutated gene in Cushing's disease and provides insights into the therapeutics of this disease.

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