Dataset Information

A human ACTH-secreting corticotroph tumoroid model: Novel Human ACTH-Secreting Tumor Cell in vitro Model.

ABSTRACT:

Background

Cushing disease (CD), although rare, is a life-threatening disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary adenoma, which leads to excess adrenal-derived cortisol. Efficacious and safe medical therapies that control both hormonal hypersecretion and pituitary corticotroph tumor growth remain an unmet need in the management of CD. Translational research in pituitary tumors has been significantly hampered by limited quantities of surgically resected tissue for ex vivo studies, and unavailability of human pituitary tumor cell models.

Methods

To characterize human corticotroph tumors at the cellular level, we employed single cell RNA-sequencing (scRNA-seq) to study 4 surgically resected tumors. We also used microarrays to compare individualized paired consecutive culture passages to understand transcriptional shifts as in vitro cultures lost ACTH secretion. Based on these findings, we then modified our in vitro culture methods to develop sustained ACTH-secreting human corticotroph tumoroid cultures.

Findings

scRNA-seq identified 4 major cell populations, namely corticotroph tumor (73.6%), stromal (11.2%), progenitor (8.3%), and immune cells (6.8%). Microarray analysis revealed striking changes in extracellular matrix, cell adhesion and motility-related genes concordant with loss of ACTH secretion during conventional 2D culture. Based on these findings, we subsequently defined a series of crucial culture nutrients and scaffold modifications that provided a more favorable trophic and structural environment that could maintain ACTH secretion in in vitro human corticotroph tumor cultures for up to 4 months.

Interpretation

Our human corticotroph tumoroid model is a significant advance in the field of pituitary tumors and will further enable translational research studies to identify critically needed therapies for CD.

Funding

This work was partly funded by NCI P50-CA211015 and the Warley Trust Foundation.

SUBMITTER: Zhang D

PROVIDER: S-EPMC8024915 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Abstract Cushing Disease (CD) is a life-threatening condition with suboptimal medical treatment. To identify drugs that not only inhibit ACTH secretion to attain eucortisolemia but also inhibit tumor growth, we conducted a high throughput screen employing a novel “gain of signal” ACTH AlphaLISA assay. From a kinase inhibitor library containing 430 compounds, we identified the dual PI3K/HDAC inhibitor, CUDC-907, as a potent inhibitor of both in vitro and in vivo corticotroph tumor ACTH secretion and growth. By stepwise comparison of CUDC-907 with mono-functional PI3K and HDAC inhibitors, we demonstrated that CUDC-907 exerts its inhibitory effect on ACTH secretion primarily through its inhibition of HDAC activity at the POMC transcriptional level; while PI3K-mediated inhibition of corticotroph cell viability further contributes to reduced ACTH secretion. We also used RNA-seq to characterize the global transcriptiome changes associated with CUDC-907 treatment. Hierarchical clustering showed that 1432 differentially expressed genes (DEGs, p≤0.05 and fold-change≥1.5) were altered by CUDC-907 treatment in comparison to the vehicle-treated control cells. Gene ontology (GO) analysis of 456 downregulated and 976 upregulated DEGs revealed that the most enriched biological processes were cholesterol biosynthesis (GO:0006695, p=1.977e-17) and the type I interferon signaling pathway (GO: 0060337, p=4.928e-7) respectively. Further analysis demonstrated downregulation of the membrane-bound transcription factor sterol regulatory element binding proteins (SREBPs). Downregulation of SREBP-2 by CUDC-907 as well as the several other target enzymes in the cholesterol biosynthesis and uptake pathway including IDI2, NSDHL, MVD, and HMGCR, was confirmed by real-time PCR. To further characterize a role for SREBP-2 in regulation of corticotroph tumor ACTH secretion and proliferation, we employed siRNA targeting endogenous SREBP-2 (SREBP-2 mRNA, Control vs. siRNA 1±0.03 vs. 0.6±0.08, p<0.05), and demonstrated that knockdown of SREBP-2 not only inhibited POMC mRNA expression (POMC mRNA, 1±0.03 vs. 0.7±0.01, p<0.01), and ACTH secretion (ACTH (ng/mL) 29±0.4 vs. 23±0.3, p<0.005), but also suppressed cell proliferation (Relative Proliferation Rate, 1±0.01 vs. 0.7±0.01, p<0.005). This was further confirmed by overexpression of cleaved mature SREBP-2, which led to increased POMC expression and cell proliferation. We demonstrate for the first time the role of the SREBP-mediated cholesterol biosynthesis pathway in regulation of corticotroph tumor POMC regulation and growth. Our studies identify SREBP and cholesterol biosynthesis as a therapeutic target in CD.