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A novel FOXO1-mediated dedifferentiation blocking role for DKK3 in adrenocortical carcinogenesis
Joyce Y Cheng1, Taylor C Brown1, Timothy D Murtha1
1Department of Surgery & Yale Endocrine Neoplasia Laboratory, Yale University School of Medicine, New Haven, CT, USA.
Background:
Dysregulated WNT signaling dominates adrenocortical malignancies. This study investigates whether silencing of the WNT negative regulator DKK3 (Dickkopf-related protein 3), an implicated adrenocortical differentiation marker and an established tumor suppressor in multiple cancers, allows dedifferentiation of the adrenal cortex.
Methods:
We analyzed the expression and regulation of DKK3 in human adrenocortical carcinoma (ACC) by qRT-PCR, immunofluorescence, promoter methylation assay, and copy number analysis. We also conducted functional studies on ACC cell lines, NCI-H295R and SW-13, using siRNAs and enforced DKK3 expression to test DKK3's role in blocking dedifferentiation of adrenal cortex.
Results:
While robust expression was observed in normal adrenal cortex, DKK3 was down-regulated in the majority (>75%) of adrenocortical carcinomas (ACC) tested. Both genetic (gene copy loss) and epigenetic (promoter methylation) events were found to play significant roles in DKK3 down-regulation in ACCs. While NCI-H295R cells harboring β-catenin activating mutations failed to respond to DKK3 silencing, SW-13 cells showed increased motility and reduced clonal growth. Conversely, exogenously added DKK3 also increased motility of SW-13 cells without influencing their growth. Enforced over-expression of DKK3 in SW-13 cells resulted in slower cell growth by an extension of G1 phase, promoted survival of microcolonies, and resulted in significant impairment of migratory and invasive behaviors, largely attributable to modified cell adhesions and adhesion kinetics. DKK3-over-expressing cells also showed increased expression of Forkhead Box Protein O1 (FOXO1) transcription factor, RNAi silencing of which partially restored the migratory proficiency of cells without interfering with their viability.
Conclusions:
DKK3 suppression observed in ACCs and the effects of manipulation of DKK3 expression in ACC cell lines suggest a FOXO1-mediated differentiation-promoting role for DKK3 in the adrenal cortex, silencing of which may allow adrenocortical dedifferentiation and malignancy.
Insights
Dickkopf-related protein 3 (DKK3) loss in adrenocortical carcinoma promotes dedifferentiation. Restoring DKK3 expression in adrenal cortex cancer cells inhibits malignancy by promoting differentiation via FOXO1.
Area of Science:
- Endocrinology
- Cancer Biology
- Molecular Oncology
Background:
- Adrenocortical malignancies are driven by dysregulated WNT signaling.
- Dickkopf-related protein 3 (DKK3) is a WNT negative regulator, adrenocortical differentiation marker, and tumor suppressor.
- DKK3's role in adrenal cortex dedifferentiation is investigated.
Purpose of the Study:
- To investigate if DKK3 silencing contributes to adrenal cortex dedifferentiation.
- To analyze DKK3 expression, regulation, and functional role in adrenocortical carcinoma (ACC).
Main Methods:
- Analyzed DKK3 expression and regulation in human ACC using qRT-PCR, immunofluorescence, promoter methylation, and copy number analysis.
- Functional studies in ACC cell lines (NCI-H295R, SW-13) using siRNAs and enforced DKK3 expression.
Main Results:
- DKK3 was down-regulated in >75% of ACCs due to genetic and epigenetic events.
- SW-13 cells showed altered motility and growth upon DKK3 manipulation.
- Enforced DKK3 expression in SW-13 cells impaired migration/invasion and increased FOXO1 expression.
Conclusions:
- DKK3 suppression in ACCs facilitates dedifferentiation and malignancy.
- DKK3 plays a differentiation-promoting role in the adrenal cortex, potentially mediated by FOXO1.
