Abstract
BackgroundDysregulated 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.MethodsWe 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.ResultsWhile 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.ConclusionsDKK3 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.
Highlights
IntroductionRecent findings from comprehensive genetic analyses of Adrenocortical carcinoma (ACC) confirmed a signature role for WNT dysregulation in the origin and/or progression of ACCs [4, 6, 8, 9]
Compared to the robust expression pattern in adrenal cortex, Dickkopf-related protein 3 (DKK3) protein expression was found to be nearly absent in Adrenocortical carcinoma (ACC) by indirect immunofluorescence analysis (Fig. 1b; a&h)
DKK3 silencing reduces clonogenic growth and promotes migration of ACC cells To test whether DKK3 plays a tumor suppressor role in ACC in vitro, we investigated the expression pattern and regulation of DKK3 in two ACC cell lines, SW-13 and NCIH295R
Summary
Recent findings from comprehensive genetic analyses of ACCs confirmed a signature role for WNT dysregulation in the origin and/or progression of ACCs [4, 6, 8, 9] Both canonical and non-canonical WNT signaling pathways play global and zone-specific roles in the development, differentiation, and homeostasis of the adrenal gland [10, 11]. Endocrine homeostasis of the adrenal glomerulosa and fasciculata zones is largely controlled by WNT-differentiation signaling mediated by the Wnt4-Fz1/2-Dvl3-β-Catenin-SF1 axis [12,13,14,15,16] Regulatory components of this proposed adrenal cortex-specific Wnt axis include the secretory factors, frizzled-related protein 1 (SFRP1) and the putative tumor suppressor, DKK3 [14, 17, 18]. Other WNT regulatory mutations found in ACCs include PRKAR1A [23] and recently identified KREMEN1 and ZNRF3 gene deletions [8, 24]
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