Methyltransferase Inhibition Enables Tgfβ Driven Induction of CDKN2A and B in Cancer Cells

Abstract

CDKN2A/B deletion or silencing is common across human cancer, reinforcing the general importance of bypassing its tumor suppression in cancer formation or progression. In rhabdomyosarcoma (RMS) and neuroblastoma, two common childhood cancers, the three CDKN2A/B transcripts are independently expressed to varying degrees, but one, ARF, is uniformly silenced. Although TGFβ induces certain CDKN2A/B transcripts in HeLa cells, it was unable to do so in five tested RMS lines unless the cells were pretreated with a broadly acting methyltransferase inhibitor, DZNep, or one targeting EZH2. CDKN2A/B induction by TGFβ correlated with de novo appearance of three H3K27Ac peaks within a 20 kb cis element ∼150 kb proximal to CDKN2A/B. Deleting that segment prevented their induction by TGFβ but not a basal increase driven by methyltransferase inhibition alone. Expression of two CDKN2A/B transcripts was enhanced by dCas9/CRISPR activation targeting either the relevant promoter or the 20 kb cis elements, and this “precise” manipulation diminished RMS cell propagation in vitro. Our findings show crosstalk between methyltransferase inhibition and TGFβ-dependent activation of a remote enhancer to reverse CDKN2A/B silencing. Though focused on CDKN2A/B here, such crosstalk may apply to other TGFβ-responsive genes and perhaps govern this signaling protein’s complex effects promoting or blocking cancer.

Keywords:

• CDKN2A/B
• Cis enhancers
• chromosome 9p21
• EZH2
• histone methyltransferase

SUPPLEMENTAL MATERIAL

Supplemental data for this article can be accessed online at https://doi.org/10.1080/10985549.2023.2186074

ACKNOWLEDGEMENTS

The authors acknowledge technical assistance provided by J. Aguayo and J. Liu, and many helpful comments by other members of the Skapek laboratory.

DATA AVAILABILITY STATEMENT

Copy-number variation (CNV) data were obtained from the National Cancer Institute (NCI) TCGA program using the Genomic Data Commons Data Portal (https://portal.gdc.cancer.gov/). CDKN2A/B expression in RMS and neuroblastoma was extracted through RNA-seq data from 42 specimens downloaded from The European Genome-phenome Archive (EGA) with accession number EGAD00001000878 (for RMS), and from 195 cases NCI Gabriella Miller Kids’ First program (https://commonfund.nih.gov/kidsfirst) (for neuroblastoma).

Additional information

Funding

We acknowledge financial support from the Cancer Prevention and Research Institute of Texas [RP180319] and the National Cancer Institute Cancer Center Support Grant [P30CA142543].