IER5, a DNA-damage response gene, is required for Notch-mediated induction of squamous cell differentiation

Abstract

Notch signaling regulates normal squamous cell proliferation and differentiation and is frequently disrupted in squamous cell carcinomas, in which Notch is a key tumor suppressive pathway. To identify the direct targets of Notch that produce these phenotypes, we introduced a conditional Notch transgene into squamous cell carcinoma cell lines, which respond to Notch activation in 2D culture and in organoid cultures by undergoing differentiation. RNA-seq and ChIP-seq analyses show that in squamous cells Notch activates a context-specific program of gene expression that depends on lineage-specific regulatory elements, most of which lie in longrange enhancers. Among the direct Notch target genes are multiple DNA damage response genes, including IER5, which is regulated by Notch through several enhancer elements. We show that IER5 is required for Notch-induced differentiation in squamous carcinoma cells and in TERT-immortalized keratinocytes. Its function is epistatic to PPP2R2A, which encodes the B55a subunit of PP2A, and IER5 interacts with B55a in cells and in purified systems. These results show that Notch and DNA-damage response pathways converge in squamous cells and that some components of these pathways promote differentiation, which may serve to eliminate DNA-damaged cells from the proliferative pool in squamous epithelia. Crosstalk involving Notch and PP2A may enable Notch signaling to be tuned and integrated with other pathways that regulate squamous differentiation. Our work also suggests that squamous cell carcinomas remain responsive to Notch signaling, providing a rationale for reactivation of Notch as a therapeutic strategy in these cancers. ImpactOur findings highlight context-specific crosstalk between Notch, DNA damage response genes, and PP2A, and provide a roadmap for understanding how Notch induces the differentiation of squamous cells.

Hydroxylation of the NOTCH1 intracellular domain regulates Notch signaling dynamics

“…Regarding the pathophysiological context, our findings are particularly relevant to Notch-driven leukemias, T-cell acute lymphoblastic leukemia (T-ALL), chronic lymphocytic leukemia (CLL), for which NOTCH1 mutations been previously described [56,57]. They could also play a role in the setting of solid cancer such as breast cancer and squamous cell carcinoma, where active Notch signaling has been described [4,9,58]. Future genome-wide analyses should not only focus on HIF1α but also on Notch-dependent gene regulation.…”

Oh, the Mutations You’ll Acquire! A Systematic Overview of Cutaneous Squamous Cell Carcinoma

“…Additionally, engineered pulses of NOTCH1 activation rapidly induce spinous differentiation and the DNA damage response, but induction of terminal differentiation is delayed. NOTCH1 activation also fails to downregulate basal markers, such as p63 [76]. Together these results show that NOTCH1 is necessary, but not sufficient for induction of epidermal differentiation.…”