Nuclear amphiregulin and lamin A contribute to genomic stability and the response to replication stress via regulation of heterochromatin.

The plasma membrane EGFR ligand, amphiregulin (AREG), is key to mammary luminal progenitor differentiation and governs estrogen-induced ductal elongation during puberty. However, AREG has also been detected in the nucleus of several epithelial cancers, although the physiological stimulus and nuclear function are not known. Using immortalized normal and BRCA2mut/+ mammary epithelial cells (MECs), we have discovered that endogenous AREG is required for maintenance of constitutive heterochromatin. Replication stress (RS) transiently increased prelamin A association with AREG in the cytoplasm, followed by incorporation into the nuclear membrane (NM), resulting in increased H3K9me3 heterochromatin in an ATR-dependent manner. Enforced nuclear AREG increased, while siRNA-mediated depletion of AREG reduced HP1α and SUV39h1 proteins, accompanied by global decompaction and reduction in H3K9me3 heterochromatin despite the presence of exogenous soluble AREG. Depletion of AREG also impacted the nuclear membrane, including dissipation of the Ran-GTPase gradient and formation of nuclear ruptures and invaginations. Loss of H3K9me3 with AREG knockdown increased replication origin usage and depleted nucleotide while enhancing global transcription. Reduced endogenous AREG also amplified RS-induced DNA damage, underscored by increased senescence of AREG-depleted BRCA2mut/+ MECs. Overall, this study reveals a novel and fundamental role for nuclear AREG in heterochromatin maintenance and genome stability in proliferating MECs.