Nuclear-Trafficked Amphiregulin Regulates H3K9me3 Heterochromatin Formation Facilitating Replication Control and Mitigating Replication Stress-Associated DNA Damage in Mammary Epithelial Cells

Abstract

The epidermal growth factor receptor (EGFR) ligand, amphiregulin (AREG) is widely expressed in epithelial cells. It is synthesized as a precursor protein that undergoes proteolytic shedding from the plasma membrane. In mammary epithelial cells (MECs), AREG engages in paracrine signaling where it functions as an important differentiation factor during mammary development at puberty. Interestingly, AREG ectodomain shedding also stimulates a lesser known, nuclear trafficking pathway, wherein it bypasses the lysosome and interacts with lamin A/C at the inner nuclear membrane (INM) and promotes H3K9me3 heterochromatin formation. However, the physiological stimulus, role, and molecular mechanism underlying AREG-mediated H3K9me3 maintenance have not been elucidated. Here, we show that in response to replication stress (RS), AREG undergoes retrograde trafficking to the nucleus in a p38-dependent manner via early endosomes in hTERT-immortalized MECs. We found that endocytosed AREG interacts with prelamin A in the cytoplasm and nucleus correlated with a transient increase in H3K9me3. siRNA-mediated AREG knockdown (KD) resulted in the downregulation of both suppressor of variegation 3-9 homolog 1 (SUV39H1), and heterochromatin protein 1 alpha (HP1α) accompanied by the reduction and decompaction of H3K9me3. Notably, AREG depletion resulted in increased DNA damage in BRCA2+/+ hTERT-MECs that was exacerbated in BRCA2mut/+ hTERT-MECs, following the inducing of RS. AREG-KD in these MECs increased DNA lesions associated with perturbed replication dynamics including slowing of the replication fork and enhanced firing of new origins. Depletion of AREG also disrupted the Ran-gradient, weakened nuclear membrane integrity resulting in the appearance of cytoplasmic DNA and activation of interferon (IFN) pathway associated genes, ultimately leading to senescence. Conversely, ectopic expression of AREG-ΔC11 lacking the C-terminal 11 amino acids, which traffics exclusively to the nucleus, increased prelamin A levels at the nuclear periphery coinciding with an increase in SUV39H1 and HP1α. Together, these experiments demonstrated a non-canonical role for AREG in maintaining heterochromatin, possibly by facilitating nuclear import of prelamin A. This process is strongly enhanced by RS, resulting in accumulation of unprocessed nuclear prelamin A and a transient increase in heterochromatin. Integrating our findings and the known ovarian hormone-dependent regulation of AREG and its role in promoting mammary progenitor differentiation, we propose that AREG safeguards genomic stability and lineage fidelity during reproductive years. After menopause, a decline in extracellular AREG may contribute to the loss of lineage fidelity and reduced heterochromatin maintenance in some MEC subpopulations. This reduction in chromatin integrity promotes DNA damage in proliferative MECs and activates IFN pathway leading to senescence in both mature and cycling MECs. Collectively, these alterations create a pro-tumorigenic environment that may contribute to the marked increase in incidences of breast cancer in postmenopausal women, a population that accounts for 70% of all breast cancer cases.