SOX9 and SEMA7A regulate cell plasticity in the postpartum mammary gland with implications for breast cancer

Postpartum mammary gland involution is a coordinated process of cell death and remodeling that returns the tissue to a near pre-pregnant state following lactation and weaning. In models of postpartum breast cancer, defined as breast cancer diagnosed in women under age 45 and within 10 years of recent childbirth, involution induces durable phenotypes in breast tumor cells that promote progression and are associated with increased risk for therapeutic resistance, metastasis, and death in patients. SRY-Box Transcription Factor 9 (SOX9), a known regulator of mammary stem and progenitor cells, also promotes resistance to therapy and metastasis in breast cancers. Yet the contribution of SOX9 to the involution process is not well understood. We utilized single-cell RNA sequencing of mouse mammary glands during involution to delineate Sox9 -expressing cell populations during lactation and involution. We found that Sox9 mRNA is primarily expressed in luminal progenitor cells that are largely absent during lactation and present during early involution. We also reveal that Sox9 is involved in a shift in cell state from lactational to non-lactational and is expressed in the surviving cells during involution. Prior work revealed that Semaphorin-7a (SEMA7A) also promotes cancer stem cell and pro-survival phenotypes in luminal progenitor cells during involution, and we observe a population of luminal progenitor cells that co-expresses Sox9 and Sema7a during involution. Mechanistically, we demonstrate that knockdown of Sox9 in cultured mammary epithelial cells results in increased SEMA7A expression, mesenchymal phenotypes, and loss of lactogenic differentiation capacity, identifying a potential regulatory axis where SOX9 balances SEMA7A expression in normal mammary epithelium and that disruption of this balance results in a dedifferentiated state that resembles mesenchymal cells. We validated a spatial relationship between SOX9 and SEMA7A proteins in a unique set of breast tissue samples from healthy human donors to show co-expression during early involution. In breast cancer datasets, we observe elevated expression of SOX9 and SEMA7A in triple-negative breast cancers, as well as in the mesenchymal subtype of triple-negative breast cancers, suggesting disruption of this regulatory axis in breast cancer. Finally, we observe that co-expression increases metastatic risk in both estrogen receptor-negative and -positive breast cancers. Collectively, these findings define a novel SOX9–SEMA7A relationship in healthy mammary tissues and illustrate how studies of normal progenitor cell phenotypes can delineate cellular mechanisms that contribute to breast tumor progression.