The nature of nurture: Investigating the role of Notch Signaling and Vacuolar ATPase in mammary gland development
Authors: Pamarthy Sahithi , Mao Liquin , Katara Gajendra K , jaiswal Mukesh K , Kulshrestha Arpita , Gilman‐Sachs Alice , Beaman Kenneth D 
Published: November 2023

Among all tissues and organs, the mammary gland is unique as most of its development occurs in adulthood. The gland is transformed from a nonfunctional organ into a mature, milk‐producing gland evolved to nurture the newborn. Recent reports suggest that alteration in breast biology during reproductive life influences breast cancer risk. Notch Signaling plays a major role in mammary gland development and has been implicated in breast cancer. Notch pathway is triggered by ligand‐receptor binding resulting in activation of target genes that are involved in cell fate determination and proliferation. The Vacuolar ATPase (V‐ATPase) is a rotary nanomotor, which maintains cellular pH by pumping hydrogen ions into the lumen of acidic vesicles. V‐ATPase is located on intracellular vesicles of normal cells and is translocated to the plasma membrane of most cancers. We have previously reported that a2V‐ATPase, an isoform of ‘a’ subunit of V‐ATPase regulates lysosomal degradation of Notch receptor Notch1 and alters Notch signaling in breast cancer. However, the role of V‐ATPase in relation to Notch Signaling in mammary gland development is not known. To study the effect of a2V‐ATPase on Notch Signaling and gland development, we generated mice with conditional knockout of a2V‐ATPase (a2V KO) utilizing the Cre‐Lox technology driven by mammary gland specific MMTV promoter. To represent distinct phases of gland development, inguinal mammary fat pads were harvested from post‐pubertal, nulliparous, early‐pregnant, late‐pregnant, lactating and involuting mice. In wild‐type mice, the basal mRNA and protein expression of a2V‐ATPase increased from puberty to lactation and then decreased during involution. The basal levels of Notch1 increased from puberty to early pregnancy and decreased during lactation. Pups from litters of a2V KO mice weighed significantly less compared to litters from wild‐type mice (p<0.001) suggesting that a2V‐ATPase is important for sufficient lactation. To investigate this further, we examined gland morphology in mammary fat pad whole mounts and demonstrated that a2V KO mice have diminished ductal outgrowth of epithelial cells that produce the milk. Indeed, the expression of beta‐casein, a marker for lactation, was lower in a2V KO mice. Using IHC and RT‐PCR, we demonstrate that loss of a2V increased Notch1 during late pregnancy and lactation, affecting steady‐state levels of Notch Signaling, which could account for insufficient lactation. Together, our results suggest that a2V‐KO mice display dysregulated Notch Signaling and improper ductal morphogenesis of the mammary gland. In summary, this is a first‐time report establishing V‐ATPase as a major developmental signal in the mammary gland. Our findings on dysregulated V‐ATPase and Notch in mammary gland have broader implications in increased susceptibility for breast cancer.Support or Funding InformationThis study is funded by the Clinical Immunology Laboratory, Rosalind Franklin University, North Chicago, IL