An essential role of CBL and CBL-B ubiquitin ligases in mammary stem cell maintenance

Abstract

The ubiquitin ligases CBL and CBL-B are negative regulators of tyrosine kinase signaling with established roles in the immune system. However, their physiological roles in epithelial tissues are unknown. Here, we used MMTV-Cre-mediated Cbl gene deletion on a Cbl-b null background, as well as a tamoxifen-inducible mammary stem cell (MaSC)-specific Cbl and Cbl-b double knockout (Cbl/Cbl-b DKO) using Lgr5-EGFP-IRES-CreERT2, to demonstrate a mammary epithelial cell-autonomous requirement of CBL and CBL-B in the maintenance of MaSCs. Using a newly engineered tamoxifeninducible Cbl and Cbl-b deletion model with a dual fluorescent reporter (Cbl flox/flox ; Cbl-b flox/flox ; Rosa26-CreERT; mT/mG), we show that Cbl/Cbl-b DKO in mammary organoids leads to hyperactivation of AKT-mTOR signaling with depletion of MaSCs. Chemical inhibition of AKT or mTOR rescued MaSCs from Cbl/Cbl-b DKO-induced depletion. Our studies reveal a novel, cell-autonomous requirement of CBL and CBL-B in epithelial stem cell maintenance during organ development and remodeling through modulation of mTOR signaling.

mTOR signaling in stem and progenitor cells

“…4F). Organoid formation could be rescued by administration of an AKT inhibitor or rapamycin, indicating restoration of MaSC function (Mohapatra et al, 2017). More work is required to fully understand the role of mTOR signaling in MaSCs, but this initial report indicates that mTOR regulates a switch between MaSC self-renewal and differentiation, and that mTORC1 activity must be tightly regulated to prevent MaSC exhaustion.…”

“…However, despite our growing understanding of MaSCs, relatively little is known about mTOR in these cells. Recent work has demonstrated that deletion of the E3 ubiquitin ligases Cbl and Cblb impairs mouse mammary gland development in vivo, and abrogates MaSC function in vitro (Mohapatra et al, 2017). Cbl ubiquitin ligases limit receptor tyrosine kinase (RTK) signaling by mediating the proteosomal degradation of RTKs (Petrelli et al, 2002;Soubeyran et al, 2002).…”

“…Cbl ubiquitin ligases limit receptor tyrosine kinase (RTK) signaling by mediating the proteosomal degradation of RTKs (Petrelli et al, 2002;Soubeyran et al, 2002). Notably, the deletion of Cbl and Cblb increases EGFR activation and mTOR activity in MaSC organoids, and impairs organoid formation (Mohapatra et al, 2017) (Fig. 4F).…”

“…There is a growing appreciation that stem cells exist within complex environments composed of distinct cell populations (Scadden, 2014), and with that has come the realization that mTOR activity within these populations is different. Indeed, although stem cells require restrained mTORC1 activity to maintain the capacity for self-renewal and differentiation (Lee et al, 2010b(Lee et al, , 2017Hobbs et al, 2010Hobbs et al, , 2015Mohapatra et al, 2017), many progenitor cells require elevated mTORC1 activity to support their expansion and differentiation (Hobbs et al, 2015;Liu et al, 2017;Yang et al, 2015b). Continued work on the heterogeneity of stem cell niches will likely reveal that distinct stem and progenitor cell populations have unique mTOR signaling requirements.…”

CBL mutations promote activation of PI3K/AKT signaling via LYN kinase

“…Along these lines, hematopoietic stem and progenitor cells from Cbl mutant mice showed increased PI3K/AKT, MAP kinase and STAT5 signaling, though there were differences in the signaling effects depending on the subpopulation analyzed (24). Third, two studies evaluated the effects of CBL mutants on a Cbl -/-/Cblb -/genetic background (35,41), which has the potential to augment or alter the signaling phenotype conferred by CBL mutant protein due to functional redundancy between Cbl and Cblb in mice (42)(43)(44). In patients with myeloid malignancies, CBLB mutations are uncommon and co-mutation of CBL and CBLB has not been described, suggesting that recurrent mutations in CBLB do not contribute significantly to oncogenesis.…”