Tristetraprolin (TTP) Expression Is Required For Maintenance of the Mammary Progenitor Cell Population

STEDILE, MICAELA; BECKERMAN, INÉS; GODDIO, MARÍA VICTORIA; PÉREZ CUERVO, LOURDES; GARCÍA SOLÁ, MARTÍN; MEDINA, MARÍA VIRGINIA; RAIMONDI, ANA; COSO, OMAR; KORDON, EDITH CLAUDIA

Washington DC

Encuentro; ASCB-EMBO 2019 meeting; 2019

American Society for Cell Biology (ASCB) y European Molecular Biology Organization (EMBO)

Messenger RNA (mRNA) stability is regulated mainly by proteins that bind sequences enriched in adenine and uracil in their 3´untranslated regions, called collectively AU‐binding proteins (AUBPs). Tristetraprolin (TTP) is an AUBP that promotes mRNA degradation of proteins involved in inflammation, proliferation and tumor invasiveness. We have previously reported that TTP expression is down‐regulated in breast cancer compared to normal mammary gland, where TTP reaches its highest levels during lactation. In addition, bitransgenic females WAP‐Cre x TTPfl/fl mice (named MG TTP‐KO), showed that reduction of TTP levels in the lactating mammary gland resulted in premature involution associated to the increase of TNFα, IL‐6 and LIF expression, STAT3 activation and massive cell death. Therefore, we concluded that TTP expression in the mammary epithelium is required for lactation maintenance. Then, as it has been reported that WAP‐expressing cells behave as a pregnancy‐induced mammary progenitor subpopulation and analysis of RNA‐seq data indicates that TTP is up‐regulated in mammary progenitor cells, our next goal was to determine whether expression of this AUBP is relevant for the maintenance of the mammary stem cell compartment. Our results showed that upon successive pregnancies MG TTP‐KO mice exhibited underdeveloped lactating glands that also presented decreased pre‐neoplastic lesions when crossbred with WAP‐Cre/RasG12D mice. Moreover, mammary glands from total TTP‐KO mice displayed underdeveloped ductal network upon transplantation into syngeneic mammary cleared fat pads. This suggested that diminished expression of TTP in the mammary progenitor compartment caused impairment of mammary gland development and differentiation. To verify this hypothesis, we generated TTP knockdown cells (TTP‐KD) by stable transfection of stem‐like HC11 mammary epithelial cells with specific TTP‐shRNA constructs. Interestingly, these cells exhibited impaired survival rates (by MTS analysis), increased apoptosis (by TUNEL), and induction of pro‐apoptotic proteins (by Western blot, WB). Besides, TTP‐KD cells also displayed high expression of inflammatory cytokines (analyzed by RT‐qPCR), increased levels of STAT3 and P‐STAT3 as well as p65/RelA and p38 phosphorylation, but inhibition of ERK 1/2 activation (studied by WB and treatment with specific inhibitors). Importantly, the TTP‐KD cells revealed a substantial decreased capacity to form mammospheres in 3D culture and to repopulate cleared fat pads of virgin BALB/c female mice. Taking together, our results indicate that TTP expression is required for mammary progenitor cell survival by preventing spontaneous pro‐inflammatory and stress‐associated events, which are able to induce mammary stem cell death.