CBIO-36p21 REGULATES STEM CELL FREQUENCY IN A SEX-SPECIFIC FASHION

Brain cancers affect more males for unknown reasons. Male prevalence in brain tumors exists for multiple tumor types across all ages, including young children. Thus sex differences in brain tumor rates cannot primarily be a consequence of acute sex hormone effects. We showed sex differences in GBM might be partly explained by sexually dimorphic RB regulation. In our model, male and female astrocytes were rendered null for neurofibromin and p53 function, and treated with EGF. Male astrocytes exhibited more rapid and higher magnitude RB phosphorylation and activation of E2F-dependent transcription. These differences were correlated with differences in proliferation, clonogenicity, soft agar colony formation, and in vivo tumorigenicity. The current study investigated the molecular basis for sex differences in RB regulation. We first surveyed the expression levels of three major RB regulators, p21/CIP1, p27/KIP1 and p16 in Nf1-/-;DNp53 astrocytes. Both P21 and P27, but not p16, were expressed. To examine p21 and p27 functions we deleted each in Nf1-/-;DNp53 astrocytes using CRISPR/Cas9. Using growth and stem cell assays, we found that while p27 loss had no effect on stem cell frequency, depletion of p21 greatly increased stem cell abundance, but in female cells only. These findings indicated that in female, but not male Nf1-/-;Dnp53 astrocytes, p21 performs a critical tumor suppressor function. In order to gain more insight into sex differences in p21 regulation we measured its induction after treatment with etoposide. Etoposide treatment resulted in significantly greater induction of p21 expression in female compared to male Nf1-/-;DNp53 astrocytes. We corroborated sex differences in p21 expression in a panel of human GBM specimens. Together these data suggest that p21 is differentially regulated by a p53 independent mechanism in male and female Nf1-/-;DNp53 astrocytes resulting in unequal numbers of clonogenic stem cells. These findings are relevant to sex differences in GBM.