PTEN-Mediated Apical Segregation of Phosphoinositides Controls Epithelial Morphogenesis through Cdc42

Abstract

PTEN controls three-dimensional (3D) glandular morphogenesis by coupling juxtamembrane signaling to mitotic spindle machinery. While molecular mechanisms remain unclear, PTEN interacts through its C2 membrane-binding domain with the scaffold protein b-Arrestin1. Because b-Arrestin1 binds and suppresses the Cdc42 GTPase-activating protein ARHGAP21, we hypothesize that PTEN controls Cdc42-dependent morphogenic processes through a b-Arrestin1-ARHGAP21 complex. Here, we show that PTEN knockdown (KD) impairs b-Arrestin1 membrane localization, b-Arrestin1-ARHGAP21 interactions, Cdc42 activation, mitotic spindle orientation and 3D glandular morphogenesis. Effects of PTEN deficiency were phenocopied by b-Arrestin1 KD or inhibition of b-Arrestin1-ARHGAP21 interactions. Conversely, silencing of ARHGAP21 enhanced Cdc42 activation and rescued aberrant morphogenic processes of PTEN-deficient cultures. Expression of the PTEN C2 domain mimicked effects of full-length PTEN but a membrane-binding defective mutant of the C2 domain abrogated these properties. Our results show that PTEN controls multicellular assembly through a membrane-associated regulatory protein complex composed of b-Arrestin1, ARHGAP21 and Cdc42.

Disruption of epithelial architecture caused by loss of PTEN or by oncogenic mutant p110α/PIK3CA but not by HER2 or mutant AKT1

“…In the NMuMG cultures, lumen formation appeared to occur at around the three day stage and significant luminal apoptosis was not observed in cells analysed between 3 and 9 days (Figure 1g and Figure S1 and data not shown). Immunofluorescent analysis of 3D polarised NMuMG cell colonies implied a modest enrichment of PTEN near the apical surface of these cells ( Figure S2) that is similar but weaker than the localisation of the the apical membrane marker atypical PKC ( Figure S1) and consistent with previous data from MDCK and T84 cells [24] and from the chick epiblast [35].…”

“…Therefore we feel our data provide further support for the significance of polarity regulation in tumour development driven by PTEN loss and PIK3CA mutation, but not HER2 amplification. Here it seems significant that either loss of PTEN or oncogenic activation of p110 not only increase PtdInsP 3 levels but also remove spatiotemporal control over localisation-dependent functions for this lipid signal [2,15,24,31].…”

“…In these models, several types of primary and immortal mammary epithelial cells give rise to spherical colonies with a single hollow lumen. However, in separate studies, cells lacking PTEN [24], expressing oncogenic mutants of p110 [25] or AKT1 [26], or over-expressing HER2 [27], have each been found to display aberrant tissue architecture and fail to form a single hollow lumen. On the other hand, expression of mutant AKT1 E17K from the endogenous gene in engineered MCF10A cells did not cause morphological changes, in contrast to mutant PIK3CA expression [28].…”

“…Because lumen formation in different cell models appears to proceed both via spatial cell separation at a nascent apical surface and via apoptosis of residual luminal cells [29], the mechanisms by which PI3K pathway transformation affects lumen formation is unclear. Since there is an expectation that druggable steps downstream of PI3K, particularly AKT and perhaps mTOR may be responsible for the aberrant luminal cell survival, but other poorly characterised pathways may be responsible for the spatial co-ordination of cell separation [24,30,31], these issues could influence cancer treatment.…”

The microenvironmental determinants for kidney epithelial cyst morphogenesis

“…Nevertheless, both protrusive structures underscore the involvement of cellular protrusive activity during cyst formation and/or development. Both filopodia-like protrusive structure and cyst-released subcellular structure may be captured in the images in recent publications (Zeng et al, 2006;Martin-Belmonte et al, 2007;Tanimizu et al, 2007;) though they did not catch the attentions in those studies. Interestingly, MDCK cysts formed on Matrigel do not stay static but actually rotate dynamically on Matrigel (our unpublished data) as earlier reported (Zeng et al, 2006), which may explain the existence of microprotrusion structures at the cyst outer surface which is not in direct contact with Matrigel (Fig.…”