Aberrant PI3K signaling has been studied extensively in cancer. This study provides evidence that PIK3CA mutations may contribute to tumorigenicity through both AKT-dependent and AKT-independent mechanisms. In the absence of AKT activation, PDK1 may transmit an alternative signal that engages alternative downstream substrates such as SGK3 in PIK3CA-mutant cancer cells. This study thereby nominates both PDK1 and SGK3 as key oncogenic effectors downstream of activating PIK3CA mutations.
Our AKT signaling results differ from studies in which mutant PIK3CA
was expressed ectopically in cell culture/chick embryo models (Isakoff et al., 2005
; Kang et al., 2005
; Zhao et al., 2005
) or introduced by knock-in methods into immortalized breast epithelial cells (Gustin et al., 2009
). On the other hand, multiple experimental lines of evidence presented herein suggest that steady-state AKT signaling is reduced in many malignant contexts where PIK3CA
-mutations are present in situ. Moreover, the AKT and PDK1 activation patterns that we observe are consistent across many human cell lines and clinical breast tumor specimens, in line with published observations (Stemke-Hale et al., 2008
). Of course, we cannot completely exclude the possibility that AKT signaling operates at very low levels in some PIK3CA
-mutant cancers, even when poorly detectable by conventional methods. However, our findings suggest that in some settings the functional “output” of PIK3CA
mutations differs importantly from that of deregulated PI3 kinase activity observed in PTEN-null cells.
Studies that employ selective small molecule AKT inhibitors may help clarify the nature of AKT dependency in PIK3CA
-mutant cancers. Consistent with our results, sensitivity in vitro to inhibition by small molecule allosteric AKT1/2 inhibitors has been correlated strongly with AKT phosphorylation in human cancer cell lines (She et al., 2008
). In the study by She et al., (2008)
, p-AKT was detectable in MCF7 cells and suppressed by an AKT inhibitor. Nonetheless, MCF-7 cells exhibited an attenuated sensitivity to pharmacologic AKT1/2 inhibition compared to several cancer cell lines with markedly elevated p-AKT levels (EC50 ~1 uM; She et al., 2008
). In our hands, AKT phosphorylation occasionally becomes detectable in MCF-7 cells after prolonged cultivation in vitro
(e.g., ~1 year; K.M.V., unpublished observations), suggesting that variances in cell culture conditions may influence p-AKT levels in some cases. Other studies have found PIK3CA
-mutant breast cancer cell lines to be generally more sensitive than PIK3CA
-mutant colon cancer cell lines to small molecule AKT inhibition, suggesting that cell lineage effects may also modulate this pharmacologic sensitivity (Greshock, 2008
). Altogether, these results endorse the notion that high p-AKT levels denote an AKT dependency in PIK3CA
-mutant cancer cells, while allowing for the possibility of AKT-independent signaling in settings where steady-state p-AKT is reduced.
Both “upstream” activation (through receptor tyrosine kinases, RAS signaling, or deficiencies in feedback regulation) and PTEN function provide important modes of PI3K regulation in cancer. Interestingly, several PIK3CA
–mutant breast cancer cell lines with high p-AKT examined herein have been shown previously to harbor either ERBB2
overexpression/amplification (MDA-MB-453 and HCC-1954) (Blend et al., 2003
; Miller et al., 1996
) or high levels of activated EGFR (BT-20) (Zhang et al., 2002
). Also, PIK3CA
-mutant cells and human breast tumors with low p-AKT tend to express wild-type PTEN and reduced 3’-phoshpatidylinositides, whereas PTEN knockdown results in robust AKT phosphorylation. Thus, many PIK3CA
-mutant cancers that depend on AKT signaling may contain concomitant upstream signal deregulation (e.g., RTK/RAS activation or loss of feedback regulation) or PTEN deficiency (Oda et al., 2005
). However, PIK3CA
mutation by itself is neither necessary nor sufficient for full AKT pathway activation when it occurs in situ.
-mutant cells exhibit robust p-PDK1 expression and membrane recruitment, regardless of AKT signaling. PDK1 exists in a constitutively active conformation that is not known to be further augmented by upstream signals (Casamayor et al., 1999
). Oncogenic membrane recruitment of PDK1 appears to depend at least partially on mutant PIK3CA
(). Differential membrane localization of PDK1 compared to AKT may relate in part to differing PH-domain phosphatidylinositide binding affinities, since prior studies suggest that PDK1 may exhibit ~20 fold higher affinity for PI(3,4,5)P3
than AKT (Stephens et al., 1998
; (Currie et al., 1999
). PDK1 recruitment might also be aided by a kinase-independent function of mutant PIK3CA
, such as stabilization of a RTK-adapter protein complex that permits phosphatidylinositol-independent PDK1 membrane localization. Towards this end, PDK1 was shown to bind the Grb14 adapter protein in a PH-domain independent fashion (King and Newton, 2004
). Thus, while AKT membrane localization (and subsequent activation) depends critically on elevated PI(3,4)P2
levels, PDK1 localization may require a lesser degree of phosphatidylinositide accumulation.
-mutant cancer cells with low AKT signaling exhibit a selective dependency on SGK3 for viability. The SGK family of AGC kinases shares more than 50% identity with the AKT kinase domain (Tessier and Woodgett, 2006
). SGK proteins become direct PDK1 substrates as a result of C-terminal hydrophobic motif (HM) phosphorylation (Frodin et al., 2002
; Sarbassov et al., 2005
). Recent evidence suggests that HM phosphorylation within SGK is mediated by the TORC2 complex (Garcia-Martinez and Alessi, 2008
), and that SGK proteins may function as critical mediators of cell growth downstream of rictor/TORC2 (Jones et al., 2009
; Soukas et al., 2009
). In accordance with previous findings, here we show that PDK1-dependent SGK3 activation is under PI3K regulation in PIK3CA
-mutant cancer cells. The SGK3 PX domain exhibits a particular affinity for phosphatidylinositol 3’ phosphate (Tessier and Woodgett, 2006
), which localizes SGK3 to endosomal membranes (Figure S9A
) (Virbasius et al., 2001
). SGK3 recruits PDK1 to endosomes upon PI-3 kinase activation (Slagsvold et al., 2006
). These phenomena raise the intriguing possibility that mutant p110α may convey its oncogenic signal from or within endosomal compartments.
The precise oncogenic signal elaborated by SGK3 remains obscure, as few endosomal SGK3 substrates have been identified. The E3 ubiquitin ligase AIP4 was identified as an endosomal SGK3 substrate whose phosphorylation leads to stabilization of CXCR4 and promotes breast cancer metastasis (Slagsvold et al., 2006
). The mammalian homologue of Drosophila Flightless I (FLI-I) is another putative SGK3 substrate (Xu et al., 2009
). FLI-I phosphorylation may protect hematopoietic cells from death induced by cytokine withdrawal. Additional studies should inform whether AIP4 or FLI-I confer tumorigenic signals relevant to the PIK3CA
Together, our observations offer a modified conceptual framework for oncogenic PI3K signaling. In the setting of PTEN deficiency, excess upstream activation, or defective feedback regulation, PIK3CA-mutant cancers may elaborate sufficient membrane 3’-phosphatidylinositols to recruit both AKT and PDK1 to the plasma membrane. When this occurs, tumors will exhibit a robust (and usually “addictive”) AKT-dependent signal. On the other hand, if PTEN function remains intact and upstream or feedback pathways are not fully dysregulated, PIK3CA-mutations may transduce an AKT-independent signal that engages PDK1 and SGK3. Unlike AKT activation, which requires simultaneous and sufficient membrane recruitment of multiple proteins following 3’-phosphatidylinositol synthesis, a moderate degree of PDK1 (endosomal) membrane localization may be all that is required to activate SGK3 in a PI3K-dependent manner.
In summary, this study has uncovered an AKT-independent signal transmitted downstream of many PIK3CA
-mutant cancers. These results provide a mechanistic basis for recent observations suggesting biological differences between PIK3CA
and PTEN mutation in human tumors (Saal et al., 2007
; Stemke-Hale et al., 2008
). Moreover, they suggest that inhibition of PI3K, PDK1 and downstream effectors may in some cases be more effective than inhibition of AKT. Several PI3K pathway inhibitors have entered clinical trials. Knowledge of the differential signaling pathways could thus inform the design of clinical trials in cancers defined by PI3K pathway mutations.