1. Department of Cancer Biology and,
2. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA
3. Department of Pathology and,
4. Department of Pathology and,
5. Department of Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA
6. These authors contributed equally to this work.

Correspondence to: Thomas M. Roberts^1,3Jean J. Zhao^1,3,5 Correspondence and requests for materials should be addressed to T.M.R. (Email: thomas_roberts@dfci.harvard.edu) or J.J.Z. (Email: jean_zhao@dfci.harvard.edu)

Class IA PI(3)Ks are heterodimeric lipid kinases consisting of a p110 catalytic subunit complexed to one of several regulatory subunits, collectively called p85 (refs 4, 5). In response to stimulation by growth factor, p110 subunits catalyse the production of the lipid second messenger phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P₃) at the membrane^{1, 2, 3, 4}. This second messenger in turn activates the serine/threonine kinase Akt and other downstream effectors^{9, 10}. Knockout mice for either p110 or p110 die early in embryonic development^{11, 12}. However, recent studies using conditional knockout strategies⁸ and isoform-specific small molecule inhibitors⁷ showed that p110 is important in growth-factor signalling, whereas a kinase-inactive knock-in mouse model showed that insulin responses depended on the catalytic activity of p110 (ref. 6).

To investigate the role(s) of p110 in cell, tissue and organismal physiology and to examine it as a potential therapeutic target in cancer, we generated mice carrying a conditional Pik3cb allele (Supplementary Fig. 1). We first investigated the role of p110 in insulin action. Because liver is the major insulin-responsive organ, we examined the effects of p110 loss on hepatic insulin function. To achieve liver-specific deletion of p110, we injected the tail veins of p110^flox/flox mice with adenoviruses expressing -galactosidase (Ade-LacZ) or Cre recombinase (Ade-Cre) to generate matched cohorts of control mice and mice with hepatocyte-specific deletion of p110. Additional cohorts of wild-type animals were subjected to Ade-Cre or Ade-LacZ, allowing us to rule out potential non-specific Cre effects (data not shown). A more than 90% decrease in p110 protein was seen in the livers of Ade-Cre-injected mice, whereas p110 expression remained unchanged in the livers of the control mice and muscle tissues from both groups as measured by western blotting (Supplementary Fig. 2a, b). Consistent with previous findings^{6, 7} that the kinase activity of p110 has only a minor function in insulin signalling, we saw no significant change in Akt phosphorylation in response to insulin challenge in livers lacking p110 (Supplementary Fig. 2a). However, mice deficient in hepatic p110 had higher levels of insulin in the blood than control animals when fasted (Fig. 1a). These animals also showed a lower tolerance of glucose and sensitivity to insulin on challenge by intraperitoneal injection of glucose or insulin (Fig. 1b, c). Mice deficient in hepatic p110 produced more glucose than control animals did in a pyruvate challenge test (Fig. 1d). An analysis of lipogenesis showed no significant changes in serum triglycerides, fatty acids and cholesterol levels when p110 was deleted from liver (Supplementary Fig. 3), but leptin levels were elevated compared with those in control animals, as was seen in p110 kinase-dead knock-in animals⁶ (Supplementary Fig. 3). Of a panel of gluconeogenic genes, only that encoding phosphoenolpyruvate carboxykinase (PEPCK) was increased in p110-deficient livers (Supplementary Fig. 4). PEPCK promotes the production and synthesis of glucose in liver, resulting in a greater release of glucose into blood. This result therefore provides at least a partial explanation for the metabolic phenotypes observed. Although these findings indicate that p110 might contribute to metabolic regulation through a kinase-independent mechanism, we cannot rule out the involvement of the catalytic role of p110 in insulin responses. Our observations are in line with earlier work⁷ in which a p110-specific small-molecule inhibitor was used to demonstrate that acute blockage of the kinase activity of p110 had little effect on insulin action. In addition, another study¹³ found that mice doubly heterozygous for knockout of p110 and p110 showed decreased sensitivity to insulin with no apparent changes in Akt phosphorylation.

Figure 1: Mice with liver-specific deletion of p110 show resistance to insulin and intolerance of glucose.

Mice 8–10 weeks old were injected with adenoviruses expressing LacZ or Cre recombinase. Two weeks after injection, metabolism was analysed as follows: fasted serum insulin levels (a); glucose tolerance test (b); insulin tolerance test (c) (results represent blood glucose concentrations as a percentage of starting value at time zero); pyruvate challenge (d). Data are shown as means and s.e.m. Asterisk, P < 0.05; two asterisks, P < 0.01; three asterisks, P < 0.001 (t-test).

High resolution image and legend (81K)

To obtain cells for detailed signalling studies, mouse embryonic fibroblasts (MEFs) were isolated from floxed embryos and their wild-type littermates, as described in Supplementary Information (Supplementary Fig. 5a–c). MEFs lacking p110 proliferated significantly more slowly than parental (p110^flox/flox) or wild-type (p110^+/+ after Cre) MEFs (Fig. 2a). To obtain a second, more easily renewed supply of knockout cells, we established immortalized p110^flox/flox and p110^+/+ MEFs by means of infection with a retrovirus encoding a dominant-negative form of p53 (DNp53)¹⁴. We also generated an add-back line by introducing haemagglutinin (HA)-tagged human p110 to DNp53-immortalized p110^flox/flox MEFs. These immortalized MEFs were then treated with Ade-Cre to yield the following MEF lines: KO (from p110^flox/flox) and KO+ (from the add-back). For wild-type control MEFs, designated WT, we used DNp53-immortalized p110^flox/flox cells without Cre treatment interchangeably with Cre-treated DNp53-immortalized p110^+/+ MEFs, because no significant differences were ever seen between these two possible controls. Deletion of p110 had no obvious negative effect on the phosphorylation of Akt in either primary MEFs or DNp53-immortalized MEFs in response to stimulation by insulin, epidermal growth factor (EGF) and platelet-derived growth factor (Fig. 2c and Supplementary Fig. 6a–c). However, a moderate diminution in the phosphorylation of the S6 ribosomal protein (S6RP) at Ser 235/Ser 236 was detected in these KO cells in response to insulin or serum (Supplementary Fig. 7). Previous studies have implicated p110 in signalling elicited by G-protein-coupled receptors (GPCRs)^{15, 16}. We found consistently that both phospho-Akt and phospho-S6RP levels were decreased in response to lysophosphatidic acid (LPA) in cells lacking p110 (Fig. 2d and Supplementary Fig. 8a).

Figure 2: Analyses of the effects of p110 deletion on cell growth and signalling.

a, Loss of p110 retards cell growth of primary MEFs. Representative data are shown from triplicate experiments. b, Loss of endogenous p110 protein in immortalized MEFs. IP, immunoprecipitation. c, Loss of p110 has no negative effect on insulin signalling. d, Loss of p110 impairs LPA-induced signalling. e, Comparison of the responses of KO, KO and WT MEFs to stimulation with EGF, insulin-like growth factor (IGF) or LPA. f, BrdU incorporation assay. g, Transferrin uptake in various MEF lines is shown as indicated. Transferrin is labelled red and 4,6-diamidino-2-phenylindole blue.

High resolution image and legend (117K)

To dissect the potential kinase-dependent and kinase-independent roles of p110, we reconstituted KO MEFs with a kinase-inactive allele of HA-tagged human p110, using the previously reported K805R mutation (KR)¹⁷ to generate the KO+KR MEF line. Though the expression of KR was lower than that of the WT add-back construct, it was expressed at a level slightly higher than endogenous p110, and expression levels of p110 were unchanged (Supplementary Fig. 9a, and data not shown). Loss of lipid kinase activity in the KR cells was confirmed by lipid kinase assay^{8, 18} after immunoprecipitation with anti-p110 (Supplementary Fig. 9b). We then examined the effect of WT or KR add-back on the altered signalling seen after the loss of p110. The decrease in both phospho-Akt and phospho-S6RP in response to stimulation with LPA observed in KO cells was restored by adding back WT but not the KR allele of p110 (Fig. 2d and Supplementary Fig. 8b), suggesting a catalytic function for p110 in LPA signalling. This seems to be unique to p110, because loss of p110 has no obvious effect on LPA signalling (Fig. 2e). The lower phospho-S6RP levels in KO cells were restored by both WT and KR add-backs in response to insulin or fetal bovine serum (FBS) (Supplementary Fig. 7, and data not shown), suggesting a scaffolding role of p110 in signalling by insulin and growth factors. However, our MEF data do not rule out a role for p110 in classical signalling by PI(3)K in other circumstances. For instance, when we ablated p110 in our earlier work, residual phosphorylation by Akt was observed in response to growth factors⁸. Because MEFs express p110 and p110 and not other class I PI(3)Ks, this residual signal was presumably transduced by p110. We also note that p110 ablation removes the protein as a competitor for p110 on receptors, which may allow any decrease in signalling caused by p110 loss to be masked or compensated for by an increased signal flux through p110.

To test the kinase-dependent and/or kinase-independent effects of p110 on cell proliferation, we studied cell cycle kinetics by first synchronizing cells by serum starvation and then measuring the proportion of cells in S phase with the incorporation of bromodeoxyuridine (BrdU) after re-feeding. Whereas KO cells had a delayed peak of BrdU incorporation, KR reconstituted cells showed a similar incorporation of BrdU to that of WT and KO+ cells (Fig. 2f). Consistently, KO+KR MEFs showed proliferation rates similar to those of WT and KO+ cells (Supplementary Fig. 9c), suggesting a kinase-independent role of p110 in cell proliferation.

Because previous studies have found p110 associated with members of the Rab family of small G proteins and clathrin-coated vesicles¹⁹, we measured transferrin uptake in KO MEFs and found it to be defective compared with that in WT and KO+ MEFs (Fig. 2g). Interestingly, normal uptake of transferrin was restored by the KR construct (Fig. 2g). Although there is ample published evidence indicating the importance of transferrin uptake for the growth of a variety of cell types²⁰, it is not clear whether the defect in transferrin uptake is a primary cause of the growth defect observed here.

Class IA PI(3)Ks have been clearly implicated in cancer^{21, 22, 23, 24}, with much recent work delineating the role of p110 in cancer^{25, 26, 27}. To study a potential role of p110 in oncogenic transformation, we performed focus formation assays by infecting monolayers of DNp53-immortalized MEFs with retroviruses expressing various oncogenes. Oncogenic HRas-G12V and EGFR-Del (L747–E749, A750P) retroviruses efficiently raised foci in WT cells but failed to transform KO MEFs (Fig. 3a). The decreases in foci seen in KO MEFs were actually more pronounced than those seen in p110 KO MEFs (Supplementary Fig. 10). Transformation was fully restored in KO+ cells but partly restored in KO+KR cells (Fig. 3a), suggesting that both the kinase activity and kinase-independent functions of p110 may have a function in oncogene-induced transformation.

Figure 3: Kinase activity of p110 contributes to transformation both in vitro and in vivo.

a, Focus formation assay in KO and reconstituted MEFs. Results are shown as means and s.e.m. for four independent experiments (asterisk, P < 0.05; two asterisks, P < 0.01, t-test). b, Effects of p110 or p110 ablation on tumorigenesis caused by PTEN loss in the anterior prostate. c, Quantification of the weight (means and s.e.m.) of anterior prostate tissues of the indicated strain (n = 10 per group; asterisk, P < 0.001, t-test). d, A model for the elevation of basal PtdIns(3,4,5)P₃ signals derived from p110 catalytic activity induced by PTEN loss.

High resolution image and legend (149K)

PTEN, a lipid phosphatase, functions to oppose class IA PI(3)K kinase activity. Loss of Pten expression is a common event in many solid tumours²⁸. The key challenge is to identify which p110 isoform's catalytic activity is unshackled by Pten loss in any given tumour. To test for a role of p110 in tumorigenesis driven by Pten loss, we generated mice that carried the Pten^flox/flox (ref. 29) and p110^flox/flox alleles, as well as a probasin-driven Cre transgene³⁰, to specifically delete Pten and p110 in prostatic epithelium. Prostates had a normal appearance in the absence of p110 (Fig. 3b, c). Prostate tissue lacking Pten expression showed universal high-grade prostatic intraepithelial neoplasia in the anterior lobe by 12 weeks. Ablation of p110 blocked the tumorigenesis caused by Pten loss in the anterior prostate (Table 1 and Fig. 3b, c). The loss of Pten was confirmed by genomic DNA analysis after laser-capture-assisted microdissection of single epithelial layers and by western blotting (Supplementary Fig. 11, and data not shown). Whereas Cre-mediated loss of Pten efficiently activated Akt in the prostate as judged by its phosphorylation on Ser 473, additional ablation of p110 diminished the phospho-Akt levels (Fig. 3b), suggesting that p110 catalytic activity contributes to tumorigenesis. More surprisingly, when we performed the same set of experiments using p110 ablation, we saw no changes either in tumour formation or in Akt phosphorylation (Table 1 and Fig. 3b, c). Again, the complete excision of p110 in tumour tissues was confirmed by multiple measures (Supplementary Fig. 12, and data not shown). It has been suggested that p110 and p110 generate distinct pools of PtdIns(3,4,5)P₃ (ref. 7). In response to insulin or other stimuli, an acute flux of PtdIns(3,4,5)P₃ is produced largely by p110 and is efficiently coupled to Akt phosphorylation. In contrast, p110 has been proposed to generate a basal level of PtdIns(3,4,5)P₃ with little effect on Akt phosphorylation⁷. It was shown that Akt phophorylation induced by Pten loss in vitro was sensitive to p110-specific inhibitors⁷. We propose that it is this basal PtdIns(3,4,5)P₃ signal that has been enhanced to drive transformation and Akt activation by Pten loss in the murine prostate (Fig. 3d). Alternatively, the differential effects of p110 and p110 ablation may arise because the signal activating PI(3)K is generated by an as yet unidentified GPCR or a receptor tyrosine kinase that functions through p110.

Table 1: Effects of ablation of p110 or p110 on prostate tumorigenesis induced by PTEN loss

Full table

Thus, our data suggest distinct functions for p110 and p110 in cell signalling and transformation. We have showed that p110 has an important physiological function in metabolic regulation and glucose homeostasis, perhaps involving a kinase-independent mechanism. A kinase-independent function of p110 was further suggested in controlling cell proliferation and trafficking in p110 KO MEFs and MEFs reconstituted with a WT or kinase-dead allele of p110. It would clearly be a mistake to overlook the contributions of p110 as a kinase. The basal PtdIns(3,4,5)P₃ pool catalysed by p110 seems to be 'silent' in response to stimulation by insulin and other growth factors, but becomes a 'powerhouse' to drive oncogenic transformation in the absence of PTEN, as evident in our mouse prostate tumour model. Taken together, our findings indicate that p110 may be an attractive target for kinase inhibitors in cancer treatment with minor metabolic disturbances.

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Supplementary Information

Supplementary information accompanies this paper.

Acknowledgements

We thank C. D. Stiles and J. D. Iglehart for advice, and H. Wu for providing floxed PTEN mice. This work was supported by grants from the National Institutes of Health (M.L., T.M.R. and J.J.Z.,), the Department of Defense for Cancer Research (J.J.Z.), the V Foundation (J.J.Z.) and the Claudia Barr Program (J.J.Z.). In compliance with Harvard Medical School guidelines, we disclose the consulting relationships: Novartis Pharmaceuticals, Inc. (M.L., T.M.R. and J.J.Z.).

Author Contributions Z.L., S.Z. and S.L. generated the floxed p110 mouse. S.J. carried out mouse tumorigenesis studies. Z.L and S.Z. performed MEF studies. P.L. performed in vivo metabolic studies. L.Z. performed transferrin uptake assays. J.Z. assisted in focus formation and BrdU incorporation experiments. S.S. and M.L. performed and interpreted pathological analyses of mouse prostate tumors. T.M.R. and J.J.Z. supervised the research, interpreted the data and wrote the paper. S.J., Z.L., S.Z., P.L., L.Z., S.L. and M.L. participated in the writing of the paper.

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