PIK3CA Cooperates with Other Phosphatidylinositol 3¶-Kinase Pathway Mutations to Effect Oncogenic Transformation

Mutations in genes functioning in different pathways frequently occur together in the same cancer, whereas mutations in the same pathway tend to be mutually exclusive. However, the majority of colon, breast, and endometrial cancers that possess mutations in PIK3CA, the catalytic subunit p110alpha of phosphatidylinositol 3'-kinase (PI3K), also possess mutations or alterations in genes upstream of PI3K such as Ras, ERBB2/ERBB3, or PTEN. PIK3CA mutations occur almost exclusively in invasive tumors, whereas upstream mutations occur as frequently in early-stage and late-stage tumors, suggesting that PIK3CA mutation is a late-stage event that may augment earlier activation of the PI3K pathway. Consistent with this, we find that levels of p-AKT (Ser(473)) induced by mutant Ras or knockdown of PTEN were dramatically increased by addition of mutant PIK3CA. Soft agar assays revealed that anchorage-independent growth induced by mutant Ras was greatly increased in the presence of mutant PIK3CA. In breast, colon, and endometrial cancers in which the PI3K pathway is activated by a combination of mutant PIK3CA and alterations in Ras, ERBB2/3, or PTEN, signaling to downstream elements such as Akt was mediated exclusively by the p110alpha isoform, rather than a combination of different PI3K isoforms. Our data therefore suggest that in tumors with co-occurring mutations in multiple components of the PI3K pathway, selective inhibition of the alpha isoform of p110 is an attractive therapeutic strategy, especially for late-stage tumors.

Class I PI3Ks are activated by receptor tyrosine kinases and comprise one of several signaling activities induced by activated Ras.Their activity is required for Ras contribution to cellular transformation (21) and is produced by four isoforms of the class I catalytic subunit (p110a, p110h, and p110y for class IA, and p110g for class IB; ref. 22).Although p110a is the only subunit in which activating mutations have been reported, the other three isoforms also have oncogenic potential (23).Indeed, p110h is more strongly activated than p110a in a PTEN-null prostate cancer cell line (PC-3; ref. 24).Thus, the activity of non-PIK3CA (non-p110a) isoforms, induced by upstream signaling events, might be functioning in early stages of tumor formation and augmented by activating mutations in PIK3CA to potentiate total PI3K signaling during malignant progression.Alternatively, the majority of PI3K oncogenic activity might always occur via PIK3CA, with a mutational event in this gene enforcing signaling duration or intensity requisite for later transformation events.
In this study, we first identify patterns of naturally occurring coexistent alterations in the PI3K pathway in endometrial and breast cancer.Second, we show that PI3K signaling is weakly activated by various upstream PI3K pathway alterations alone and is augmented when mutant p110a is coexpressed.Third, we show that mutant p110a, in combination with mutant Ras, contributes to efficient malignant transformation of immortalized human mammary epithelial cells (HMEC).Lastly, we show that the p110a isoform is most responsible for PI3K pathway activation in tumors with coexistent PIK3CA mutations and other PI3K activating alterations.

Materials and Methods
Cell lines and retroviral infection.The culture condition of cell lines is described in Supplementary data.HMLE (HMECs with LT and hTERT) cells and HMLE cells with pBabe-zeo-H-Ras (G12V) and pBabe-puro-H-Ras (G12V) were a kind gift from Dr. Robert A. Weinberg.pBabe-zeo-H-Ras (G12V) clone expressed H-Ras at a lower level than the pBabe-puro-H-Ras (G12V) clone (7.2-versus 12-fold in comparison with endogenous level; ref. 25).
To create amphotropic retroviruses, Phoenix cells were transfected with retroviral vectors using Lipofectamine 2000 (Invitrogen): pFB-neo-p110a [wild-type (WT) and mutant], pFB-neo-GFP (green fluorescent protein), and pLXSP3-puro-K-Ras (G12V).The resulting supernatants were used to infect PAE, U2OS, and HMLE cells.Drug selection was used to purify cell populations after infections.Cells were selected in neomycin (500-1,500 Ag/mL, 7 d) and puromycin (0.5-1.5 Ag/mL, 3 d).Retroviral vectors carrying only drug resistance genes were used as controls.PTEN knockdown was achieved by infection of retrovirus expressing PTEN-specific short hairpin RNA (shRNA; Open Biosystems) and drug selection in puromycin (0.5-1.5 mg/mL, 3 d).We successfully introduced mutant p110a (H1047R) and GFP with neomycin selection at 1,500 Ag/mL in HMECs, although neomycin had been used at 200 Ag/mL for LT selection (most of the cells were GFP positive in the neo-GFP clone; data not shown).
Endometrial tumor samples and genomic DNA.The clinical characteristics and genomic DNA extraction of 66 endometrial tumors were described previously (11,26).All of the patients provided informed consent for the research use of their samples, and the collection and use of tissues for this study were approved by the appropriate institutional ethics committees.
Statistical analysis.The association of variables was evaluated by the Fisher's exact test.P < 0.05 was considered to be significant in all the tests.
Quantification of protein and mRNA levels.A semiquantitative measurement of PTEN protein levels, p-ERBB2 (Tyr 1248 ), p-ERBB3 (Tyr 1289 ), total ERBB2, and total ERBB3 in breast cancer cell lines was determined from TIFF files of Western blots using Scion Image software. 5Protein levels across the Westerns were normalized using the common controls within each blot.The PTEN expression is considered ''reduced'' when the expression level is <25% compared with the average from positive cell lines.ERBB2 and ERBB3 are considered overexpressed when the mRNA level in expression array hybridization is more than 4-and 15-fold, compared with that of the nontumorigenic cell line MCF10A, respectively.''Intermediate'' ERBB3 expression corresponds to a 10-to 15-fold increase from that in MCF10A.Small interfering RNA transfection.Small interfering RNA (siRNA) was used to inhibit the expression of ERBB2, ERBB3, p110a, or p110h.The targeted sequences of ERBB2, p110a, and p110h siRNAs are 5 ¶-AAGGTGCTTGGATCTGGCGCT-3 ¶, 5 ¶-AAGGAGCCCAAGAATGCACAA-3 ¶, and 5 ¶-AAAGGGAGCGAGTGCCTTTTA-3 ¶, respectively.siRNA to ERBB3 (siGenome) was purchased from Dharmacon RNA Technologies.Cells were seeded at 2.0 Â 10 5 per six-well plate 24 h before transfection and transfected with 60 to 120 nmol/L siRNA duplexes using Lipofectamine 2000 (Invitrogen).Cells were collected 72 h after transfection and analyzed by immunoblotting.Suppression of p110a and p110h by siRNA was confirmed by real-time PCR.
Cluster analysis.Unsupervised Cluster analysis was done on an Affymetrix U133A-based breast tumor cell line data set (27) using Cluster and Treeview software.Average linkage clustering results using the 15,000 array elements, which exhibit the most variance across the data set, are reported.Red squares represent high and green squares represent low relative expression levels of ERBB2 and ERBB3.
Soft agar assays.PAE, U2OS, and HMLE cells were seeded at 1 Â 10 3 or 4 Â 10 3 per well in six-well dishes with a bottom layer of 0.6% agar in DMEM and a top layer of 0.35% agar in DMEM ( for PAE and U2AOS) or mammary epithelium growth medium ( for HMLEs).Fresh medium (0.5 mL; DMEM for PAE and U2OS, mammary epithelium growth medium for HMLEs) was added after 1.5 wk.Colonies (>0.2 mm in diameter) were counted after 2 to 3 wk.At least two independent assays were done in triplicate.

Results
PIK3CA mutations coexist with K-ras and PTEN mutations in endometrial carcinomas.We screened 79 endometrial carcinomas (66 clinical specimens and 13 cell lines) for mutations of K-ras, PIK3CA, and PTEN.The mutational status of 13 endometrial cancer cell lines is shown in Table 1A.Overall, the mutation frequency in 79 endometrial cancers is 19% for K-ras, 57% for PTEN, and 37% for PIK3CA.Tumors with K-ras mutation exhibited a tendency to carry PIK3CA mutation more frequently (9 of 15, 60%) than tumors without K-ras mutation (21 of 64, 33%), although statistical significance was not reached (P = 0.075, Fisher's exact test).We did not find any association between PTEN mutations and K-ras mutations (9 of 47 versus 6 of 32, P = 1.0;Table 1B ), suggesting that mutation of these two genes occurs independently.As shown in Table 1B, 24 of 30 (80%) tumors with PIK3CA mutations possess K-ras and/or PTEN mutations.
PIK3CA mutations are associated with ERBB2 and/or ERBB3 overexpression in breast cancer cell lines.We screened a panel of 47 breast cancer cell lines for activators of PI3K signaling.The mutational status of PIK3CA was determined by examination of the COSMIC database (Sanger Center).6 ERBB2, ERBB3, PTEN, and p-Akt (Ser 473 ) levels were assessed by expression array hybridization and/or Western blotting (Fig. 1A; summarized in Supplementary Table S2).We focused on ERBB2 and ERBB3 expression levels in breast cancer because (a) the association between PIK3CA mutations and ERBB2 overexpression is still controversial (28); (b) the ERBB2/ERBB3 heterodimer functions as an oncogenic unit (29); and (c) ERBB3 overexpression in breast cancer is also common (30).In addition, ERBB3 contains six docking and activation sites for PI3K, and p-ERBB3 is shown to activate the PI3K pathway (31).Micro-RNAs miR-125a and miR-125b, which suppress both ERBB2 and ERBB3 expression, are down-regulated in breast cancers (32), suggesting that ERBB2/ ERBB3 signaling is important for breast tumorigenesis.Relative to expression levels found in the nontumorigenic cell line MCF10A, we found ERBB2 and/or ERBB3 overexpression in 19 of 47 (40%) cell lines; 10 with ERBB2 overexpression and 13 with ERBB3 overexpression, including four (BT474, HCC202, UACC-812, and SUM225CEM) with overexpression of both molecules.PIK3CA mutations were positive in 13 (28%) cell lines (Fig. 1A ; Supplementary Table S2).Nine of 13 (69%) with PIK3CA mutations harbor ERBB2 and/or ERBB3 overexpression (P = 0.020, Fisher's exact test; Fig. 1B).On the other hand, reduced PTEN expression (11 of 47, 23%) was less common in cells with PIK3CA mutations (1 of 13, 8%), compared with those without PIK3CA mutations (10 of 34, 29%; P = 0.15).In addition, reduced PTEN expression was inversely correlated with ERBB2 and/or ERBB3 overexpression (P = 0.032).Elevated phosphorylation of Akt (20 of 47, 43%) was significantly associated with PIK3CA mutations and/or reduced PTEN expression (P = 0.019).In contrast to endometrial carcinomas, loss of PTEN expression is reported to be very rare in noninvasive breast cancer (ductal carcinoma in situ) and more common in advanced stage tumors (33).Thus, loss of PTEN may be a late event in breast cancer.Semiquantitative analysis of phospho-ERBB2 (Tyr 1248 ) and phoshpho-ERBB3 (Tyr 1289 ) levels (27) showed that overexpression of each molecule was well associated with the level of its phosphorylation and p-Akt (Fig. 1A; Supplementary Table S3).Six of nine PIK3CA mutated tumors showed high p-ERBB2 and/or p-ERBB3 levels (Supplementary Table S3).Our data indicate that PIK3CA mutations frequently coexist with active ERBB2/ERBB3 signaling in breast carcinomas.
Mutant p110A is more potent in phosphorylation of Akt than mutant Ras or reduced PTEN expression.According to our previous report that multiple alterations in the PI3K pathway could further activate PI3K signaling in endometrial cell lines (11), we hypothesized that the presence of the upstream PI3K activation might cooperate with mutant p110a to increase PI3K activity, and that the presence of mutant p110a might be important for oncogenic conversion or progression.Therefore, we introduced mutant K-(or N-) Ras (G12V), mutant p110a (H1047R), shRNAs that targeted PTEN, and combinations of these alleles into PAE (porcine aortic endothelial) and U2OS osteosarcoma cells by retrovirus-mediated gene transfer.Both PAE cells and U2OS cells do not harbor any mutations in Ras, PTEN, and PIK3CA, resulting in very low basal p-Akt levels.Either PTEN knockdown or mutant Ras expression effected the p-Akt at a much lower level than did mutant p110a alone in both cells (Fig. 2A and B).The phosphorylation level of FoxO1, FoxO3a, and GSK3h was enhanced by either mutant K-Ras or PTEN silencing when combined with mutant p110a (Fig. 2A).Transient introduction of mutant Ras increased the p-Akt level in U2OS cells stably expressing mutant p110a (Fig. 2C).In summary, either mutant Ras or PTEN silencing alone has limited effect on the activation of the PI3K pathway, but the effect is much more drastic when combined with mutant p110a.
Anchorage-independent growth is induced by mutant p110A in PAE and U2OS cells.We tested whether mutant p110a might induce oncogenic behavior (anchorage-independent growth) in the PAE and U2OS cell lines because both PAE (untransformed) and U2OS cells are unable to grow without attachment to a substratum (34).Stable expression of mutant p110a (H1047R) resulted in growth of colonies in soft agar in both cells (Supplementary Fig. S1A and B), suggesting that the higher PI3K activity via mutant p110a is involved in anchorageindependent growth in these cells.
Mutant Ras and p110A efficiently induced oncogenic transformation in immortalized HMEC cells.Because alterations of Ras or ERBB2 are observed in noninvasive tumors of the colorectum, breast, and endometrium, we hypothesized that activation of multiple Ras effectors is useful for tumor initiation and that higher PI3K activation via additional mutations in p110a

Cancer Research
Cancer Res 2008; 68: (19).October 1, 2008 is helpful for malignant progression of noninvasive tumors.In addition, because PIK3CA mutations without other alterations are rare in these tumor types, mutant p110a alone might be insufficient for oncogenic transformation.Mutant H-Ras (G12V) transforms immortalized HMECs only when it is highly overexpressed (25); we assumed that additional mutant p110a might increase transformation efficiency in cells expressing lower levels of mutant Ras.To test this hypothesis, we obtained HMECs previously immortalized with large T-antigen and TERT (HMLE), with and without various alleles of activated H-Ras (25), and introduced activating alleles of p110a, K-Ras, or PTEN-silencing shRNAs in various combinations to determine their relative transformation potential (summarized in Supplementary Table S4).
Akt was more potently activated by introduction of mutant p110a than by mutant (H-or K-) Ras or PTEN shRNA (Supplementary Fig. S2A and B).The transformation potential of these stable cell lines was then assessed by soft agar assay.Mutant p110a (H1047R) alone failed to cause anchorageindependent growth in the parental HMLE (Fig. 3A).In addition, mutant p110a plus partial reduction of PTEN expression by shRNA did not cause anchorage-independent growth.However, in HMLE sublines, which constitutively express zeo-H-Ras (G12V) or puro-H-Ras (G12V), addition of mutant p110a (H1047R) doubled the colony number relative to control GFP (Fig. 3A).Unlike mutant H-Ras, introduction of mutant K-Ras (G12V) alone did not induce anchorage-independent growth; however, the combination of mutant p110a (H1047R) and mutant K-Ras enabled cells to grow in soft agar (Fig. 3B).
p110A is the predominant form of PI3K activity in breast cancer cells with coexistent mutant p110A and overexpression of ERBB2 and/or ERBB3.To determine which p110 isoforms were activated by upstream PI3K pathway activating alterations in cells with PIK3CA mutation, we examined the effect of p110 isoformselective inhibitors in five breast cancer cell lines with coexistent PIK3CA mutations and ERBB2 and/or ERBB3 overexpression (SUM185PE, MDA-MB361, and T47D: ERBB3 overexpression; HCC202 and BT474: both ERBB2 and ERBB3 overexpression).Four types of p110 inhibitors were tested on each cell line at 2 or 5 Amol/L for 1 to 6 h.These include the selective p110h inhibitor TGX-286; the selective p110y inhibitor PIK39; PIK85, which selectively inhibits p110a, p110g, and p110y; and the pan-selective p110 inhibitor PI-103.The structure of these four compounds is available from ref. 35 and Supplementary Fig. S3.The IC 50 values of the four compounds were listed in Supplementary Table S5.Both PIK39 (p110y inhibitor) and TGX-286 (p110h inhibitor) failed to suppress PI3K signaling as assessed by phosphorylation of Akt (Ser 473 ) and GSK-3h (Ser 9 ), whereas PIK85 (p110a, p110g, and p110y inhibitor) drastically suppressed phosphorylation of these molecules to a level similar to that of PI-103, wortmannin, and Ly294002 (pan-selective agents) in all these breast cancer cell lines (Fig. 4A and B, and data not shown).The p110g hybridization signal is low to undetectable in the analysis of Affymetrix gene expression profiling data (27) in breast cancer cell lines, indicating that little, if any, of this mRNA is expressed in the breast, whereas p110a, p110h, and p110y are expressed at comparable and more robust levels.These data suggest that breast cancer cell lines with mutant p110a and overexpression of ERBB2 and/or ERBB3 are dependent on p110a activity, rather than other isoforms, for activation of the PI3K pathway.Transfection with p110a-specific siRNA, but not with p110h-specific siRNA, suppresses Akt phosphorylation in BT474 cells (Fig. 4C).The p-Akt level was also decreased in SUM185PE cells by p110a-specific siRNA (Fig. 4C).These data also suggest that p110a is the major activated form in these cells.
To identify the contribution of ERBB2 and/or ERBB3 overexpression on the activity of the PI3K pathway, we used siRNA knockdown to reduce the expression of ERBB2 or ERBB3 in BT474.Attenuating the expression of these proteins partially reduces the level of p-Akt (Fig. 4D).ERBB3 suppression reduced the p-Akt level in SUM185PE cells (Fig. 4D).In addition, serum starvation decreased the p-Akt level in all five cell lines not shown).These data indicate that activation of PI3K signaling depends not only on mutant p110a but also on ERBB2/ERBB3 overexpression and/or serum-derived growth factors.
p110A is predominantly active in colorectal cancer cells with double mutations of p110A and Ras.The pattern of coexistent alterations in the PI3K pathway in colorectal cancer is double mutations of PIK3CA and K-ras.To determine whether PI3K activity with these coexistent mutations also relies primarily on p110a, we tested the p110 isoform-selective inhibitors in two colorectal cancer cell lines with these double mutations (DLD1 and HCT116).The phosphorylation of Akt was sensitive to PIK85 and PI-103 but resistant to TGX-286 and PIK39 (Supplementary Fig. 4A  and B) in these two cells.These data suggest that double mutations of K-ras and PIK3CA predominantly activate the p110a isoform in colorectal cancer.

Discussion
These results highlight the important role of PIK3CA mutations during oncogenic transformation in multiple epithelial tumor types.In this study, we addressed the meaning of multiple alterations in the PI3K pathway by analyzing (a) the patterns of coexistent alterations, (b) the efficiency of PI3K activation by single alterations, (c) the oncogenic transformation by coexistent alterations, and (d) the p110 isoform selectivity.These issues are discussed in more detail below.
Coexistent PIK3CA mutations and other alterations in endometrial, breast, and colorectal cancers.Endometrial, breast, and colorectal cancers are three major tumor types that show a high frequency of PIK3CA mutations (8,11,36).Curiously, the partners of PIK3CA mutations differ between these tissues: PTEN mutations in endometrium, ERBB2 overexpression in breast, and K-ras mutations in colorectum were reported to commonly coexist with PIK3CA mutations (10)(11)(12).In this study, we further identified double mutations of K-ras and PIK3CA in endometrial cancer and the coexistence of PIK3CA mutation and ERBB2/ERBB3 signaling in breast cancer cell lines.Various patterns of coexistent alterations of PIK3CA and the genes upstream of PI3K suggest that the role of mutant PIK3CA might be different from the other alterations.Ras and ERBB2/ERBB3 are involved in various signaling pathways, whereas PTEN also has PI3K-independent functions.Therefore, the co-occurring alterations in the PI3K pathway may have two major roles: augmentation of the PI3K pathway and activation of other pathways.
Enhanced activation of the PI3K pathway by additional mutant p110A.Combined PIK3CA mutation and other alterations in the Ras-PI3K pathway (PTEN and/or K-ras mutations in endometrium, ERBB2 and/or ERBB3 overexpression in breast, and K-ras mutation in colorectum) have two common features: First, these other alterations already exist in noninvasive tumors, whereas PIK3CA mutation is rarely observed in noninvasive tumors.Second, the combined ratio (z40%) of other alterations is higher than that of PIK3CA mutations (20-40%) in these cancers (Table 1B; Supplementary Table S1).These data suggest that activation of the PI3K pathway via any WT p110 isoform is not sufficiently high in tumors with single alterations, although those tumors occasionally progress to invasive tumors without an additional PIK3CA mutation (possibly via other unknown alterations or compensation from other pathways).In our experiments, sole expression of mutant Ras or knocking down PTEN was less effective than mutant p110a, and addition of mutant p110a drastically increased p-Akt levels in PAE, U2OS, and HMLE cells.Samuels and colleagues (38) established isogenic HCT116 and DLD1 colorectal cell lines in which either the WT or mutant alleles of PIK3CA were disrupted.Because both HCT116 and DLD1 possess mutant K-Ras (G13D), their analysis compared mutant K-Ras alone versus double mutants of K-Ras and PIK3CA.The PIK3CA mutant clones showed higher levels of p-Akt and p-FoxO1/p-FoxO3a compared with PIK3CA WT clones in both cell lines.Their data implied that mutant Ras alone is not sufficient for full activation of the PI3K pathway and that addition of the endogenous level of mutant p110a enhances the pathway, including some downstream effectors such as FoxO1/FoxO3a.
Efficient oncogenic transformation by combined mutant Ras and mutant p110A.Oncogenic transformation by mutant p110a was reported in chicken embryo fibroblasts in vitro and in vivo (37,39).In addition, we found anchorage-independent growth by mutant p110a in PAE and U2OS cells (Supplementary Fig. S1A and B).However, it has not been clear whether mutant p110a alone is sufficient for malignant transformation in immortalized human epithelial cells.In HMLEs, mutant p110a failed to induce anchorage-independent growth (Fig. 3A), suggesting that activation of the PI3K pathway alone is insufficient for transformation.This is compatible with the observation that mutant p110a frequently coexists with other alterations in the Ras-PI3K pathway.Transformation by mutant Ras is well analyzed in various types of immortalized human epithelial cells (25,(40)(41)(42).However, low levels of mutant H-Ras alone failed to confer a full malignant phenotype (Fig. 3A; refs.25,40) or required another input in the PI3K pathway [constitutively active form of Akt (41) or SV40 small t antigen (42)].In addition, mutant K-Ras did not induce anchorage-independent growth in HMLE (Fig. 3B).These observations suggest that endogenous levels of mutant Ras alone are insufficient for full transformation in immortalized human epithelial cells.However, mutant p110a significantly increased the number of colonies in soft agar when combined with mutant H-Ras or K-Ras.In our experiments, mutant Ras itself activated the PI3K pathway at a weaker level, but additional mutant p110a clearly enhanced the level of p-Akt, suggesting that the activity of the PI3K pathway was more strengthened.Our data indicate that additional mutant p110a plays a significant role in tumorigenicity and suggest that the enhanced PI3K activity might be responsible for the oncogenic activity.
The role of PTEN alterations in tumorigenesis is still unclear and may be tissue type specific.PTEN knockdown was incomplete in HMLE cells in our soft agar assay, although p-Akt was enhanced.That might be why the transformation efficiency might not be affected by PTEN shRNA.In addition, reduced PTEN expression in breast cancer is rare in noninvasive tumors and is instead associated with tumor invasion (33).However, PTEN mutations are common in atypical hyperplasia of the endometrium, as well as K-ras mutations (19,43), and also frequent in ovarian endometriosis (44).In mouse models, either mutant K-Ras or conditional PTEN deletion within the ovarian surface gives rise to preneoplastic ovarian (endometriosis like) lesions and leads to invasive endometrioid ovarian cancer when combined (45).These observations indicate that PTEN alterations may have a similar effect with mutant K-Ras in tumor initiation and cooperate with mutant p110a in tumorigenesis of the endometrium and the ovary.Another possibility is that PI3K-independent PTEN functions (i.e., phosphatase-independent function) may be important in the tumorigenesis.Therefore, each mutation of K-ras, PTEN, and PIK3CA may have different meanings in endometrial tumorigenesis.
The model of tumorigenesis in colorectal, breast, and endometrial tumors with coexistent PIK3CA and other alterations is summarized in Fig. 5D.Mutant Ras (or ERBB2 overexpression, etc.) is important for tumor initiation, which might be achieved through multiple Ras effector pathways, including the mitogen-activated protein kinase, Ral guanine nucleotide exchange factor, and PI3K pathways (21,46,47).However, the PI3K activation is not saturated in these noninvasive tumors.Once PIK3CA is mutated during the latent phase, the PI3K pathway could be activated to a greater level, which may result in efficient malignant transformation of noninvasive tumors.Without PIK3CA mutations, the noninvasive tumors may need another input (possibly PI3K pathway independent) to become transformed or they may need a longer latent phase.It is still unclear whether the augmented PI3K pathway activation alone is responsible for the transformation efficiency in PIK3CA mutated tumors.Mutant p110a may have any functions of cross talk with the other pathways, which are already activated by the earlier alterations.In addition, the magnitude of PI3K pathway activation is not always linked to the transformation efficiency.In PAE cells, mutant K-Ras induced more colony numbers than mutant p110a in soft agar assay (data not shown), suggesting that the requirement for PI3K pathway enhancement depends on the cellular context.
Predominant p110A activation in tumors with coexistent mutant p110A and other PI3K-activating alteration(s).Our observations with p110 isoform-selective inhibitors highlighted the importance of p110a activity in tumors with mutant p110a and other alterations.We used PIK-85 to inhibit p110a, p110g, and p110y.p110g expression is suggested to be exclusively restricted to hematopoietic cells (48), and the expression in cancer cell lines was also at low to undetectable levels.PIK39 was useful to exclude p110y activation, as it is a highly selective p110y inhibitor.We had assumed that the higher PI3K activation seen with coexistent mutations in the PI3K pathway might be due to the activation of multiple p110 isoforms.In fact, we observed that several cancer cell lines with PTEN alterations alone (without PIK3CA mutations) in breast and endometrium showed some sensitivity to TGX-286 or PIK39 (data not shown), suggesting that loss of PTEN function is not always associated with activation of p110a.One possible explanation is that other alterations without a PIK3CA mutation may activate the PI3K pathway through multiple (or non-a) p110 isoform(s), and that isoform dependency might be switched to p110a once p110a is mutated.To clarify the mechanism of p110 isoform selectivity, cells from invasive and noninvasive tumors that do not harbor PIK3CA mutations but have alterations in other PI3K pathway elements might be studied to address this question.
Predominant p110a activation in PIK3CA mutant tumors indicates that p110a-selective inhibition might be a good therapeutic target in tumors with mutant p110a.Trastuzumab (Herceptin) is a well-established targeted therapy in breast cancer (49), but our data suggest that trastuzumab might not be sufficient to suppress the PI3K pathway in tumors with double alterations of ERBB2 and PIK3CA.p110a isoform-selective inhibition may be less toxic than pan-selective PI3K inhibition and may be more effective than trastuzumab in tumors with mutant p110a.

Conclusion
We confirmed that PIK3CA mutations frequently coexist with other alterations in the PI3K pathway in endometrium, breast, and colorectum.All cell lines with coexistent mutant p110a and other alterations showed p110a dependency and enhanced activity of the PI3K signaling pathway.Mutant p110a cooperates with mutant Ras to efficiently transform HMECs.Taken together, the high activity of p110a by a coexistent PIK3CA mutation with other alterations can be a critical step for malignant transformation.

Figure 2 .
Figure 2. Stably expressed mutant p110a strongly phosphorylates Akt and its substrates.A, stably expressed mutant Ras, mutant p110a, and PTEN shRNA and combination of these were established in PAE cell line.The phosphorylation level of Akt and its substrates was compared among these stable clones.B, stable clones were also established in the U2OS cell line, and p-Akt level was evaluated by Western blotting.C, mutant K-(or N-) Ras was transiently expressed in U2OS stable clones and the synergistic effect of mutant p110a and mutant Ras was examined by p-Akt level.

Figure 3 .
Figure 3. Anchorage-independent growth is induced by the combination of mutant Ras and mutant p110a in immortalized HMEC cells.A, soft agar assay in HMECs expressing mutant H-Ras and/or mutant p110a.One thousand cells were seeded and cultured for 14 d and stained with neutral red.Columns, mean number of soft agar colonies from three experiments; bars, SD.B, soft agar assay in HMECs expressing mutant K-Ras and/or mutant p110a.Four thousand cells were seeded and treated as described in A. Columns, mean number of soft agar colonies from three experiments; bars, SD.

Figure 4 .
Figure 4. Biochemical responses of various breast cancer cell lines with PIK3CA mutation and ERBB2 and/or ERBB3 overexpression to p110 isoform-selective inhibition.Cells were cultured for the times indicated and Western blotting extracts were prepared with selective inhibitors of p110 isoform(s).SUM185PE (A) and HCC-202 (B) were cultured in six-well dishes.The indicated concentrations of TGX-286, PI-103, PIK85, and PIK39 were added to the culture medium, and 1 or 6 h later cells were harvested into lysis buffer.C and D, silencing of p110a, p110h, ERBB2, or ERBB3 by siRNA in BT474 and SUM185PE cells.Cells were transfected with 60 to 120 nmol/L siRNA duplexes targeted against p110a, ERBB2, or ERBB3.siRNA duplexes that do not match known genes were used as a control [control (nonsilencing) siRNA; Qiagen, Inc.].Lysates of cells were immunoblotted with the indicated antibodies.siRNA concentrations: ERBB3-(1), 60 nmol/L; p110a, p110h, ERBB2, ERBB3-(2), and control, 120 nmol/L.

Figure 5 .
Figure 5. Sensitivity to p110a-selective inhibitors in various endometrial cancer cell lines with double mutations of PIK3CA and PTEN .HHUA (A ), HEC-59 (B ), and HEC-6 (C ) were treated with the indicated concentrations of PIK39, TGX-286, PIK85, and PI-103.Cells were harvested 3 h later into lysis buffer and lysates were immunoblotted with the indicated antibodies.D, model of tumorigenesis in tumors of endometrium, colon, and breast with PIK3CA mutations and other alterations.Addition of mutant p110a in noninvasive tumors with other upstream PI3K pathway input(s) causes more efficient transformation.

Table 1 .
Coexistent mutations of PIK3CA and K-Ras and/or PTEN in endometrial cell lines A. Mutational status of PIK3CA, K-Ras, and PTEN in 13 endometrial cancer cell lines Potential Conflicts of Interest K. Oda: commercial research grant, Daiichi-Sankyo Pharmaceutical Co. Ltd.F. McCormick: commercial research grant and consultant, Daiichi-Sankyo Pharmaceutical Co. Ltd.; speaker honoraria, Novartis; ownership interest, Onyx, Exelixis, and Nexgenix.The other authors disclosed no potential conflicts of interest.