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Research Articles: Therapeutics, Targets, and Development

Colon carcinoma cells harboring PIK3CA mutations display resistance to growth factor deprivation induced apoptosis

Departments of ¹ Pharmacology and Therapeutics and ² Cancer Genetics, Roswell Park Center Institute, Elm and Carlton Streets, Buffalo, New York; ³ OSI Pharmaceuticals, Broadhollow Bioscience Park, Farmingdale, New York; and ⁴ University of Texas Southwestern Medical Center at Dallas, Dallas, Texas

Requests for reprints: Michael G. Brattain, Department of Pharmacology and Therapeutics, Roswell Park Center Institute, Elm and Carlton Streets, Buffalo, NY 14263. Phone: 716-845-3044; Fax: 716-845-8857. E-mail: michael.brattain{at}roswellpark.org

Abstract

PIK3CA, encoding the p110 catalytic subunit of phosphatidylinositol 3-kinase (PI3K), is mutated in a variety of human cancers. We screened the colon cancer cell lines previously established in our laboratory for PIK3CA mutations and found that four of them harbored gain of function mutations. We have now compared a panel of mutant and wild-type cell lines for cell proliferation and survival in response to stress. There was little difference in PI3K activity between mutant PIK3CA-bearing cells (mutant cells) and wild-type PIK3CA-bearing cells (wild-type cells) under optimal growth conditions. However, the mutant cells showed constitutive PI3K activity during growth factor deprivation stress (GFDS), whereas PI3K activity decayed rapidly in the wild-type cells. Importantly, constitutively active PI3K rendered the mutant cells resistant to GFDS-induced apoptosis relative to the wild-type cells, indicating a biological advantage under stress conditions that is imparted by the mutant enzymes. Compared with the wild-type cells, the mutant cells were hypersensitive to the apoptosis induced by the PI3K inhibitor LY294002. In addition, PIK3CA small interfering RNA significantly decreased DNA synthesis and/or induced apoptosis in the mutant cells but not in the wild-type cells. Furthermore, ecotopic expression of a mutant PIK3CA in a nontumorigenic PIK3CA wild-type cell line resulted in resistance to GFDS-induced apoptosis, whereas transfection of wild-type PIK3CA or empty vector had little effect. Taken together, our studies show that mutant PIK3CA increases the capacity for proliferation and survival under environmental stresses, such as GFDS while also imparting greater dependency on the PI3K pathway for proliferation and survival. [Mol Cancer Ther 2007;6(3):1143–50]

Introduction

Phosphatidylinositol 3-kinase (PI3K) is a lipid kinase that catalyzes the transfer of the -phosphate from ATP to the D3 position of the phosphatidylinositol, generating phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (1). Class I PI3Ks are heterodimers consisting of two subunits: an adaptor/regulatory subunit and a catalytic subunit (p110). The regulatory subunit is a 50-kDa or 85-kDa protein that is tightly associated with the p110 catalytic subunit. PI3Ks are activated through receptor tyrosine kinases or through G protein–coupled receptors. Class I_A PI3Ks are very diverse in mammals; they have three catalytic p110 isoforms (p110, ß, and ), each encoded by a separate gene, and seven adaptor proteins generated by expression and alternative splicing of three different genes (p85, ß, and p55; ref. 2).

PI3K activation has been shown to have important roles in sustaining processes important to malignancy, including cell proliferation, adhesion, survival, and motility. There are several downstream effectors of PI3K, including PDK1, Rac, p70S6K, certain isoforms of protein kinase C, and AKT, which is most relevant to cell survival (1). Phosphorylation of phosphatidylinositol-4,5-bisphosphate by PI3K generates phosphatidylinositol 3,4,5-trisphosphate, which then recruits PDK1 to the cell membrane. PDK1 subsequently phosphorylates and activates AKT. Activated AKT can then phosphorylate a variety of substrates, such as Bad, FKHR, caspase-9, and GSK3, that participate in a variety of cellular processes, including cell survival and proliferation. Phosphorylation of Bad and caspase-9 by AKT inhibits the ability of these proteins to induce apoptosis. Therefore, the PI3K pathway has been shown to inhibit apoptotic processes and has been linked to inappropriate cell survival by malignant cells in response to stress (3, 4).

Overexpression of AKT has an antiapoptotic effect in many cell types, resulting in a delay of cell death during stress. AKT was found to be amplified in human breast (5, 6), ovarian (7), pancreatic (8), and prostate cancer (9), suggesting the specific involvement of AKT in the onset and/or propagation of cancer. In addition, the genomic locus encoding the p110 catalytic subunit PIK3CA was found to be amplified in a high percentage of various cancers. PIK3CA gene amplification has been shown to be associated with the progression of several types of tumors to an invasive phenotype (10–12). Recent studies have shown that PIK3CA is mutated in approximately one-third of 234 colorectal cancers but only in 2 of 76 adenomas (13). It is also mutated at a high frequency in other cancers, including breast, ovarian, and hepatocellular carcinomas (14–16), making it one of the most common mutations described to date in human cancers. Expression of one of the PIK3CA mutants in NIH3T3 cells conferred significantly more lipid kinase activity compared with the wild-type protein, suggesting the gain-of-function nature of the mutations (13). In addition, some of the most frequent mutants of PIK3CA showed transforming activity with high efficiency in chicken embryo fibroblasts, as well as in NIH3T3 cells (17, 18). Analogy to gain-of-function epidermal growth factor receptor (EGFR) mutants in lung cancers suggests that these PIK3CA mutations may be associated with "oncogenic addiction" and are, therefore, hypersensitive to PI3K inhibition. Thus, mutant PI3K may be a promising target for PI3K small molecule inhibitors. Exploration of the roles of mutant PI3K in cancer growth, survival, and metastasis will help unveil the mechanisms of malignant progression and facilitate development of drugs for cancer treatment.

This study compares a panel of unmodified PIK3CA mutant and wild-type colon cell lines for proliferation and survival under stress condition. We found that mutant PIK3CA-bearing cells were more resistant to growth factor deprivation stress (GFDS)–induced apoptosis than the wild-type PIK3CA-bearing cells, probably due to the constitutively active PI3K and its downstream effectors. In addition, the mutant cells were hypersensitive to a potent PI3K inhibitor, LY294002, as reflected by significantly increased apoptosis compared with the wild-type cells. PIK3CA small interfering RNA dramatically decreased DNA synthesis and induced apoptosis in the mutant cells but not in the wild-type cells. Ecotopic expression of mutant, but not wild-type PI3K in the wild-type cells, led to resistance to GFDS-induced apoptosis. Our results suggest that mutant PIK3CA confers increased growth and/or survival capacity to colon cancer cells and that the mutant cells possess an increased requirement for the PI3K signaling pathway. Therefore, the constitutively active PI3K/AKT signaling in the mutant PI3K cells might provide an additional therapeutic target for colon cancer treatment.

Our study is complementary to a recent study by Samuels et al., in which they knocked out the wild-type or mutant PIK3CA allele in HCT116 or DLD1 colon cancer cells and showed that haploid PIK3CA cells, bearing only gain-of-function mutant PIK3CA, had reduced cellular dependence on growth factors and were more sensitive to PI3K inhibition by LY294002 relative to haploid wild-type PIK3CA-bearing cells (19). Consequently, some of the results observed in the Samuels study could have reflected haploid insufficiency. In contrast, our study was done with intact diploid cells, thus reducing the potential effects resulting from PIK3CA haplo-insufficient cells. It has been shown that expression of mutant PI3K enhances lipid kinase activity and induces constitutive activation of AKT signaling and oncogenic transformation in chicken embryo fibroblasts, as well as in NIH3T3 cells (17, 18). Because environmental restriction on growth seems to be common in solid tumors (20), the ability of malignant cells to withstand these stresses is considered a key factor in tumor development and progression. Our finding that the differential cell survival characteristics between the mutant and wild-type cells under stress conditions, in which the wild-type PI3K activity is more severely down-regulated, provides an explanation for the irrelevance of the wild-type PI3K activity in terms of the advantage provided by the mutant enzyme. We also found that ecotopic expression of the mutant PI3K in the wild-type PI3K-bearing colon cancer cells led to increased resistance to GFDS-induced apoptosis. This result indicates that gain-of-function mutant PIK3CA may contribute to the ability to withstand the environmental stresses that are important in tumor development and/or progression.

Materials and Methods

Cell Culture and Reagents
Human colon carcinoma cells used in this study were established in vitro from primary tumors, as described previously (21). HCT116, FET, GEO, CBS, and RCA were cultured in SM medium [McCoy's 5A serum-free medium (Sigma, St. Louis, MO) with pyruvate, vitamins, amino acids, and antibiotics] supplemented with 10 ng/mL epidermal growth factor, 20 µg/mL insulin, and 4 µg/mL transferrin, as described previously (22). RKO and TENN were cultured in SM medium supplemented with 10% fetal bovine serum. Vaco481 were cultured in MEM basal medium (MEM medium with glutamine and amino acids) supplemented with 100 µg/mL insulin, 20 µg/mL transferrin, 8.6 ng/mL selenium, and 2% fetal bovine serum. Cells were maintained at 37°C in a humidified atmosphere of 5% CO₂. When the cells were under GFDS, they were cultured in SM medium or MEM basal medium without growth factor or serum supplements for the indicated time periods without medium change in between.

Antibodies for caspase-3, caspase-9, poly (ADP-ribose) polymerase (PARP), Akt, phosphorylated (p) Akt (Ser⁴⁷³), PDK1, pPDK1 (Ser²⁴¹), pBAD (Ser¹³⁶), and pFKHR (Thr²⁴) were obtained from Cell Signaling Technology (Beverly, MA). Actin antibody was from Sigma. LY294002 was purchased from Calbiochem (La Jolla, CA).

PCR, Sequencing, and Mutational Analysis
Human colon carcinoma cell lines were screened for PIK3CA mutations by direct sequencing of all 20 exons and splice junction regions. Primer pairs for each exon were designed using the Primer3 program and positioned at least 25 bp upstream of the 3' and 5' splice junction site (details available on request). PCR amplification of the individual exons was done through 35 cycles using standard procedures. PCR products were sequenced in both directions using an ABI Prism 3100.

PI3K Assay
The cells were washed with PBS and lysed in lysis buffer [137 mmol/L NaCl, 20 mmol/L Tris (pH 7.4), 1 mmol/L MgCl₂, 1 mmol/L CaCl₂, 1% NP40, 100 µmol/L Na₃VO_4, and 1 mmol/L phenylmethylsulfonyl fluoride]. Protein concentrations were determined by bicinchoninic acid, a protein assay reagent (Pierce, Rockford, IL). Lysates (400 µg of protein) were incubated with p85 antibody (Upstate, Charlottesville, VA) at 4°C overnight, followed by further incubation with protein A-agarose for 2 h. Immune complexes were washed twice with each wash buffer: PI3K wash 1, PBS 1% NP40/Na₃VO₄; PI3K wash 2, 100 mmol/L Tris (pH 7.4) and 5 mmol/L LiCl/Na₃VO₄; PI3K wash 3, 10 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, and 5 mmol/L EDTA/Na₃VO₄. After the last wash was removed, PI3K assays were done as described previously (23). Briefly, samples were resuspended in 50 µL of PI3K buffer [20 mmol/L Tris (pH 7.5), 100 mmol/L NaCl, and 0.5 mmol/L EGTA], and 10 µg of phosphatidylinositol were added. After 10 min at room temperature, 10 µCi of [³²P] ATP and MgCl₂ to a final concentration of 20 µmol/L was added. After 10 min at room temperature, lipids were extracted: first extraction with 150 µL of CHCl₃/methanol/HCl (10:20:0.2) and 100 µL of pure CHCl₃; and second extraction with 80 µL of methanol/1 N HCl (1:1). Samples were spotted on 1% potassium oxalate–treated TLC plates (Analtech, Newark, DE) and developed in CHCl₃/methanol/NH₄OH/H₂O (129:114:15:21).

Western Blot Analysis
Cells were lysed in NP40 lysis buffer [50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 0.5% NP40, 50 mmol/L NaF, 1 mmol/L Na₃VO₃, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L DTT, 25 µg/mL aprotinin, 25 µg/mL trypsin inhibitor, and 25 µg/mL leupeptin] at 4°C. The supernatants were cleared by centrifugation. Protein (30–100 µg) was fractionated on an acrylamide denaturing gel and transferred onto a nitrocellulose membrane (Amersham, Piscataway, NJ) by electroblotting. The membrane was blocked with 5% nonfat dry milk in TBST [50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 0.05% Tween 20] for 1 h at room temperature or overnight at 4°C and washed in TBST. The membrane was then incubated with primary antibodies for 1 h at room temperature or overnight at 4°C. After washing with TBST for 15 min, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (Amersham) for 1 h at room temperature. After further washing in TBST for 15 min, the proteins were detected by the enhanced chemiluminescence system (Amersham).

Apoptosis Assays
Apoptosis assays were done to study the effect of LY294002 (Invitrogen, Carlsbad, CA) on the mutant and wild-type cells. Cells were seeded in 96-well plates. LY294002 was added at indicated concentrations on the next day. Apoptosis assays were done using a DNA fragmentation ELISA kit as described in the manufacturer's protocol (Invitrogen) 72 h later.

Knockdown of p110 Protein Using Small Interfering RNAs
PIK3CA smartpool consisting of four small interfering RNA duplexes (Dharmacon Research, Inc., Lafayette, CO) was used against PIK3CA. A scrambled small interfering RNA duplex (Qiagen, Valencia, CA) was used as a control. HCT116, RKO, and CBS cells were plated in six-well plates. The next day, the cells were 60% confluent and transfected with 50 nmol/L small interfering RNA duplexes using LipofectAMINE 2000 (Invitrogen). The cells were lysed for Western blot analyses or subject to DNA synthesis assays or DNA fragmentation assays 72 h later, as described above.

DNA Synthesis Assays
[³H]thymidine incorporation was used to determine cell proliferation of the mutant and wild-type cells after transfection of scrambled or PIK3CA small interfering RNA. The cells were seeded in six-well tissue culture plates and grown to 60% confluence. At 72 h after small interfering RNA tarnsfection, the cells were labeled with [³H]thymidine (7 µCi, 46 Ci/mmol; Amersham) for 1 h. DNA was then precipitated with 10% trichloroacetic acid and solubilized in 0.2 mol/L NaOH. The amount of [³H]thymidine that was incorporated was analyzed by liquid scintillation counting in a Beckman LS7500 scintillation counter.

Ecotopic Expression of Wild-Type and Mutant PIK3CA
The wild-type and mutant PIK3CA (H1047R) cDNAs were kind gifts from Dr. Vogelstein (Johns Hopkins University Medical Institutions), which were then subcloned into a pBabe-based retroviral vector. The 293GP packaging cells (Clontech, Mountain View, CA) were cotransfected with pVSV-G and empty retroviral vector or retroviral vectors containing PIK3CA wild-type or mutant cDNA. The viruses were harvested 48 h later and used to infect FET cells. Puromycin (3.0 µg/mL) was used to select infected cells.

Results

Mutations of PIK3CA Imparted Constitutive PI3K Activity in Colon Carcinoma Cell Lines
Human colon carcinoma cell lines were screened for PIK3CA mutations by direct sequencing of all 20 exons and splice junction regions. Four cell lines were identified as bearing putative gain of function mutations (H1047R in HCT116 and RKO, C378R in TENN, and Q546P in Vaco481) previously described by Samuels et al. as being present in cancer specimens (13). The mutations were verified by sequencing. These mutations were mainly located in the helical or kinase domains. Meanwhile, a cohort of cell lines bearing only the wild-type PIK3CA alleles (FET, GEO, CBS, and RCA) was identified and verified by sequencing for comparison to the mutants.

We first examined whether the mutant PI3K was constitutively active. To determine this, the cells were subjected to GFDS with the expectation that if the mutant PI3K was constitutively active, PI3K activity would remain high in the mutant cells despite the absence of exogenous growth factors, whereas the removal of growth factors from the wild-type PI3K cells would result in the impairment of its activity. As shown in Fig. 1A , the cell lines carrying the mutant PI3K had relatively higher sustained PI3K activity after 4 days of growth factor and nutrient deprivation, whereas the enzyme activity was significantly reduced in the wild-type cells during GFDS. Taken together with the fact that p110 expression levels did not change under GFDS in both mutant and wild-type cells (data not shown), these results indicated that the mutant cells, but not the wild-type cells, had constitutively active enzyme activity during GFDS. Phosphorylation at Ser²⁴¹ and Ser⁴⁷³ was used to reflect the activity of PDK1 and AKT, respectively. The constitutive activation of AKT, which acts downstream of PI3K, further supported the constitutive PI3K activity of the mutant cells observed in Fig. 1A. In contrast, cells with only wild-type PI3K showed rapid decay of phosphorylation of both PDK1 and AKT during GFDS (Fig. 1B and C).

View larger version (34K): [in this window] [in a new window]   Figure 1. Constitutive activation of PI3K, PDK1, and AKT in mutant PIK3CA-bearing colon cancer cells during GFDS. The mutant and wild-type cells were deprived of growth factors or serum for 0, 48, and 96 h after growth to 80% confluence as described in Materials and Methods. A, cells were harvested at the indicated times, and the lysates were subjected to PI3K assays as described in Materials and Methods. B and C, cells were harvested at the indicated times and Western blot analyses were done with pPDK1 (Ser241; pPDK1 S241) or pAKT [Ser473; pAKT (S473)] antibody. Actin was used as a loading control.

View larger version (11K): [in this window] [in a new window]   Figure 2. Mutant PIK3CA confers resistance to GFDS-induced apoptosis. The mutant and wild-type cells were grown to 80% to 90% confluence, and the cells were then deprived of growth factor or serum for 48 h. Cells were harvested and Western blot analyses were done with PARP antibody. Actin was used as a loading control.

View larger version (16K): [in this window] [in a new window]   Figure 3. Effect of LY294002 (LY) on apoptosis. A, cells were grown to 80% confluence and were then deprived of growth factor or serum for 48 h although treated with increasing concentrations of LY294002. Cells were harvested, and Western blot analyses were done with pAKT (Ser473) antibody. Actin was used as a loading control. B, cells were seeded in 96-well plates and treated with 25 µmol/L LY294002 for 72 h. DNA fragmentation assays were done as described in Materials and Methods. C, cells were grown to 80% confluence and were then deprived of growth factor or serum for 48 h although treated with increasing concentrations of LY294002. Cells were harvested, and Western blot analyses were done with pFKHR or pBAD antibodies. Actin was used as a loading control.

To show that the mutant cells, but not the wild-type cells, are strictly dependent on constitutive activation of PI3K, two of the mutant cell lines, HCT116 and RKO, were challenged with PIK3CA small interfering RNAs. Transfection of PIK3CA small interfering RNA into both cell lines resulted in significantly reduced expression of p110 with a concomitant decrease of AKT phosphorylation (Fig. 4A ), indicating that PIK3CA small interfering RNA knocked down p110 expression and inhibited PI3K downstream signaling. As a result, there was increased PARP cleavage and caspase-9 activation in HCT116 p110 knockdown cells compared with the scrambled small interfering RNA transfected control cells. There was, however, no increased PARP cleavage or caspase-9 activation in RKO cells transfected with PIK3CA small interfering RNA compared with the scrambled small interfering RNA transfected control cells (Fig. 4A). Consistent with the PARP cleavage Western blot results, DNA fragmentation assays also showed that PIK3CA small interfering RNA induced apoptosis in HCT116 cells but not in RKO cells. We further investigated whether knockdown of p110 had any effect on cell proliferation. DNA synthesis was determined 72 h after transfection of PIK3CA or scrambled small interfering RNA into the cells. The PIK3CA small interfering RNA–transfected cells showed >50% inhibition of DNA synthesis compared with the scrambled small interfering RNA–transfected cells in both HCT116 and RKO cells (Fig. 4C), indicating that both HCT116 and RKO cells are dependent on the PI3K pathway for DNA synthesis and proliferation and that HCT116, but not RKO, cells are dependent on the PI3K pathway for survival. Transfection of PIK3CA small interfering RNA into one of the wild-type cell lines, CBS, knocked down p110 expression to the similar level as that in HCT116 or RKO cells (Fig. 4A). However, PIK3CA knockdown did not have much effect on apoptosis or DNA synthesis compared with the scrambled small interfering RNA transfection (Fig. 4A–C). Taken together, these results showed that, compared with the wild-type cells, the PIK3CA mutant-bearing cells are hypersensitive to PI3K inhibition and display dependence on constitutive PI3K activity for proliferation and/or survival.

View larger version (15K): [in this window] [in a new window]   Figure 4. Effect of PIK3CA knockdown on DNA synthesis and apoptosis. HCT116, RKO, and CBS cells were seeded in six-well plates and transfected with PIK3CA or scrambled small interfering RNA. A, cells were harvested 72 h later, and Western blot analyses were done with p110, pAKT (Ser473), PARP, or caspase-9 antibodies. Actin was used as a loading control. DNA fragmentation assays (B) or DNA synthesis assays (C) were done on the transfected cells 72 h later.

View larger version (20K): [in this window] [in a new window]   Figure 5. Mutant PIK3CA rendered FET cells more resistant to GFDS-induced apoptosis. A, FET cells were infected with retroviruses encoding mutant or wild-type PIK3CA. After drug selection, the infected cells were harvested and Western blot analyses were done with p110 or pAKT (Ser473) antibodies. Actin was used as a loading control. B, vector, wild-type, or mutant PIK3CA–infected cells were deprived of growth factor or serum for 48 or 96 h. Cells were harvested and Western blot analyses were done with pAKT (Ser473) or AKT antibodies. Actin was used as a loading control. C, vector, wild-type, or mutant PIK3CA–infected cells were deprived of growth factor or serum for 48 or 96 h. Cells were harvested, and Western blot analyses were done with PARP or caspase-3 antibodies. Actin was used as a loading control.

We have identified a subset of colon cancer cell lines with PIK3CA mutations. Taken together with the demonstration that PIK3CA mutations confer increased PI3K activity and high-transforming activity in NIH3T3 and chicken embryo fibroblasts, respectively, (13, 17, 18) and that Iressa response in nonsmall cell lung cancer is closely associated with gain of function mutations of the EGFR (24), we hypothesized that mutant PIK3CA conferred increased growth and survival capacity in colon cancer cells and that mutant PIK3CA-bearing cells were highly dependent upon PI3K signaling for their malignant phenotype. The results from this study are consistent with this hypothesis. Mutant PIK3CA-bearing cells displayed constitutive activation of PI3K and its downstream mediators during GFDS, which is associated with increased resistance to GFDS-induced apoptosis in the mutant cells relative to the wild-type cells. In addition, the mutant cells were hypersensitive to a potent PI3K inhibitor, LY294002, as reflected by significantly increased apoptosis compared with the wild-type cells and/or hypersensitivity to inhibition of proliferation. PIK3CA small interfering RNA dramatically decreased DNA synthesis and induced apoptosis in the mutant cells. These studies indicate that human colon cancer cells with PIK3CA mutations display an increased dependence, sometimes called addiction, on the PI3K signaling pathway in cell proliferation and/or survival. This raises the prospect of targeting the PI3K pathway and perhaps targeting the mutant PI3K active site in selective fashion to treat those colon cancer patients with PI3K mutations. Our data provide a biological basis for the treatment of such cancers. Future in vivo efficacy studies in relevant animal models will allow for further assessment of the therapeutic potential of small molecular inhibitors against PI3K/AKT signaling pathway in colon cancer.

Similar to the gain-of-function EGFR mutants in lung cancers (24), our studies indicate that the cells expressing mutant PIK3CA are hypersensitive to PI3K inhibition and more dependent on aberrant PI3K signaling, therefore suggesting that PIK3CA mutations are associated with oncogenic addiction (26). It has been previously shown that PTEN-deficient tumors had enhanced sensitivity to inhibition of FRAP/mTOR (27) and that PI3K inhibitors selectively inhibited the anchorage-independent growth of ErbB2-overexpressing human mammary carcinoma cells, thereby indicating that those cells displayed an increased dependence on PI3K signaling (28). Thus, there seems to be multiple mechanisms for inactivating the normal regulation of PI3K signaling pathway, including PIK3CA mutation. Many investigators have noted from these types of studies that an important role of PI3K in carcinogenesis is implied and suggest that identification of the subset of patients harboring PIK3CA abnormalities and developing adequate drugs for targeting the PI3K pathway might be an efficient way to treat this subset of cancers.

There is a recent study demonstrating that there was no difference of PI3K activity and downstream signaling between the wild-type and mutant PIK3CA carrying colon cancer cells under normal cell culture conditions (29). We also observed this in our study. When we examined the PI3K activity or phosphorylation of AKT and PDK1 under normal conditions (GFDS, 0 h), PI3K activity and signaling are very similar between the wild-type and mutant cells. However, there was significant difference in PI3K signaling between the wild-type and mutant cells under GFDS (GFDS, 48 or 96 h; Fig. 1A–C). These results indicated that PI3K activity was sustained in the mutant cells under GFDS, whereas it deceased in the wild-type cells. Sustained PI3K activity under stress conditions would enable mutant cells to survive environmental stresses. Because environmental restriction on growth seems to be common in solid tumors due to inadequate vascularization (20, 30), the ability of PI3K mutant cells to withstand these stresses is considered a key factor in tumor development and progression. Therefore, mutational activation of PIK3CA is likely to be one of the mechanisms of tumor progression in colon carcinomas.

Among the mutant PIK3CA-expressing cells, only RKO cells were insensitive to apoptosis induced by PI3K inhibition, either through LY294002 treatment (Fig. 3B) or PIK3CA small interfering RNA transfection (Fig. 4A and B). However, DNA synthesis and proliferation of RKO cells were associated with hypersensitivity to LY294002 (data not shown) or PIK3CA small interfering RNA (Fig. 4C). This reflects the possibility that the PI3K pathway regulates proliferation but not survival in RKO cells. Because LY294002 had no effect on pFKHR or pBAD that plays important roles as target of LY294002-induced apoptosis in other PI3K mutant cells, this result suggested that there were defects in apoptosis pathways in RKO cells. Further studies are needed to determine the mechanism(s) of resistance to LY294002-induced apoptosis in RKO cells. On the other hand, among the wild-type cells, GEO cells were growth factor independent (data not shown) and more sensitive to LY294002-induced apoptosis compared with other wild-type cells (Fig. 3B). GEO cells have been shown to have strong autocrine heregulin activity that results in constitutive ErbB2 activation, which in turn leads to constitutive activation of the PI3K/AKT pathway (31, 32). The PI3K pathway supports both proliferation and cell survival in these cells.

Recently, Samuels et al. did somatic knockouts by eliminating one allele of either wild-type or mutant PIK3CA in HCT116 or DLD1 colon cancer cells. They showed that cells bearing only mutant PI3K had reduced dependence on growth factors and were more sensitive to PI3K inhibition by LY294002 (19). We have compared a panel of intact cell lines with the wild-type or mutant PIK3CA and native levels of PI3K activity. Because the study by Samuels et al. used somatic PIK3CA knockout cells, comparisons of cells bearing two copies of the PIK3CA gene were not possible. Thus, the work by Samuels et al. was a comparison of haploid levels of PI3K. Consequently, some of the results observed in the Samuels study could have reflected haploid insufficiency. In contrast, our study was done with native intact diploid cells, thus reducing the possibility of haploid effects. Our finding that the differential cell survival characteristics between the mutant and wild-type cells under stress conditions, in which the wild-type PI3K is severely down-regulated, provides an explanation for the irrelevance of the wild-type PI3K activity in terms of the advantage provided by the mutant enzyme.

One could argue that there might be other genetic changes contributing to the differences observed between the wild-type and mutant PI3K cells in addition to PI3K status. However, we are asking the question of whether mutant PIK3CA imparts properties that can be generalized to the mutant-bearing versus nonmutant-bearing cells, given the many additional genetic changes that can occur in those cells. Our studies indicate that we can distinguish the wild-type and mutant PI3K cells based on their different properties despite the presence of other genetic changes. For example, unlike other mutant cell lines, RKO cells are insensitive to PI3K inhibition–induced apoptosis, probably due to other defects in the apoptosis pathways. However, they are still hypersensitive to PI3K inhibition–induced growth inhibition. Therefore, although there are exceptions in the phenotypes of the wild-type or mutant PI3K cells due to other genetic changes, the overall differences between these two cell types are recognizable. In addition, we show that these properties in the mutant cells can be gained when the nonmutant cells are transfected with mutant PIK3CA.

Acknowledgments

We thank David Chervinsky for his expert assistance with the preparation of samples for DNA sequencing.

Footnotes

Grant support: NIH grants CA 34432, CA54807, and CA 16056.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 9/ 8/06; revised 12/21/06; accepted 1/31/07.

References

1. Wymann MP, Pirola L. Structure and function of phosphoinositide 3-kinases. Biochim Biophys Acta 1998;1436:127–50.[Medline]
2. Vanhaesebroeck B, Alessi DR. The PI3K-PDK1 connection: more than just a road to PKB. Biochem J 2000;346:561–76.[CrossRef][Medline]
3. Yao R, Cooper GM. Growth factor-dependent survival of rodent fibroblasts requires phosphatidylinositol 3-kinase but is independent of pp70S6K activity. Oncogene 1996;13:343–51.[Medline]
4. Yao R, Cooper GM. Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science 1995;267:2003–6.[Abstract/Free Full Text]
5. Nakatani K, Thompson DA, Barthel A, et al. Up-regulation of Akt3 in estrogen receptor-deficient breast cancers and androgen-independent prostate cancer lines. J Biol Chem 1999;274:21528–32.[Abstract/Free Full Text]
6. Bellacosa A, de Feo D, Godwin AK, et al. Molecular alterations of the AKT2 oncogene in ovarian and breast carcinomas. Int J Cancer 1995;64:280–5.[Medline]
7. Cheng JQ, Godwin AK, Bellacosa A, et al. AKT2, a putative oncogene encoding a member of a subfamily of protein-serine/threonine kinases, is amplified in human ovarian carcinomas. Proc Natl Acad Sci U S A 1992;89:9267–71.[Abstract/Free Full Text]
8. Cheng JQ, Ruggeri B, Klein WM, et al. Amplification of AKT2 in human pancreatic cells and inhibition of AKT2 expression and tumorigenicity by antisense RNA. Proc Natl Acad Sci U S A 1996;93:3636–41.[Abstract/Free Full Text]
9. Graff JR, Konicek BW, McNulty AM, et al. Increased AKT activity contributes to prostate cancer progression by dramatically accelerating prostate tumor growth and diminishing p27Kip1 expression. J Biol Chem 2000;275:24500–5.[Abstract/Free Full Text]
10. Woenckhaus J, Steger K, Werner E, et al. Genomic gain of PIK3CA and increased expression of p110 are associated with progression of dysplasia into invasive squamous cell carcinoma. J Pathol 2002;198:335–42.[CrossRef][Medline]
11. Ma YY, Wei SJ, Lin YC, et al. PIK3CA as an oncogene in cervical cancer. Oncogene 2000;19:2739–44.[CrossRef][Medline]
12. Shayesteh L, Lu Y, Kuo WL, et al. PIK3CA is implicated as an oncogene in ovarian cancer. Nat Genet 1999;21:99–102.[CrossRef][Medline]
13. Samuels Y, Wang Z, Bardelli A, et al. High frequency of mutations of the PIK3CA gene in human cancers. Science 2004;304:554.[Free Full Text]
14. Campbell IG, Russell SE, Choong DY, et al. Mutation of the PIK3CA gene in ovarian and breast cancer. Cancer Res 2004;64:7678–81.[Abstract/Free Full Text]
15. Lee JW, Soung YH, Kim SY, et al. PIK3CA gene is frequently mutated in breast carcinomas and hepatocellular carcinomas. Oncogene 2005;24:1477–80.[CrossRef][Medline]
16. Bachman KE, Argani P, Samuels Y, et al. The PIK3CA gene is mutated with high frequency in human breast cancers. Cancer Biol Ther 2004;3:772–5.[Medline]
17. Kang S, Bader AG, Vogt PK. Phosphatidylinositol 3-kinase mutations identified in human cancer are oncogenic. Proc Natl Acad Sci U S A 2005;102:802–7.[Abstract/Free Full Text]
18. Ikenoue T, Kanai F, Hikiba Y, et al. Functional analysis of PIK3CA gene mutations in human colorectal cancer. Cancer Res 2005;65:4562–7.[Abstract/Free Full Text]
19. Samuels Y, Diaz LA, Jr., Schmidt-Kittler O, et al. Mutant PIK3CA promotes cell growth and invasion of human cancer cells. Cancer Cell 2005;7:561–73.[CrossRef][Medline]
20. Hajra KM, Liu JR. Apoptosome dysfunction in human cancer. Apoptosis 2004;9:691–704.[CrossRef][Medline]
21. Brattain MG, Levine AE, Chakrabarty S, Yeoman LC, Willson JK, Long B. Heterogeneity of human colon carcinoma. Cancer Metastasis Rev 1984;3:177–91.[CrossRef][Medline]
22. Boyd DD, Levine AE, Brattain DE, McKnight MK, Brattain MG. Comparison of growth requirements of two human intratumoral colon carcinoma cell lines in monolayer and soft agarose. Cancer Res 1988;48:2469–74.[Abstract/Free Full Text]
23. Jackson JG, St Clair P, Sliwkowski MX, Brattain MG. Blockade of epidermal growth factor- or heregulin-dependent ErbB2 activation with the anti-ErbB2 monoclonal antibody 2C4 has divergent downstream signaling and growth effects. Cancer Res 2004;64:2601–9.[Abstract/Free Full Text]
24. Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004;350:2129–39.[Abstract/Free Full Text]
25. Vlahos CJ, Matter WF, Hui KY, Brown RF. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem 1994;269:5241–8.[Abstract/Free Full Text]
26. Weinstein IB. Cancer. Addiction to oncogenes—the Achilles heal of cancer. Science 2002;297:63–4.[Free Full Text]
27. Neshat MS, Mellinghoff IK, Tran C, et al. Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR. Proc Natl Acad Sci U S A 2001;98:10314–9.[Abstract/Free Full Text]
28. Hermanto U, Zong CS, Wang LH. ErbB2-overexpressing human mammary carcinoma cells display an increased requirement for the phosphatidylinositol 3-kinase signaling pathway in anchorage-independent growth. Oncogene 2001;20:7551–62.[CrossRef][Medline]
29. Morrow CJ, Gray A, Dive C. Comparison of phosphatidylinositol-3-kinase signalling within a panel of human colorectal cancer cell lines with mutant or wild-type PIK3CA. FEBS Lett 2005;579:5123–8.[CrossRef][Medline]
30. Goldie JH, Coldman AJ. A mathematic model for relating the drug sensitivity of tumors to their spontaneous mutation rate. Cancer Treat Rep 1979;63:1727–33.[Medline]
31. Venkateswarlu S, Dawson DM, St Clair P, Gupta A, Willson JK, Brattain MG. Autocrine heregulin generates growth factor independence and blocks apoptosis in colon cancer cells. Oncogene 2002;21:78–86.[CrossRef][Medline]
32. Hu YP, Venkateswarlu S, Sergina N, et al. Reorganization of ErbB family and cell survival signaling after knock-down of ErbB2 in colon cancer cells. J Biol Chem 2005;280:27383–92.[Abstract/Free Full Text]

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