The gene of phosphatidylinositol 3-kinase catalytic subunit α (PIK3CA) has been implicated as an oncogene in ovarian cancer [L. Shayesteh et al., Nat. Genet., 21: 99–102, 1999]. In this study, we examined the expression of PIK3CA mRNA and its p110α protein product in human ovarian carcinoma and investigated its role in regulating angiogenesis via vascular endothelial growth factor (VEGF). PIK3CA mRNA was detected in 66.6% of stage I and 93.9% of advanced stage ovarian cancer specimens and in all 17 ovarian cancer cell lines. PIK3CA mRNA levels were significantly higher in invasive carcinomas compared with benign and low malignant potential neoplasms (P = 0.007), but no significant difference was seen between early and advanced stage carcinomas (P = 0.812). Strong expression of immunoreactive p110α was detected in tumor cells and/or stroma endothelium. PIK3CA expression in vivo positively correlated, both at the mRNA and the protein level, with the expression of VEGF as well as with the extent of microvascular development. Furthermore, PIK3CA mRNA overexpression positively correlated with increased proliferation and decreased apoptosis of tumor cells in vivo. In vitro, PIK3CA expression positively correlated with the expression of VEGF in ovarian cancer cells, whereas the phosphatidylinositol 3′-kinase inhibitor Ly294002 reduced both the constitutive and inducible expression of hypoxia-inducible factor-1α at the mRNA and protein levels and abrogated VEGF up-regulation by glucose starvation. Furthermore, Ly294002 suppressed cell proliferation and, at higher doses, induced marked apoptosis in ovarian cancer cells. Collectively, these data strongly indicate that PIK3CA supports ovarian cancer growth through multiple and independent pathways affecting cell proliferation, apoptosis and angiogenesis, and plays an important role in ovarian cancer progression.

PI3k3 is a novel intracellular transducer with lipid substrate specificity, which is involved in many cancer-associated signaling pathways (1, 2, 3, 4). PI3k comprises a Mr 110,000 catalytic subunit (PIK3CA) and a regulatory subunit of either Mr 85,000, Mr 55,000, or Mr 50,000 (5). The gene encoding PIK3CA is located at 3q26, a region with reportedly increased copy numbers in >40% of ovarian and other cancer specimens by comparative genomic hybridization (6, 7). PIK3CA gene amplification has also been found in a variety of human solid tumors (8, 9, 10, 11, 12, 13, 14, 15). Recently, PIK3CA has been implicated as an oncogene in ovarian cancer (1). Increased DNA copy numbers have been frequently found in human ovarian cancer cell lines, which was associated with increased PIK3CA transcription and expression of the p110α protein (1). Importantly, PIK3CA gene amplification leads to increased p110α-p85 heterodimer formation and increased PI3k activity in vitro(1). However, current knowledge about the role of PIK3CA as an oncogene in ovarian cancer has been derived from in vitro experiments and very little information is available on the expression and role of PIK3CA in ovarian cancer in vivo.

A critical effect of oncogene activation is to promote aberrant cellular mitogenesis. However, accumulating evidence indicates that oncogenes such as ras, src, myc, HER-2/neu, as well as human papilloma virus E6(16, 17, 18, 19, 20, 21, 22, 23) or tumor suppressor genes such as p53, pVHL, and PTEN(24, 25, 26, 27, 28, 29, 30, 31) are implicated in the control of tumor angiogenesis via HIF and the downstream VEGF (32, 33). PI3k pathway may be involved in the physiological regulation of VEGF and may serve as an angiogenesis regulatory gene (4, 34, 35, 36, 37, 38, 39, 40, 41). In endothelial cells, PI3k partakes in several growth factor-dependent pathways regulating VEGF expression and angiogenesis (4, 36) via HIF-1α. Direct in vitro or in vivo evidence for the involvement of PIK3CA in the regulation of VEGF in human tumors is lacking to date.

In this study, we show that PIK3CA overexpression is indeed highly prevalent in ovarian cancer and occurs early in the process of malignant transformation. Its expression positively correlates with the expression of VEGF, both at the mRNA and the protein level. Additionally, we demonstrate that PI3k is directly implicated in the control of VEGF expression in ovarian carcinoma, an effect that is mediated via HIF-1α. Finally, we show that PIK3CA overexpression correlates with increased proliferation and decreased apoptosis of tumor cells in ovarian cancer in vivo, an effect that may be mediated jointly through an increase in angiogenesis as well as a direct effect of PI3k. Collectively, these data strongly support a role of PIK3CA in ovarian cancer progression.

Ovarian Tumors.

Two normal human ovaries, 4 benign cystadenomas, 4 LMPs, and 42 malignant tumors, including 9 stage I and 33 stage III or IV carcinomas, were randomly selected from the specimen bank existing in our lab (44, 45) and analyzed by quantitative real-time RT-PCR. Thirty-nine advanced stage tumors from the same specimen bank were analyzed by immunostaining.

Cell Lines and Cell Culture.

A total of 17 human ovarian cancer cell lines (45) was cultured in 5% CO2 atmosphere at 37°C and in DMEM supplemented with 10% fetal bovine serum and 100 units/ml penicillin. Primary human ovarian surface epithelium cultures were established as described previously (45). In some experiments, cells were treated with the PI3k inhibitor Ly294002 (43) (10 or 50 μm; Calbiochem, La Jolla, CA). Cells were seeded in 6-well plates and cultured in complete growth media. Upon reaching 90% confluence, cells were cultured in serum-free media overnight. After washing with PBS, cells were cultured in glucose- and serum-free DMEM in the presence or absence of Ly294002. Control cells were treated with DMSO. All experiments were repeated three times.

RNA Isolation.

Total RNA was isolated from 1 × 106 cultured cells or 100–500 mg of frozen tissue with Trizol reagent (Invitrogen). After treatment with RNase-free DNase (Invitrogen), RNA was further purified with RNeasy RNA isolation kit (Qiagen, Valencia, CA). Total RNA was reverse transcribed using Superscript First-Strand Synthesis Kit for RT-PCR (Invitrogen) under conditions described by the supplier.

Quantitative Real-Time RT-PCR.

cDNA was quantified by real-time PCR on the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA) as described previously (43). Specific oligonucleotide primers and probes (Table 1) were designed based on published sequences.

IHC and Image Analysis.

IHC was performed using the Vectastain ABC kit as described by the manufacturer (Vector, Burlingame, CA). All primary antibodies, mouse antihuman p110α (1:50; PharMingen, San Diego, CA), VEGF (1:200; PharMingen), CD31 (1:30; Dako, Carpinteria, CA), rabbit antihuman Ki-67 (1:200; Dako), and cytokeratin (1:1000; Dako), were incubated on sample sections for 1 h. The prevalence of immunoreactive VEGF and p110α in tumors was scored semiquantitatively and blindly by two independent investigators and confirmed by a third investigator as following: 0 = all cells in tumor islets were negative; 1 = <20% of cells in tumor islets were positive; 2 = between 20 and 80% of the cells in tumor islets were positive; and 3 = >80% of cells in tumor islets were positive. Ki-67 staining was quantified by image analysis. Images were collected through Cool Snap Pro color digital camera (Media Cybernetics), and staining index was analyzed using Image-Pro Plus 4.1 software (Media Cybernetics). Immunofluorescent double staining was performed as described previously (47).

Western Blotting.

Cultured cells were lysed in 1 ml of lysis buffer containing 50 mm Tris-HCl (pH 7.4), 150 mm NaCl, and 1% Triton X-100. Protein was separated by 12% SDS-PAGE under denaturing conditions and transferred to nitrocellulose membrane. Membranes were incubated with an anti-HIF-1α monoclonal antibody (1:500; PharMingen), followed by incubation in rabbit antimouse secondary antibody conjugated with horseradish peroxidase (1:5000; Sigma, St. Louis, MO). Immunoreactive proteins were visualized using enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ).

In Situ TUNEL Assay.

The ApopTag in Situ detection kit (Intergen, Purchase, NY) was used to visualize apoptotic cells as described previously (47).

Proliferation Assay.

Cells were seeded at a density of 1–5 × 103/well in 96-well plates in complete culture medium. After 24 h of culture, serum-free medium was added with or without Ly294002 (10 μm). Cell proliferation was assessed 72 h later using CellTiter 96 (Promega, Madison, WI). Cell proliferation was determined as the ratio of the absorbance of treated cells to the density of control untreated cells.

Annexin-V Assay.

Annexin-V staining was detected by flow cytometry using an apoptosis detection kit (R&D Systems, Minneapolis, MN) as described previously (47).

Statistical Analysis.

Statistical analysis was performed using the SPSS statistics software package (SPSS, Chicago, IL). All results were expressed as mean ± SE, and P < 0.05 was used for significance.

PIK3CA Is Overexpressed in Ovarian Carcinoma.

We used quantitative real-time RT-PCR to assess mRNA levels in ovarian neoplasms, including benign cystadenomas (n = 4), LMP tumors (n = 4), and invasive ovarian carcinomas at early (I, n = 9) or advanced stage (III or IV, n = 33). PIK3CA mRNA was detectable in 6 of 9 (66.6%) stage I and 31 of 33 (93.9%) advanced-stage ovarian cancer specimens. As a whole, malignant ovarian tumors significantly overexpressed PIK3CA mRNA compared with the group of benign and LMP neoplasms (P = 0.007). No significant difference was seen between early and advanced stage carcinomas (P = 0.812; Fig. 1,A). Interestingly, mRNA levels of PIK3CA were similar in whole normal postmenopausal ovaries (n = 2) and invasive carcinomas (data not shown). PIK3CA mRNA was also detected in all 17 human ovarian cancer cell lines analyzed (Fig. 2 A). The mean expression of PIK3CA in ovarian cancer cell lines was 2.7-fold higher compared with primary cultures of human ovarian surface epithelium.

To examine the expression and localization of p110α protein in ovarian carcinoma, we performed IHC. Immunoreactive p110α was detectable in 22 of 39 (56.4%) ovarian cancer specimens, with moderate to strong staining localized to tumor cells of islets (Figs. 1, B and C, and 2, C and F). Weak or no staining was found in tumor cells in the remaining 17 samples. Immunostaining was localized mainly to the cytoplasm of tumor cells but not to their nucleus (Figs. 1, B and C, and 2, C and F). Interestingly, p110α expression was also detected in endothelial cells of medium caliber vessels of tumor stroma in 28 of 37 evaluable (75.7%) samples (Fig. 1,D), whereas it was absent from vessels in the remaining 9 (24.3%) samples (Fig. 1,C). In addition, there was no relationship of the p110α expression between tumor cells and tumor-associated endothelial cells (Fig. 1,E). p110α expression was not detectable in other types of stroma supporting cells (Figs. 1,B–D and 2, C and F). Interestingly, in normal postmenopausal ovaries, p110α immunoreactivity was localized exclusively to the muscularis layer of blood vessels but not to their endothelium (data not shown).

PIK3CA Levels Correlate with VEGF Expression and Angiogenesis.

In ovarian cancer cell lines, PIK3CA amplification leads to increased PI3k activity (1). Because PI3k has been shown to affect the expression of HIF-1α, an upstream regulator of VEGF (35), we investigated whether the observed overexpression of PIK3CA correlated with overexpression of VEGF in ovarian cancer. We analyzed the mRNA levels of PIK3CA and VEGF in 33 advanced stage ovarian cancer specimens, as well as 17 established human ovarian cancer cell lines using quantitative real-time RT-PCR. A significantly positive correlation was found between VEGF and PIK3CA mRNA levels in both tissue specimens (P = 0.008), as well as established cell lines (P = 0.026, Fig. 2 A).

To confirm the correlation between PIK3CA and VEGF expression, we examined the expression of p110α and VEGF protein by IHC in serial sections of ovarian cancer specimens. VEGF immunolocalized predominantly to cells located within tumor islets but not in tumor stroma (Fig. 2, D and G). Select tumors exhibited strong immunostaining for VEGF. A significant correlation (P < 0.01) was found between the semiquantitative score of VEGF and that of p110α protein expression (Fig. 2,B), with two representative cases shown in Fig. 2,C–G. Furthermore, microvascular development, as assessed by CD31 staining, was markedly more pronounced in the tumors with high p110α expression as compared with tumors with low p110α expression. Two representative cases are shown in Fig. 2, E and H. These data collectively suggest a possible functional relationship between PIK3CA kinase activity and VEGF expression in ovarian cancer.

PI3k Regulates VEGF Expression via HIF in Ovarian Cancer Cells.

To test whether PI3k is involved in the regulation of VEGF expression in ovarian cancer, we examined the ability of the PI3k inhibitor Ly294002 (43) to block VEGF or HIF in established cell lines. Our previous work demonstrated that glucose starvation can dramatically up-regulate VEGF expression in ovarian cancer cell lines (46). We therefore subjected cells to glucose starvation in the presence or absence of Ly294002 (10 μm) for 24 h. VEGF was analyzed by quantitative real-time RT-PCR and IHC. In all of the five cell lines tested, VEGF mRNA was up-regulated by glucose starvation; this effect was largely negated by the presence of Ly294002 (Fig. 3,A). These results were confirmed at the protein level by IHC (Fig. 3,B). To test whether PI3k regulates VEGF via HIF, cells were treated at resting conditions with 10 μm Ly294002 for 36 h. Incubation with the inhibitor significantly suppressed the mRNA levels of HIF-1α as assessed by quantitative real-time RT-PCR in all of the five ovarian cancer cell lines (Fig. 3,C). In addition, Ly294002 suppressed the induction of HIF-1α by glucose starvation (data not shown). These results were additionally confirmed by Western blot analysis. HIF-1α protein levels markedly decreased after exposure of A2008 or A2780 cells to Ly294002 (Fig. 3 D). Collectively, these data indicate that PIK3CA might play an important role in the regulation of the constitutive as well as induce expression of HIF-1α and VEGF in ovarian cancer.

Overexpressed PIK3CA Is Associated with Enhanced Proliferation and Reduced Apoptosis of Tumor Cells.

Because PIK3CA promotes angiogenesis and because it is a putative oncogene in ovarian cancer (1), we hypothesized that PIK3CA overexpression is associated with enhanced tumor cell survival and proliferation in vivo. Using the median of PIK3CA mRNA level (21.8 relative expression units) as the cutoff point, all advanced stage tumors were separated into two groups: one with relatively high and one with relatively low expression of PIK3CA mRNA. Cell proliferation was detected by Ki-67 staining. Ki-67-positive cells were found predominantly in tumor islets and, occasionally, in tumor stroma. Double immunofluorescent staining of Ki-67 and cytokeratin revealed that 99% of Ki-67-positive cells were also cytokeratin positive and were therefore tumor cells (Fig. 4,A), whereas <1% of cells within tumor islets were Ki-67 positive and cytokeratine negative. The ratio of Ki-67-positive cells over total cells was used to compute the tumor proliferation index. Three random fields of tumor islets/tumor were analyzed by computer-assisted imaging analysis (examples of tumor with low or high PIK3CA expression are provided in Fig. 4, C and D, respectively). The proliferation index was significantly higher in tumors with high PIK3CA expression compared with those with low PIK3CA expression (P < 0.05, Fig. 4,B). We additionally assessed the viability of tumor cells by examining cell proliferation and apoptosis in the same specimens by combined fluorescent immunostaining and ApopTag in Situ TUNEL assay. Consistent with the result from IHC, higher prevalence of proliferating cells and lower prevalence of apoptotic cells were detected in tumors with high PIK3CA expression compared with those with low PIK3CA expression (Fig. 4, E and F).

To additionally confirm the effects of PIK3CA-signaling pathway on ovarian cancer cells, we treated human ovarian cancer cell lines A2008, A1847, A2780, and its platinum-resistant clone A2780/CP70, with Ly294002. It was found that Ly294002 significantly decreased cell proliferation in vitro at a low dose (10 μm; Fig. 4,G) while potently induced cell apoptosis at a high dose (50 μm; Fig. 4 F). Collectively, these data are consistent with the reports from other groups (1), supporting the function of PIK3CA as an important oncogene in ovarian carcinoma.

Although in vitro evidence strongly suggests that PIK3CA functions as an important oncogene in ovarian cancer (1), the prevalence of PIK3CA overexpression in vivo, especially in early stage ovarian cancer, remains to date unknown. Furthermore, the effects of PIK3CA overexpression on the tumor microenvironment have not been elucidated. In this study, we report that PIK3CA overexpression is highly prevalent in ovarian carcinoma. Our results are in agreement with the previously reported increased copy number of PIK3CA gene and increased PIK3CA transcription in ovarian cancer cell lines (1). Our findings also suggest that PIK3CA overexpression may occur early during malignant transformation in the ovary because mRNA levels were similar in early- and late-stage tumors. These findings are in agreement with findings that gain in 3q26 region, where PIK3CA is located, are encountered frequently in early-stage ovarian carcinoma (48, 49).

In this study, we provide direct evidence that PIK3CA overexpression induces increased angiogenesis in ovarian carcinoma because of up-regulation of VEGF via HIF. These data provide an explanation for the reported complete abrogation of ascites during in vivo treatment with Ly294002 in a xenograft i.p. model of ovarian carcinoma (50) because VEGF is directly implicated in ascites formation in ovarian carcinoma. These data are also in agreement with evidence that PIK3CAdel/del embryos demonstrated areas of defective angiogenesis (51). PIK3CA is therefore added to the list of other oncogenes overexpressed in ovarian carcinoma, which may initiate angiogenesis through HIF activation.

Interestingly, immunoreactive p110α was strongly expressed in tumor stroma endothelial cells in 75.7% of tumors examined. Tumor vasculature claims a morphologically and functionally unique phenotype, greatly distinct in growth properties, functions, and responses to angiogenic factors (52). The PI3k-signaling pathway plays an important role in endothelial cell migration and proliferation during angiogenesis (4, 36, 39, 40, 41). Expression of p110α at levels detectable through IHC may therefore reflect activation of tumor endothelial cells and enhanced angiogenesis. In fact, vascular endothelium from postmenopausal ovaries was negative for p110α expression. Conversely, strong expression of p110α was detected in the muscularis layer of vessels from postmenopausal ovaries, possibly reflecting a physiological role of PIK3CA in menopause-induced vascular smooth muscle changes (53, 54).

The PI3k-signaling pathway is involved in multiple aspects of malignant transformation (1, 2, 3, 55, 56, 57, 58). We found that PIK3CA overexpression was associated with increased cell proliferation and decreased apoptosis, suggesting an important association with overall enhanced tumor growth. This effect may be partly attributed to the observed effect of PIK3CA on angiogenesis because increased angiogenesis and microvascular density closely correlates with tumor cell survival and proliferation and tumor growth (59). In agreement with a previous article (1), we found that PIK3CA promotes also cell survival and proliferation directly in ovarian cancer in vitro.

In summary, this study provides strong evidence that PIK3CA overexpression plays an important role in ovarian cancer. Collectively, our data indicate that PIK3CA promotes tumor growth through at least two distinct pathways: it enhances cell survival and proliferation through a direct mechanism and it up-regulates VEGF via HIF to promote angiogenesis. PI3k and its associated pathway have been considered as a potential antitumor therapeutic target (4, 50, 60, 61, 62, 63), and PI3k blockade has shown efficacy in a xenograft model of ovarian carcinoma (50, 62). Our data support the notion that therapy strategies targeting PI3k or directly PIK3CA (e.g., antisense or interfering RNA therapy) should contribute not only to the inhibition of oncogenic signaling pathways but also to the inhibition of tumor angiogenesis.

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.

1

Supported by American Association of Obstetricians and Gynecologists’ Foundations, National Cancer Institute Ovarian Grant SPORE P01-CA83638, institutional funding from the Abramson Family Cancer Center and Cancer Research Institute, and the Department of Obstetrics and Gynecology at the University of Pennsylvania. The laser capture microdissection facility was supported by a generous grant from the Fannie Rippel Foundation.

3

The abbreviations used are: PI3k, phosphatidylinositol 3′-kinase; PIK3CA, PI3k catalytic subunit α; HIF, hypoxia-inducible factor; VEGF, vascular endothelial growth factor; LMP, low malignant potential; RT-PCR, reverse transcription-PCR; IHC, immunohistochemistry; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; FIGO, International Federation of Gynecologists and Obstetricians.

Fig. 1.

PIK3CA is overexpressed in ovarian carcinoma. A, PIK3CA mRNA quantitation by real-time RT-PCR in human ovarian tumors: group 1, benign (n = 4) and LMP tumors (n = 4); group 2, FIGO stage I ovarian cancer (n = 9); and group 3, FIGO stage III and IV ovarian cancer (n = 33). Data are shown as mean ± SE. B–D, p110α protein detection by IHC in stage III and IV ovarian cancer. p110α was detectable in 22 of 39 (56.4%) advanced stage ovarian cancer samples, where it localized to the cytoplasm of tumor cells (B and C). p110α expression was also detected in vascular endothelial cells of stroma in 28 of 39 cases (71.8%; D). In 9 of 39 (23.1%) specimens, p110α was absent from stroma vessels (C). E, summary of p100α expression in advanced stage ovarian cancer. There was no correlation between the expression of p110α in tumor cells and endothelial cells.

Fig. 1.

PIK3CA is overexpressed in ovarian carcinoma. A, PIK3CA mRNA quantitation by real-time RT-PCR in human ovarian tumors: group 1, benign (n = 4) and LMP tumors (n = 4); group 2, FIGO stage I ovarian cancer (n = 9); and group 3, FIGO stage III and IV ovarian cancer (n = 33). Data are shown as mean ± SE. B–D, p110α protein detection by IHC in stage III and IV ovarian cancer. p110α was detectable in 22 of 39 (56.4%) advanced stage ovarian cancer samples, where it localized to the cytoplasm of tumor cells (B and C). p110α expression was also detected in vascular endothelial cells of stroma in 28 of 39 cases (71.8%; D). In 9 of 39 (23.1%) specimens, p110α was absent from stroma vessels (C). E, summary of p100α expression in advanced stage ovarian cancer. There was no correlation between the expression of p110α in tumor cells and endothelial cells.

Close modal
Fig. 2.

PIK3CA expression correlates with VEGF expression and angiogenesis. A, PIK3CA and VEGF mRNA levels were quantified by real-time RT-RCR. PIK3CA mRNA levels significantly correlated with VEGF mRNA levels in advanced stage (FIGO stage III/IV) ovarian carcinoma (n = 33, P = 0.008) and in 17 cultured human ovarian cancer cell lines (n = 17, P = 0.026). B–H, immunohistochemical staining of p110α, VEGF, and CD31 was performed in advanced stage ovarian cancer samples. B shows the correlation of p110α with VEGF staining score (n = 39, P < 0.01). C–E show representative results in a tumor with low p110α expression; F–H show representative results in a tumor with high p110α expression. p110α expression is associated with markedly increased VEGF expression (D and G) and increased microvascular development (E and H).

Fig. 2.

PIK3CA expression correlates with VEGF expression and angiogenesis. A, PIK3CA and VEGF mRNA levels were quantified by real-time RT-RCR. PIK3CA mRNA levels significantly correlated with VEGF mRNA levels in advanced stage (FIGO stage III/IV) ovarian carcinoma (n = 33, P = 0.008) and in 17 cultured human ovarian cancer cell lines (n = 17, P = 0.026). B–H, immunohistochemical staining of p110α, VEGF, and CD31 was performed in advanced stage ovarian cancer samples. B shows the correlation of p110α with VEGF staining score (n = 39, P < 0.01). C–E show representative results in a tumor with low p110α expression; F–H show representative results in a tumor with high p110α expression. p110α expression is associated with markedly increased VEGF expression (D and G) and increased microvascular development (E and H).

Close modal
Fig. 3.

PI3k regulates VEGF expression via HIF in ovarian cancer cell lines. A and B, blocking of PI3k by Ly294002 abolishes glucose starvation-induced VEGF up-regulation in ovarian cancer cell lines. VEGF mRNA levels in control cells and cells incubated in glucose-free media in the absence or presence of Ly294002. VEGF mRNA was quantified by real-time RT-PCR (A). Intracellular VEGF expression by IHC in control cells and cells incubated in glucose-free media in the absence or presence of Ly294002 (B). C and D, blocking of PI3k by Ly294002 significantly decreased HIF-1α expression in ovarian cancer cell lines. Ly294002 treatment reduced HIF-1α mRNA level. HIF-1α mRNA was quantified by real-time RT-PCR (C). HIF-1α protein levels, detected by Western blot, were decreased after exposure to Ly294002 (D). Experiments were repeated three times, and data are shown as mean ± SE.

Fig. 3.

PI3k regulates VEGF expression via HIF in ovarian cancer cell lines. A and B, blocking of PI3k by Ly294002 abolishes glucose starvation-induced VEGF up-regulation in ovarian cancer cell lines. VEGF mRNA levels in control cells and cells incubated in glucose-free media in the absence or presence of Ly294002. VEGF mRNA was quantified by real-time RT-PCR (A). Intracellular VEGF expression by IHC in control cells and cells incubated in glucose-free media in the absence or presence of Ly294002 (B). C and D, blocking of PI3k by Ly294002 significantly decreased HIF-1α expression in ovarian cancer cell lines. Ly294002 treatment reduced HIF-1α mRNA level. HIF-1α mRNA was quantified by real-time RT-PCR (C). HIF-1α protein levels, detected by Western blot, were decreased after exposure to Ly294002 (D). Experiments were repeated three times, and data are shown as mean ± SE.

Close modal
Fig. 4.

PIK3CA overexpression is associated with increased tumor cells proliferation and low prevalence of apoptosis. A, more than 99% of Ki-67-positive cells (red, Texas Red) in tumor islets are tumor cells, as assessed by expression of cytokeratin (green, FITC). B, using the median value of PIK3CA mRNA level (= 21.8 relative expression units) as the cutoff point, 39 stage III tumors were separated in two groups with low and high PIK3CA mRNA expression. The Ki-67 proliferation index in tumors with high PIK3CA mRNA expression was significantly higher than in tumors with low PIK3CA mRNA expression (P < 0.05). C and D, show Ki-67 staining (brown, 3,3′-diaminobenzidine) in two representative specimens with low or high PIK3CA expression, respectively. E and F, using TUNEL in situ apoptosis assay combined with double immunofluorescent staining, apoptotic tumor cells and proliferating tumor cells were detected in the same sections. E and F show the results of two representative specimens with low or high PIK3CA expression, respectively. Green (FITC) shows TUNEL-positive apoptotic cells, blue (7-amino-4-methylcoumarin-3-acetic acid) shows Ki-67-positive proliferating cells, and red (Texas Red) shows cytokeratin-positive tumor cells. A higher number of proliferating cells and lower number of apoptotic cells were found in tumors with high PIK3CA mRNA expression (F) as compared with tumors with low PIK3CA mRNA expression (E). G and H, Ly294002 (10 μm) significantly decreased proliferation of ovarian cancer cells in vitro (G). At a high dose (50 μm), Ly294002 induced apoptosis in ovarian cancer cells, as assessed by annexin-V assay (H). Experiments were repeated three times, and data are expressed as mean ± SE.

Fig. 4.

PIK3CA overexpression is associated with increased tumor cells proliferation and low prevalence of apoptosis. A, more than 99% of Ki-67-positive cells (red, Texas Red) in tumor islets are tumor cells, as assessed by expression of cytokeratin (green, FITC). B, using the median value of PIK3CA mRNA level (= 21.8 relative expression units) as the cutoff point, 39 stage III tumors were separated in two groups with low and high PIK3CA mRNA expression. The Ki-67 proliferation index in tumors with high PIK3CA mRNA expression was significantly higher than in tumors with low PIK3CA mRNA expression (P < 0.05). C and D, show Ki-67 staining (brown, 3,3′-diaminobenzidine) in two representative specimens with low or high PIK3CA expression, respectively. E and F, using TUNEL in situ apoptosis assay combined with double immunofluorescent staining, apoptotic tumor cells and proliferating tumor cells were detected in the same sections. E and F show the results of two representative specimens with low or high PIK3CA expression, respectively. Green (FITC) shows TUNEL-positive apoptotic cells, blue (7-amino-4-methylcoumarin-3-acetic acid) shows Ki-67-positive proliferating cells, and red (Texas Red) shows cytokeratin-positive tumor cells. A higher number of proliferating cells and lower number of apoptotic cells were found in tumors with high PIK3CA mRNA expression (F) as compared with tumors with low PIK3CA mRNA expression (E). G and H, Ly294002 (10 μm) significantly decreased proliferation of ovarian cancer cells in vitro (G). At a high dose (50 μm), Ly294002 induced apoptosis in ovarian cancer cells, as assessed by annexin-V assay (H). Experiments were repeated three times, and data are expressed as mean ± SE.

Close modal
Table 1

Sequence of primers used

Primer nameSequence
PIK3CA F TCA AAG GAT TGG GCA CTT TT 
PIK3CA R GCC TCG ACT TGC CTA TTC AG 
VEGF F AAC CAT GAA CTT TCT GCT GTC TTG 
VEGF R TTC ACC ACT TCG TGA TGA TTC TG 
HIF-1α F AAG CCC TAA CGT GTT ATC TGT CG 
HIF-1α R CTG CTT GAA AAA GTG AAC CAT CA 
18S F GAA ACT GCG AAT GGC TCA TTA AA 
18S R CAC AGT TAT CCA AGT GGG AGA GG 
GAPDH F CCT GCA CCA CCA ACT GCT TA 
GAPDH R CAT GAG TCC TTC CAC GAT ACC A 
GAPDH probe CCT GGC CAA GGT CAT CCA C 
Primer nameSequence
PIK3CA F TCA AAG GAT TGG GCA CTT TT 
PIK3CA R GCC TCG ACT TGC CTA TTC AG 
VEGF F AAC CAT GAA CTT TCT GCT GTC TTG 
VEGF R TTC ACC ACT TCG TGA TGA TTC TG 
HIF-1α F AAG CCC TAA CGT GTT ATC TGT CG 
HIF-1α R CTG CTT GAA AAA GTG AAC CAT CA 
18S F GAA ACT GCG AAT GGC TCA TTA AA 
18S R CAC AGT TAT CCA AGT GGG AGA GG 
GAPDH F CCT GCA CCA CCA ACT GCT TA 
GAPDH R CAT GAG TCC TTC CAC GAT ACC A 
GAPDH probe CCT GGC CAA GGT CAT CCA C 

We thank Drs. Kang-Sheng Yao and Steven Johnson (Department of Pharmacology, University of Pennsylvania) for donating the human ovarian cancer cells. We also thank Ann O’Brien-Jenkins and Alisha Mohamed-Hadley for their excellent technical support.

1
Shayesteh L., Lu Y., Kuo W. L., Baldocchi R., Godfrey T., Collins C., Pinkel D., Powell B., Mills G. B., Gray J. W. PIK3CA is implicated as an oncogene in ovarian cancer.
Nat. Genet.
,
21
:
99
-102,  
1999
.
2
Vivanco I., Sawyers C. L. The phosphatidylinositol 3-kinase AKT pathway in human cancer.
Nat. Rev. Cancer
,
2
:
489
-501,  
2002
.
3
Cantley L. C. The phosphoinositide 3-kinase pathway.
Science (Wash. DC)
,
296
:
1655
-1657,  
2002
.
4
Katso R., Okkenhaug K., Ahmadi K., White S., Timms J., Waterfield M. D. Cellular function of phosphoinositide 3-kinases: implications for development, homeostasis, and cancer.
Annu. Rev. Cell Dev. Biol.
,
17
:
615
-675,  
2001
.
5
Hiles I. D., Otsu M., Volinia S., Fry M. J., Gout I., Dhand R., Panayotou G., Ruiz-Larrea F., Thompson A., Totty N. F., et al Phosphatidylinositol 3-kinase: structure and expression of the 110 kd catalytic subunit.
Cell
,
70
:
419
-429,  
1992
.
6
Iwabuchi H., Sakamoto M., Sakunaga H., Ma Y. Y., Carcangiu M. L., Pinkel D., Yang-Feng T. L., Gray J. W. Genetic analysis of benign, low-grade, and high-grade ovarian tumors.
Cancer Res.
,
55
:
6172
-6180,  
1995
.
7
Knuutila S., Bjorkqvist A. M., Autio K., Tarkkanen M., Wolf M., Monni O., Szymanska J., Larramendy M. L., Tapper J., Pere H., El-Rifai W., Hemmer S., Wasenius V. M., Vidgren V., Zhu Y. DNA copy number amplifications in human neoplasms: review of comparative genomic hybridization studies.
Am. J. Pathol.
,
152
:
1107
-1123,  
1998
.
8
Massion P. P., Kuo W. L., Stokoe D., Olshen A. B., Treseler P. A., Chin K., Chen C., Polikoff D., Jain A. N., Pinkel D., Albertson D. G., Jablons D. M., Gray J. W. Genomic copy number analysis of non-small cell lung cancer using array comparative genomic hybridization: implications of the phosphatidylinositol 3-kinase pathway.
Cancer Res.
,
62
:
3636
-3640,  
2002
.
9
Redon R., Muller D., Caulee K., Wanherdrick K., Abecassis J., du Manoir S. A simple specific pattern of chromosomal aberrations at early stages of head and neck squamous cell carcinomas: PIK3CA but not p63 gene as a likely target of 3q26-qter gains.
Cancer Res.
,
61
:
4122
-4129,  
2001
.
10
Woenckhaus J., Steger K., Werner E., Fenic I., Gamerdinger U., Dreyer T., Stahl U. Genomic gain of PIK3CA and increased expression of p110α are associated with progression of dysplasia into invasive squamous cell carcinoma.
J. Pathol.
,
198
:
335
-342,  
2002
.
11
Hui A. B., Lo K. W., Yin X. L., Poon W. S., Ng H. K. Detection of multiple gene amplifications in glioblastoma multiforme using array-based comparative genomic hybridization.
Lab. Investig.
,
81
:
717
-723,  
2001
.
12
Hui A. B., Lo K. W., Teo P. M., To K. F., Huang D. P. Genome wide detection of oncogene amplifications in nasopharyngeal carcinoma by array based comparative genomic hybridization.
Int. J. Oncol.
,
20
:
467
-473,  
2002
.
13
Singh B., Reddy P. G., Goberdhan A., Walsh C., Dao S., Ngai I., Chou T. C., O-Charoenrat P., Levine A. J., Rao P. H., Stoffel A. p53 regulates cell survival by inhibiting PIK3CA in squamous cell carcinomas.
Genes Dev.
,
16
:
984
-993,  
2002
.
14
Ma Y. Y., Wei S. J., Lin Y. C., Lung J. C., Chang T. C., Whang-Peng J., Liu J. M., Yang D. M., Yang W. K., Shen C. Y. PIK3CA as an oncogene in cervical cancer.
Oncogene
,
19
:
2739
-2744,  
2000
.
15
Zhang A., Maner S., Betz R., Angstrom T., Stendahl U., Bergman F., Zetterberg A., Wallin K. L. Genetic alterations in cervical carcinomas: frequent low-level amplifications of oncogenes are associated with human papillomavirus infection.
Int. J. Cancer
,
101
:
427
-433,  
2002
.
16
Arbiser J. L., Moses M. A., Fernandez C. A., Ghiso N., Cao Y., Klauber N., Frank D., Brownlee M., Flynn E., Parangi S., Byers H. R., Folkman J. Oncogenic H-ras stimulates tumor angiogenesis by two distinct pathways.
Proc. Natl. Acad. Sci. USA
,
94
:
861
-866,  
1997
.
17
Rak J., Mitsuhashi Y., Sheehan C., Tamir A., Viloria-Petit A., Filmus J., Mansour S. J., Ahn N. G., Kerbel R. S. Oncogenes and tumor angiogenesis: differential modes of vascular endothelial growth factor up-regulation in ras-transformed epithelial cells and fibroblasts.
Cancer Res.
,
60
:
490
-498,  
2000
.
18
Jiang B. H., Agani F., Passaniti A., Semenza G. L. V-SRC induces expression of hypoxia-inducible factor 1 (HIF-1) and transcription of genes encoding vascular endothelial growth factor and enolase 1: involvement of HIF-1 in tumor progression.
Cancer Res.
,
57
:
5328
-5335,  
1997
.
19
Shim H., Dolde C., Lewis B. C., Wu C. S., Dang G., Jungmann R. A., Dalla-Favera R., Dang C. V. c-Myc transactivation of LDH-A: implications for tumor metabolism and growth.
Proc. Natl. Acad. Sci. USA
,
94
:
6658
-6663,  
1997
.
20
Baudino T. A., McKay C., Pendeville-Samain H., Nilsson J. A., Maclean K. H., White E. L., Davis A. C., Ihle J. N., Cleveland J. L. c-Myc is essential for vasculogenesis and angiogenesis during development and tumor progression.
Genes Dev.
,
16
:
2530
-2543,  
2002
.
21
Pelengaris S., Khan M., Evan G. I. Suppression of Myc-induced apoptosis in β cells exposes multiple oncogenic properties of Myc and triggers carcinogenic progression.
Cell
,
109
:
321
-334,  
2002
.
22
Laughner E., Taghavi P., Chiles K., Mahon P. C., Semenza G. L. HER2 (neu) signaling increases the rate of hypoxia-inducible factor 1α (HIF-1α) synthesis: novel mechanism for HIF-1-mediated vascular endothelial growth factor expression.
Mol. Cell. Biol.
,
21
:
3995
-4004,  
2001
.
23
Lopez-Ocejo O., Viloria-Petit A., Bequet-Romero M., Mukhopadhyay D., Rak J., Kerbel R. S. Oncogenes and tumor angiogenesis: the HPV-16 E6 oncoprotein activates the vascular endothelial growth factor (VEGF) gene promoter in a p53 independent manner.
Oncogene
,
19
:
4611
-4620,  
2000
.
24
An W. G., Kanekal M., Simon M. C., Maltepe E., Blagosklonny M. V., Neckers L. M. Stabilization of wild-type p53 by hypoxia-inducible factor 1α.
Nature (Lond.)
,
392
:
405
-408,  
1998
.
25
Ravi R., Mookerjee B., Bhujwalla Z. M., Sutter C. H., Artemov D., Zeng Q., Dillehay L. E., Madan A., Semenza G. L., Bedi A. Regulation of tumor angiogenesis by p53-induced degradation of hypoxia-inducible factor 1α.
Genes Dev.
,
14
:
34
-44,  
2000
.
26
Yuan A., Yu C. J., Luh K. T., Kuo S. H., Lee Y. C., Yang P. C. Aberrant p53 expression correlates with expression of vascular endothelial growth factor mRNA and interleukin-8 mRNA and neoangiogenesis in non-small-cell lung cancer.
J. Clin. Oncol.
,
20
:
900
-910,  
2002
.
27
Yu J. L., Rak J. W., Coomber B. L., Hicklin D. J., Kerbel R. S. Effect of p53 status on tumor response to antiangiogenic therapy.
Science (Wash. DC)
,
295
:
1526
-1528,  
2002
.
28
Maxwell P. H., Wiesener M. S., Chang G. W., Clifford S. C., Vaux E. C., Cockman M. E., Wykoff C. C., Pugh C. W., Maher E. R., Ratcliffe P. J. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis.
Nature (Lond.)
,
399
:
271
-275,  
1999
.
29
Turner K. J., Moore J. W., Jones A., Taylor C. F., Cuthbert-Heavens D., Han C., Leek R. D., Gatter K. C., Maxwell P. H., Ratcliffe P. J., Cranston D., Harris A. L. Expression of hypoxia-inducible factors in human renal cancer: relationship to angiogenesis and to the von Hippel-Lindau gene mutation.
Cancer Res.
,
62
:
2957
-2961,  
2002
.
30
Zundel W., Schindler C., Haas-Kogan D., Koong A., Kaper F., Chen E., Gottschalk A. R., Ryan H. E., Johnson R. S., Jefferson A. B., Stokoe D., Giaccia A. J. Loss of PTEN facilitates HIF-1-mediated gene expression.
Genes Dev.
,
14
:
391
-396,  
2000
.
31
Zhong H., Chiles K., Feldser D., Laughner E., Hanrahan C., Georgescu M. M., Simons J. W., Semenza G. L. Modulation of hypoxia-inducible factor 1α expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics.
Cancer Res.
,
60
:
1541
-1545,  
2000
.
32
Rak J., Yu J. L., Kerbel R. S., Coomber B. L. What do oncogenic mutations have to do with angiogenesis/vascular dependence of tumors?.
Cancer Res.
,
62
:
1931
-1934,  
2002
.
33
Rak J., Yu J. L., Klement G., Kerbel R. S. Oncogenes and angiogenesis: signaling three-dimensional tumor growth.
J Investig. Dermatol. Symp. Proc.
,
5
:
24
-33,  
2000
.
34
Mazure N. M., Chen E. Y., Laderoute K. R., Giaccia A. J. Induction of vascular endothelial growth factor by hypoxia is modulated by a phosphatidylinositol 3-kinase/Akt signaling pathway in Ha-ras-transformed cells through a hypoxia-inducible factor-1 transcriptional element.
Blood
,
90
:
3322
-3331,  
1997
.
35
Jiang B. H., Jiang G., Zheng J. Z., Lu Z., Hunter T., Vogt P. K. Phosphatidylinositol 3-kinase signaling controls levels of hypoxia-inducible factor 1.
Cell Growth Differ.
,
12
:
363
-369,  
2001
.
36
Jiang B. H., Zheng J. Z., Aoki M., Vogt P. K. Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of vascular endothelial growth factor in endothelial cells.
Proc. Natl. Acad. Sci. USA
,
97
:
1749
-1753,  
2000
.
37
Blancher C., Moore J. W., Robertson N., Harris A. L. Effects of ras and von Hippel-Lindau (VHL) gene mutations on hypoxia-inducible factor (HIF)-1{α}. HIF-2{α}, and vascular endothelial growth factor expression and their regulation by the phosphatidylinositol 3′-kinase/Akt signaling pathway.
Cancer Res.
,
61
:
7349
-7355,  
2001
.
38
Chen E. Y., Mazure N. M., Cooper J. A., Giaccia A. J. Hypoxia activates a platelet-derived growth factor receptor/phosphatidylinositol 3-kinase/Akt pathway that results in glycogen synthase kinase-3 inactivation.
Cancer Res.
,
61
:
2429
-2433,  
2001
.
39
Thakker G. D., Hajjar D. P., Muller W. A., Rosengart T. K. The role of phosphatidylinositol 3-kinase in vascular endothelial growth factor signaling.
J. Biol. Chem.
,
274
:
10002
-10007,  
1999
.
40
Soldi R., Mitola S., Strasly M., Defilippi P., Tarone G., Bussolino F. Role of αvβ3 integrin in the activation of vascular endothelial growth factor receptor 2.
EMBO J.
,
18
:
882
-892,  
1999
.
41
Carmeliet P., Lampugnani M. G., Moons L., Breviario F., Compernolle V., Bono F., Balconi G., Spagnuolo R., Oostuyse B., Dewerchin M., Zanetti A., Angellilo A., Mattot V., Nuyens D., Lutgens E., Clotman F., de Ruiter M. C., Gittenberger-de Groot A., Poelmann R., Lupu F., Herbert J. M., Collen D., Dejana E. Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis.
Cell
,
98
:
147
-157,  
1999
.
42
Zhang L., Yang N., Mohamed-Hadley A., Rubin S. C., Coukos G. Vector-based RNAi, a novel tool for isoform-specific knock-down of VEGF and anti-angiogenesis gene therapy of cancer.
Biochem. Biophys. Res. Commun.
,
303
:
1169
-1178,  
2003
.
43
Vlahos C. J., Matter W. F., Hui K. Y., Brown R. F. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-1 (LY294002).
J. Biol. Chem.
,
269
:
5241
-5248,  
1994
.
44
Zhang L., Conejo-Garcia J. R., Katsaros D., Gimotty P. A., Massobrio M., Regnani G., Makrigiannakis A., Gray H., Schlienger K., Liebman M. N., Rubin S. C., Coukos G. Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer.
N. Engl. J. Med.
,
348
:
203
-213,  
2003
.
45
Zhang L., Yang N., Conejo-Garcia J. R., Katsaros D., Mohamed-Hadley A., Fracchioli S., Schlienger K., Toll A., Levine B., Rubin S. C., Coukos G. Expression of endocrine gland-derived vascular endothelial growth factor in ovarian carcinoma.
Clin. Cancer Res.
,
9
:
264
-272,  
2003
.
46
Zhang L., Conejo-Garcia J. R., Yang N., Huang W., Mohamed-Hadley A., Yao W., Benencia F., Coukos G. Different effects of glucose starvation on expression and stability of VEGF mRNA isoforms in murine ovarian cancer cells.
Biochem. Biophys. Res. Commun.
,
292
:
860
-868,  
2002
.
47
Zhang L., Yang N., Garcia J. R., Mohamed A., Benencia F., Rubin S. C., Allman D., Coukos G. Generation of a syngeneic mouse model to study the effects of vascular endothelial growth factor in ovarian carcinoma.
Am. J. Pathol.
,
161
:
2295
-2309,  
2002
.
48
Suzuki S., Moore D. H., II, Ginzinger D. G., Godfrey T. E., Barclay J., Powell B., Pinkel D., Zaloudek C., Lu K., Mills G., Berchuck A., Gray J. W. An approach to analysis of large-scale correlations between genome changes and clinical endpoints in ovarian cancer.
Cancer Res.
,
60
:
5382
-5385,  
2000
.
49
Gray J. W., Suzuki S., Kuo W. L., Polikoff D., Deavers M., Smith-McCune K., Berchuck A., Pinkel D., Albertson D., Mills G. B. Specific keynote: genome copy number abnormalities in ovarian cancer.
Gynecol. Oncol.
,
88
:
S16
-S21, S22S24, discussion 
2003
.
50
Hu L., Zaloudek C., Mills G. B., Gray J., Jaffe R. B. In vivo and in vitro ovarian carcinoma growth inhibition by a phosphatidylinositol 3-kinase inhibitor (LY294002).
Clin. Cancer Res.
,
6
:
880
-886,  
2000
.
51
Bi L., Okabe I., Bernard D. J., Wynshaw-Boris A., Nussbaum R. L. Proliferative defect and embryonic lethality in mice homozygous for a deletion in the p110α subunit of phosphoinositide 3-kinase.
J. Biol. Chem.
,
274
:
10963
-10968,  
1999
.
52
Ruoslahti E. Specialization of tumor vasculature.
Nat. Rev. Cancer
,
2
:
83
-90,  
2002
.
53
Shimada T., Morita T., Nagai K., Sato F., Mori H., Campbell G. R. Morphological changes in spiral artery of the mammalian ovary with age.
Horm. Res.
,
39 (Suppl. 1)
:
9
-15,  
1993
.
54
Komalavilas P., Mehta S., Wingard C. J., Dransfield D. T., Bhalla J., Woodrum J. E., Molinaro J. R., Brophy C. M. PI3-kinase/Akt modulates vascular smooth muscle tone via cAMP signaling pathways.
J. Appl. Physiol.
,
91
:
1819
-1827,  
2001
.
55
Klippel A., Escobedo M. A., Wachowicz M. S., Apell G., Brown T. W., Giedlin M. A., Kavanaugh W. M., Williams L. T. Activation of phosphatidylinositol 3-kinase is sufficient for cell cycle entry and promotes cellular changes characteristic of oncogenic transformation.
Mol. Cell. Biol.
,
18
:
5699
-5711,  
1998
.
56
Frevert E. U., Kahn B. B. Differential effects of constitutively active phosphatidylinositol 3-kinase on glucose transport, glycogen synthase activity, and DNA synthesis in 3T3-L1 adipocytes.
Mol. Cell. Biol.
,
17
:
190
-198,  
1997
.
57
Khwaja A., Rodriguez-Viciana P., Wennstrom S., Warne P. H., Downward J. Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway.
EMBO J.
,
16
:
2783
-2793,  
1997
.
58
Kennedy S. G., Wagner A. J., Conzen S. D., Jordan J., Bellacosa A., Tsichlis P. N., Hay N. The PI 3-kinase/Akt signaling pathway delivers an anti-apoptotic signal.
Genes Dev.
,
11
:
701
-713,  
1997
.
59
Folkman J. Tumor angiogenesis: therapeutic implications.
N. Engl. J. Med.
,
285
:
1182
-1186,  
1971
.
60
Stein R. C., Waterfield M. D. PI3-kinase inhibition: a target for drug development?.
Mol. Med. Today
,
6
:
347
-357,  
2000
.
61
Razzini G., Berrie C. P., Vignati S., Broggini M., Mascetta G., Brancaccio A., Falasca M. Novel functional PI 3-kinase antagonists inhibit cell growth and tumorigenicity in human cancer cell lines.
FASEB J.
,
14
:
1179
-1187,  
2000
.
62
Hu L., Hofmann J., Lu Y., Mills G. B., Jaffe R. B. Inhibition of phosphatidylinositol 3′-kinase increases efficacy of paclitaxel in in vitro and in vivo ovarian cancer models.
Cancer Res.
,
62
:
1087
-1092,  
2002
.
63
Mills G. B., Lu Y., Kohn E. C. Linking molecular therapeutics to molecular diagnostics: Inhibition of the FRAP/RAFT/TOR component of the PI3K pathway preferentially blocks PTEN mutant cells in vitro and in vivo.
Proc. Natl. Acad. Sci. USA
,
98
:
10031
-10033,  
2001
.