Objective
To evaluate PIK3CA mutational status and c-erbB2 gene amplification in a series of primary uterine serous carcinomas (USC) cell lines. To assess the efficacy of GDC-0980, a potent inhibitor of Class I PI3 kinase and mTOR kinase (TORC1/2), against primary USC harboring HER2/neu gene amplification and/or PIK3CA mutations.
Study Design
Twenty-two primary USC cell lines were evaluated for c-erbB2 oncogene amplification by fluorescence in situ hybridization (FISH) assays and for PIK3CA gene mutations by direct DNA sequencing of exons 9 and 20. In vitro sensitivity to GDC-0980 was evaluated by flow-cytometry-based viability and proliferation assays. Downstream cellular responses to GDC-0980 were assessed by measuring phosphorylation of the 4-EBP1 protein by flow-cytometry.
Results
Five of 22 (22.7%) USC cell lines contained oncogenic PIK3CA mutations although 9 (40.9%) harbored c-erbB2 gene amplification by FISH. GDC-0980 caused a strong differential growth inhibition in FISH+ USC when compared with FISH− (GDC-0980 IC 50 mean ± SEM = 0.29 ± 0.05 μM in FISH+ vs 1.09 ± 0.20 μM in FISH− tumors, P = .02). FISH+ USC harboring PIK3CA mutations were significantly more sensitive to GDC-0980 exposure when compared with USC cell lines harboring wild-type PIK3CA ( P = .03). GDC-0980 growth-inhibition was associated with a significant and dose-dependent decline in phosphorylated 4-EBP1 levels.
Conclusion
Oncogenic PIK3CA mutations and c-erbB2 gene amplification may represent biomarkers to identify patients harboring USC who may benefit most from the use of GDC-0980.
Endometrial carcinoma is one of the most common gynecologic cancers. About 49,560 new cases of endometrial cancer will be diagnosed in the year 2013 in the United States, with approximately 8190 deaths. Type I endometrial cancers account for most of these tumors and, typically presents as well or moderately differentiated carcinomas; these tumors are endometrioid in histology, are associated with endogenous hyperestrogenic conditions or chronic exposure to unopposed estrogens and are generally highly curable with primary surgery. In contrast, type II endometrial cancers account for a minority of cases and are not associated with unopposed estrogen exposure; these tumors have often an unfavorable prognosis because of their aggressive biologic behavior and consequent high risk for recurrence. Uterine serous carcinoma (USC) represents the most aggressive variant of type II endometrial cancer. Although this tumor subtype constitutes less than 10% of all endometrial tumors, it is responsible for 40% of deaths because of endometrial cancer. The discovery of molecular signaling pathways highly active in uterine serous tumors may lead to the development of novel, specific, and more effective therapeutic strategies against this aggressive subset of uterine carcinoma.
The phosphatidylinositol 3-kinase/AKT (PIK3/AKT)-mammalian target of rapamycin (mTOR) signaling cascade plays a central role in diverse cellular responses such as proliferation, survival, mobility, metabolism, and control of malignant cellular growth. HER2/neu, a member of the erbB receptor tyrosine kinase (TK) family located upstream to the PIK3CA/AKT/mTOR pathway, is associated with cancer cell proliferation, poor survival, and resistance to therapy in multiple human tumors. HER2/neu and the PIK3CA/AKT/mTOR pathway are often constitutively activated in various human cancers secondary to gene amplifications (ie, HER2/neu) or activating mutations in the PIK3CA/AKT genes, providing novel targets for cancer therapy.
Importantly, multiple research groups including our own have recently reported presence of PIK3CA gene mutations and HER2/neu gene amplifications in a relevant number of USC by whole exome sequencing. These data provide evidence to suggest that the use of PIK3/AKT/mTOR inhibitors may provide anticancer activity in biologically aggressive Type II endometrial cancers such as USC.
GDC-0980 is a novel, potent, orally available small molecule inhibitor with selectivity for class I PIK3CA and mTOR kinase. GDC-0980 has shown remarkable ability to target a variety of human cancer cell lines through the inhibition of both PIK3CA and mTOR signaling. GDC-0980 strongly inhibited the PI3K pathways and the phosphorylation of mTORC1 substrates S6K and 4E-BP1 as well as phosphorylation of the mTORC2 substrate AKT and downstream proteins. In vivo, GDC-0980 has demonstrated potent antitumor efficacy in nude mice bearing multiple human xenografts of breast cancer, nonsmall cell lung cancer, pancreatic cancer, colon cancer, prostate cancer, and melanoma. On the basis of these promising preclinical results, GDC-0980 is currently being tested in phase II studies in patients with advanced solid tumors.
Although multiple mTOR and/or PIK3CA inhibitors are currently under evaluation in clinical trials against a variety of human cancers including endometrial cancer, to our knowledge, no study has yet analyzed and compared the in vitro sensitivity of primary USC cell lines to selective dual PIK3CA/mTOR inhibitors. More importantly, no study has yet evaluated whether biologically aggressive USC harboring PIK3CA mutations and/or HER2/neu gene amplifications are selectively sensitive to agents targeting the PIK3CA/mTOR pathway. To fill this gap in knowledge, in this study we have: (1) sequenced a large panel of primary USC cell lines for oncogenic PIK3CA mutations, (2) evaluated c-erbB2 oncogene amplification by FISH assays, (3) evaluated and compared the sensitivity to GDC-0980 in USC harboring amplification of c-erbB2 and/or PIK3CA mutations vs USC control cell lines, and (4) analyzed the baseline levels and changes in phosphoprotein 4E-BP1 expression as a downstream cellular response to GDC-0980 exposure in vitro. We report the first evidence that oncogenic PIK3CA gene mutations and HER2/neu gene amplifications may be predictive of the sensitivity of USC cell lines to GDC-0980. Class I PIK3CA/mTOR kinase inhibitors such as GDC-0980 may represent promising novel drugs in patients harboring biologically aggressive USC with PIK3CA mutations and amplification of c-erbB2 .
Materials and Methods
Establishment of USC cell lines and evaluation for HER2/neu gene amplification
Twenty-two primary uterine papillary serous carcinoma cell lines were evaluated in our study. Specimens were obtained from fresh tumor biopsies collected at the time of surgery, under approval of the institutional review board. The cells were maintained as a monolayer in RPMI-1640 medium (Mediatech, Manassas, VA) supplemented with 10% fetal bovine serum (Gemini, Woodland, CA), 1% antibiotic (penicillin, streptomycin), and 0.3% antimycotic amphotericin B (Invitrogen, Carlsbad, CA). Cells were incubated at 37°C in a humidified atmosphere of 95% air/5% CO 2 . Source-patient characteristics of the USC cell lines are described in the Table . Tumor c-erbB2 gene amplification was evaluated by FISH as previously described by our group in all cell lines and results are presented in the Table .
| Sample ID | Age | Race | FIGO stage | PIK3CA mutations Exon 9,20 | C-erbB2 FISH |
|---|---|---|---|---|---|
| USPC-ARK1 a | 62 | B | IV | 542/1068 | Amplified |
| USPC-ARK2 a | 63 | B | IV | Not detected | Amplified |
| USPC-ARK3 a | 59 | B | IV | Not detected | Amplified |
| USPC-ARK4 a | 73 | W | IV | Not detected | Not amplified |
| USPC-ARK5 a | 73 | B | IIIC | Not detected | Not amplified |
| USPC-ARK6 a | 62 | W | IB | Not detected | Not amplified |
| USPC-ARK7 a | 75 | W | IIC | Not detected | Not amplified |
| USPC-ARK8 a | 88 | W | IIIA | Not detected | Not amplified |
| USPC-ARK9 | 73 | B | IIC | 1044/1068 | Amplified |
| USPC-ARK10 | 79 | W | IVB | 1044/1068 | Amplified |
| USPC-ARK11 a | 80 | B | IIIC | Not detected | Not amplified |
| USPC-ARK12 | 80 | B | IIIC | Not detected | Not amplified |
| USPC-ARK13 | 67 | W | IVB | Not detected | Not amplified |
| USPC-ARK14 a | 73 | B | IV | 546/1068 | Not amplified |
| USPC-ARK15 a | 67 | W | IIIC | Not detected | Not amplified |
| USPC-ARK16 | 65 | W | IV | Not detected | Not amplified |
| USPC-ARK17 | 59 | W | IIIA | Not detected | Amplified |
| USPC-ARK18 | 62 | B | IIB | Not detected | Amplified |
| USPC-ARK19 a | 65 | W | IA | Not detected | Not amplified |
| USPC-ARK20 a | 42 | W | II | 1047/1068 | Amplified |
| USPC-ARK21 a | 70 | W | IA | Not detected | Amplified |
| USPC-ARK22 a | 60 | W | IVB | Not detected | Not amplified |
a USC cell lines established as long-term cultures used in chemosensitivity experiments with GDC-0980.
Polymerase chain amplification
Primers for polymerase chain reaction (PCR) amplification and sequencing were designed using the Primer3 program ( http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi ) and were synthesized by the Yale Keck facility ( http://keck.med.yale.edu/ ), based on PIK3CA sequence obtained from National Center for Biotechnology Information (accession number N M_006218). Two primer pairs were used to individually amplify exon 9 and exon 20 of PIK3CA from genomic USC primary cell lines DNA ( Table ). PCR amplification was performed in 20 μL reaction volumes that contained 100 ng of DNA, 75 mmol/L Tris-HCl, 1.5 mmol/L MgCl 2 , 50 mmol/L KCl, 20 mmol/L (NH 4 ) 2 SO 4 , 0.2 μmol/L of each primer, 0.2 mmol/L of each deoxyribonucleotide triphosphate, and 1 U of Taq DNA polymerase (BIOTOOLS; B&M Laboratories, SA, Madrid, Spain). The 2 exons were amplified with the following PCR conditions: an initial 5-minute denaturation at 94°C followed by 35 cycles of 1 minute at 94°C, 1 minute at 57°C, 1 minute at 72°C, and a final extension of 10 minutes at 72°C.
DNA sequencing
PCR products were first purified using the MinElute PCR Purification Kit (Qiagen GmbH, Hilden, Germany) and were bidirectionally sequenced using the original primer pair and Applied Biosystem Cycle Sequencing kit (Applied Biosystem Inc, Santa Clara, CA) at the Yale Keck facility ( http://keck.med.yale.edu ). Samples were analyzed on the ABI Prism 3100 Avant instrument (Applied Biosystems), using standard run parameters. The separation matrix used was POP-6 using 1× Tris-borate-EDTA buffer with EDTA running buffer (Applied Biosystems).
Fluorescent in situ hybridization
FISH analysis was performed using the PathVysion HER2 DNA FISH Kit (Abbott Molecular Inc., Abbott Park, IL) according to the manufacturer’s instructions. Fluorescent signals in at least 30 nonoverlapping interphase nuclei with intact morphology were scored. Tumor cells were scored for the number of orange (HER2) and green (chromosome 17) signals. A case was scored as amplified when the ratio of the number of fluorescent signals of HER2 to chromosome 17 was ≥2.
Drug
GDC-0980 was provided by Genentech Inc (South San Francisco, CA). GDC-0980 was dissolved in dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO) as a 10-mM stock solution and was diluted in culture medium immediately before addition to cell lines.
Primary USC growth rate analysis
The doubling time at the log phase of growth for the c-erbB2 FISH + USC cell lines was determined by counting the number of live cells at different time points after plating. Briefly, cell lines were cultured in RPMI 1640 (Mediatech, Manassas, VA) supplemented with 10% fetal bovine serum (Gemini, Woodland, CA). When cultures had grown to approximately 80% confluence, cells were harvested from the flask using 0.25% Trypsin EDTA (Invitrogen), then counted on a hemocytometer chamber and assessed for viability via Trypan blue exclusion. Cell density was adjusted to a concentration of 10,000 cells/mL, then cells were seeded into a 6-well μL plate (Corning Life Sciences, Lowell, MA) at a density of 40,000 cells per well. Three replicate wells were plated per cell line per time point. Cell plates were incubated at 37°C with 5% CO 2 for 24, 48, 72, 96, and 120 hours, at which time they were removed from the incubator, harvested from the wells using 0.25% Trypsin EDTA, counted on a hemocytometer chamber and assessed for viability via Trypan blue exclusion.
Chemo-response assay
The effect of GDC-0980 on the viability and IC 50 of cells was determined using flow cytometry assays as previously described. Briefly, tumor cells derived from 15 primary USC cell lines established as long term cultures in vitro (ie, 5 cell lines harboring HER2/neu gene amplifications vs 10 HER2/neu-negative cell lines) were plated in 6-well tissue culture plates and when in exponential growth treated with GDC-0980 at concentrations of 0.01 0.25, 0.5, 0.75, 1.0, 2.0 μM. Concentrations up to 4.0 μM were also used against the more resistant USC cell lines. After 72 hours of additional incubation, well contents were harvested in their entirety, centrifuged then stained with propidium iodide (2 μL of a 500 μg/mL stock solution in PBS with 0.1% sodium azide and 2% fetal bovine serum) for flow cytometric counts. Viable cells were then quantified using flow cytometry as percent of viable cells (mean ± SEM) after exposure to different concentrations of GDC-0980 relative to vehicle-treated cells taken as 100% viable. A minimum of 3 independent experiments per USC cell line were performed.
Flow cytometry analysis of phosphorylated 4-EBP1 intracellular levels in primary USC cell lines
A previously validated flow cytometry-based assay was used to evaluate the baseline level as well as the change in phosphorylated 4-EBP1 expression as a downstream cellular response to GDC-0980 in USC cell lines. Briefly, USC cells after 72 hours exposure to 0.5 μM, 1.0 μM, and 2.0 μM of GDC-0980 were fixed in 4% formaldehyde and permeabilized with ice-cold 100% methanol. GDC-0980 treated and untreated control cells were incubated with primary rabbit monoclonal antibody against 4-EBP1 (Cell Signaling Technology, Inc., Danvers, MA) following the protocol provided by the manufacturers and stained with a fluorescein isothiocyanate-conjugated goat antirabbit F(ab’) immunoglobulin as a secondary reagent (Chemicon International, Temecula, CA). Cells (ie, 10,000 events per sample) were analyzed on FACSCalibur, using Cell Quest software (BD Biosciences, San Jose, CA).
Statistical analysis
Statistical analysis was conducted using GraphPad Prism5 (GraphPad Software, Inc., San Diego, CA). For each independent experiment of GDC-0980 on a given cell line, the measures of growth under different dose levels were normalized to the mean of the control group receiving no drug, so that all data were expressed as a proportion of the control. Normalized data then were fit via nonlinear regression to a 3-parameter logistic response curve against the base-10 logarithms of dose in micrometers, and the resulting parameter estimates were used to calculate the value of the IC 50 (in log 10 units) for that experiment. The collection of log 10 (IC 50 )s thereby determined were compared between each cell line. For the flow cytometry experiments, changes in the phosphorylated protein 4-EBP1 levels were analyzed comparing the mean intensity of fluorecence (MFI) before and after the exposure with GDC-0980. Unpaired t test was used to compare 4-EBP1 changes in cell lines exposed with GDC-0980. Differences in all comparisons were considered significant at P values < .05.
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