Persistent mTORC1 activation is sufficient to limit BYL719 sensitivity

We next investigated whether the mTORC1 activation status was altered in cells that acquired resistance to BYL719. We chose MDA-MB-453 (herein referred as MDA453) and T47D cell lines to generate these models of acquired resistance because they were among the most sensitive lines. Both cell lines were grown in increasing concentrations of BYL719 until their proliferation rate was undisturbed by constant inhibition of p110α with 1 μM BYL719 (~6 months, Fig. 3A). At this concentration of BYL719, Akt phosphorylation was inhibited in both parental and resistant cells, suggesting that resistance was not due to lack of target inhibition. Although in the sensitive parental cells pS6 was almost undetectable after treatment with BYL719, S6 phosphorylation was present in both of the derived resistant cell lines (Fig. 3B). Similar results were observed for phosphorylated 4EBP1 (p4EBP1) expression. These results prompted us to explore whether mTORC1 was reactivated in cells with acquired resistance to GDC-0941, a molecule that inhibits all four isoforms of class I PI3K (25). We obtained MCF7 cells with acquired resistance to GDC-0941 (MCF7R) with the same strategy as that for MDA453R and T47DR cells (Fig. 3C). GDC-0941 suppressed Akt phosphorylation in both MCF7 and MCF7R cells, whereas pS6 levels were not fully suppressed in the resistant cells (Fig. 3D). These results suggest that failure to suppress mTORC1 signaling indicates a common resistance mechanism for different PI3K inhibitors. Indeed, BYL719-resistant MDA453R and T47DR cells were less sensitive to GDC-0941 treatment than were parental control cells (fig. S4A). Likewise, GDC-0941–resistant MCF7R cells were more resistant to BYL719 than were the parental counterparts (fig. S4B). Western blot analysis confirmed that neither BYL719 nor GDC-0941 prevented S6 phosphorylation in resistant cells (fig. S4).

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Fig. 3

Resistance to PI3K inhibition induced by mTORC1 activation

(A) Generation of MDA453 and T47D cell lines with acquired resistance to BYL719. (Right) Proliferation of parental and resistant (MDA453R and T47DR) cells in the presence of 1 μM BYL719. (B) Immunoblotting analysis of phosphorylated proteins in parental, MDA453R, and T47DR cell lines after 24 hours of treatment with 1 μM BYL719. (C) Generation of MCF7 cell line with acquired resistance to GDC-0941. (Right) Proliferation of parental and resistant (MCF7R) cells in the presence of 1 μM GDC-0941. (D) Immunoblotting analysis of phosphorylated proteins in MCF7 and MCF7R after 24 hours of treatment with 1 μM GDC-0941. (E) Reduction in the number of reads by individual FRAP1 shRNAs transfected into drug-resistant cells after 7 days of treatment with either BYL719 or GDC-0941 [normalized to dimethyl sulfoxide (DMSO)–treated controls]. (F) Immunoblotting analysis of phosphorylated proteins in MCF7 and T47D cells after knockdown of TSC2 with shTSC2 and treatment with 1 μM BYL719 for 2 hours. (G) Proliferation assay of MCF7 and T47D cells after TSC2 knockdown with shTSC2 in the presence of 1 μM BYL719 for 4 days. Graph shows one representative experiment of two performed. Data are means ± SEM. P value was calculated using two-sided Student's t test.

We then performed a short hairpin RNA (shRNA) screen to identify genes (fig. S5 and table S1) that could potentially be knocked down to resensitize MDA453R and T47DR cells to BYL719 or MCF7R to GDC-0941 (fig. S5). We found that the proliferation of cells transfected with shRNAs for FRAP1 (mTOR) and phosphoinositide-dependent kinase-1 (PDPK1) was strongly inhibited by BYL719 or GDC-0941 compared to DMSO-treated cells (as indicated by the reduction of shRNA reads, Fig. 3E and fig. S6). Notably, RICTOR or RAPTOR knockdown also resensitized both T47DR and MDA453R cells to BYL719 and MCF7R cells to GDC-0941 (fig. S6). These findings were corroborated by infecting the highly sensitive MCF7 and T47D cells with lentivirus encoding shRNAs targeting tuberous sclerosis protein 2 (TSC2), a direct mTOR suppressor (26, 27). This suppression of TSC2 expression abrogated BYL719-induced down-regulation of pS6 in both MCF7 and T47D cells (Fig. 3F) and was sufficient to significantly (P < 0.01) decrease their sensitivity to p110α inhibition (Fig. 3G).

Together, these results indicate that sustained activation of mTORC1 plays a pivotal role in limiting the antitumor activity of BYL719 and possibly other PI3K/Akt inhibitors.

mTORC1 inhibition correlates with clinical response in treated patients

To assess the clinical relevance of our findings, we studied the correlation between the magnitude of inhibition of S6 phosphorylation and clinical response to BYL719 in patients treated at our institutions as part of an ongoing first-in-human phase 1 clinical trial of BYL719 in patients with solid tumors harboring PIK3CA mutations (21). We used an Institutional Review Board–approved protocol to collect paired tumor biopsies before commencing BYL719 therapy and also on the 28th day of the second cycle of treatment between 4 and 6 hours after the daily drug administration. Twelve paired biopsies (before and on treatment) from six patients with advanced breast cancer were suitable for IHC analysis of pS6(240/4). Table 2 displays the breast tumor subtype, the BYL719 dose, the type of PIK3CA mutation, the amount of pS6 by H-score, the best tumor response to therapy by Response Evaluation Criteria in Solid Tumors (RECIST) criteria (28), and the tumor site from which the biopsies were taken in each patient (Table 2). A strong suppression of S6 phosphorylation was observed in three of the tumors (patients 4 to 6) that responded to therapy (>20% shrinkage of tumor size compared to baseline) (Fig. 4, A and B). In contrast, pS6 expression was only weakly inhibited in three tumors (patients 1 to 3) that did not respond to therapy (Fig. 4, A and B).

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Fig. 4

Correlation between pS6(240/4) inhibition and clinical response to BYL719 in breast cancer patients

(A) Representative sections stained by IHC for pS6(240/4) in paired biopsies obtained from tumors of patients before (untreated) and at day 28 of BYL719 treatment. Patient numbers are indicated at left in parentheses. Images were captured at ×40 magnification; scale bar, 100 μm. (B) Differences in pS6(240/4) inhibition by BYL719 between the nonresponder (n = 3) and responder (n = 3) patients. pS6(240/4) was quantified in all the tumor cells present in the pretreatment biopsies and upon BYL719 treatment for each patient. (C) Levels of pS6(240/4) in tumor samples from patients 4 and 5 collected before initiation of BYL719 therapy (left bars), at 28 days after therapy initiation (middle bars), and at disease progression (right bars). Below the bars, the change in tumor volume (% of pretreatment size) by RECIST criteria is indicated at each time point.

Furthermore, we analyzed pS6(240/4) in tumor biopsies from two patients (patients 4 and 5) who initially responded to treatment but who later (after 5.6 and 13.9 months, respectively) showed tumor progression. Strikingly, pS6 in these regrowing lesions (also obtained 4 hours after the last drug administration) was present at levels comparable to those in the baseline pretreatment tumor samples (Fig. 4C). For the third responding patient (patient 6, who progressed on BYL719 after 3.6 months of treatment), pS6 could not be measured in the regrowing lesions because we were unable to perform a biopsy 4 hours after the last drug administration.

Together, these results from patients' biopsies are consistent with our observation that inhibition of mTORC1 activity in the tumor is necessary for PIK3CA mutant cancers to respond to BYL719. These results also corroborate our observations that pS6 is reactivated upon the development of acquired resistance.

mTORC1 inhibition enhances the efficacy of p110α inhibition and delays resistance

The effects of combined mTORC1 and p110α blockade were further examined in cells and tumors intrinsically resistant to BYL719. In proliferation/viability studies, the combination of BYL719 and RAD001 was more effective than either single agent in both JIMT-1 and HCC1954 cells (Fig. 5A and fig. S7). Similarly, cell cycle analysis showed that, unlike single-agent therapy, combined therapy induces both S-phase arrest and apoptosis (Fig. 5B and fig. S8A). As expected, the combination of BYL719 and RAD001 effectively inhibited both Akt and S6 phosphorylation (fig. S8B). Using 3D cell culture assays, we examined the effect of BYL719 and RAD001, either alone or in combination, in JIMT-1 and HCC1954 cell lines grown as spheroids. Whereas change in size and induction of cell death were minimal when BYL719 and RAD001 were used as single agents, concomitant p110α and mTORC1 suppression reduced spheroid size and induced apoptosis (the effect was more evident in HCC1954 cells) (Fig. 5C and fig. S9). We also tested the effect of targeting mTORC1 in our laboratory models of acquired resistance to PI3K inhibitors. RAD001 abolished pS6 expression in the MCF7 cell line with acquired resistance to BYL719 (MCF7R) and resensitized the cells to PI3K inhibition (fig. S10). We then tested the antitumor activity of this combination in BYL719-resistant PIK3CA-mut xenografts. We treated CAL51-derived xenografts with two different doses of BYL719, a fixed dose of RAD001, or the respective combinations. Although single agents had little or no effect in preventing tumor growth, the combinations of BYL719 and RAD001 had a pronounced antitumor effect (Fig. 5D). In a similar fashion, HCC1954-derived tumors were treated with a fixed dose of BYL719 (25 mg/kg), two different doses of RAD001, or the combinations. Consistently, single agents had minimal antitumor activity, whereas both combination arms showed markedly impaired tumor growth (Fig. 5E). The superiority of the BYL719/RAD001 combination in comparison to monotherapy was further confirmed in JIMT-1 xenografts (Fig. 5F).

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Fig. 5

Antitumor effects of combined mTORC1 (RAD001) and p110α (BYL719) inhibition

(A) Proliferation of the BYL719-resistant JIMT-1 and HCC1954 cell lines in the presence of BYL719 (0.5 μM), RAD001 (1 nM), or the combination for 72 hours. (B) Cell cycle analysis of JIMT-1 and HCC1954 cells with BYL719 (0.5 μM), RAD001 (1 nM), or the combination for 48 hours. (C) Spheroid size of JIMT-1 and HCC1954 cells treated with BYL719 (1 μM), RAD001 (10 nM), or the combination for 48 hours. In vitro experiments were in triplicates and repeated three times. (D) Tumor growth curves of BYL719-resistant CAL51-derived xenografts treated with RAD001 (0.5 mg/kg), BYL719 (50 or 25 mg/kg), or the described combinations (n = 10 for each treatment condition). (E) Tumor growth curves of BYL719-resistant HCC1954 cell–based xenografts treated with BYL719 (25 mg/kg), RAD001 (0.1 or 1 mg/kg), or the described combinations (n = 10 for each treatment condition). (F) Tumor growth curves of BYL719-resistant JIMT-1 cell–based xenografts treated with RAD001 (0.5 mg/kg), BYL719 (25 mg/kg), or the described combination (n = 10 for each treatment condition). (G) Tumor growth curve of BYL719-sensitive MCF7-derived xenografts treated as indicated with BYL719 (25 mg/kg), RAD001 (0.5 mg/kg), or the combination of both. RAD001 was added to the BYL719-treated cohort at day 46 (n = 10 for each treatment condition). (H) An average of 6 to 15 images of two to four independent tumors was used to quantify IHC for pAkt and pS6 on days 0, 3, and 46 of BYL719 treatment. Quantification of IHC was performed with CellProfiler and is shown in bar graphs to the right of each panel. Images were captured at ×40 magnification; scale bar, 100 μm. Data are means ± SEM. P value was calculated using two-sided Student's t test.

Next, we determined whether combined p110α and mTORC1 blockade would also be effective in tumors treated with BYL719 that initially responded to therapy but eventually progressed. MCF7-derived xenografts, sensitive to BYL719 (Fig. 2A), were treated with either BYL719 (25 mg/kg) or RAD001 (0.5 mg/kg). Both single-agent treatments delayed tumor growth compared to tumors in control vehicle– treated mice. The effect of RAD001 used as a single agent ceased after ~20 days of treatment, whereas BYL719 suppressed tumor growth for ~40 days. However, after 46 days, tumors started to grow in these animals despite BYL719 treatment, at a rate similar to that in vehicle-treated xenografts. At that time, RAD001 was added to the BYL719 given to this group of animals, and the treatment rapidly halted the tumor progression (Fig. 5G). This effect lasted until the end of the experiment (an additional 100 days). The cohort of mice treated with the combination of the two drugs from day 0 showed immediate and consistent reduction of tumor volume, and only 4 of 10 tumors were measurable after 120 days (Fig. 5G).

To further understand the link between mTORC1 activation and tumor escape from p110α inhibition, we measured S6 phosphorylation in MCF7 xenografts treated with BYL719 over time (Fig. 5H). At day 3 after beginning treatment, both pAkt and pS6 were suppressed. However, after 46 days of treatment, pS6(240/244) was again high despite continued inhibition of pAkt. Thus, these findings are consistent with data from our cell line models of acquired resistance and the patient biopsies of acquired resistance that showed restoration of pS6 in the resistant cancers. These results further suggest that the combination of BYL719 and RAD001 can overcome both intrinsic and acquired resistance to BYL719.

Cell lines and chemical compounds

OCUB-F cell line was obtained from Riken Gene Bank. SUM185PE and SUM190PT were provided by S. P. Ethier's laboratory. All other cell lines were purchased from commercial vendors. 293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum (FCS). MDA-MB-361, MDA-MB-453, UACC893, KPL-1, MCF7, BT474, JIMT-1, BT20, CAL148, MFM223, UACC812, ZR-75-1, CAL51, MDA-MB-468, and MDA-MB-231 tumor cells were maintained in DMEM/F12 (1:1) with 10% FCS. T47D, CAMA-1, BT483, HCC1428, EFM19, HCC202, and HCC1954 tumor cells were grown in RPMI with 10% FCS. SUM185PE and SUM190PT were cultured in F12 with 5% FCS and Medium 199 without FCS, respectively. SUM185PE and SUM190PT were also supplemented with h-Insulin (5 μg/ml) (Sigma, I9278) and hydrocortisone (1 μg/ml) (Sigma, H-4001).

All cultured media contained 2 mM l-glutamine, penicillin (20 U/ml), and streptomycin (20 μg/ml). All cells were maintained at 37°C in a humidified atmosphere at 5% CO₂.

MCF7R cells were obtained after chronic exposure to increasing concentrations of GDC-0941 for ~6 months. MDA453R and T47DR were obtained after chronic exposure to increasing concentrations of BYL719 for ~6 months. The p110α inhibitor BYL719 was provided by Novartis. The mTORC1 inhibitor RAD001 and the pan-p110 inhibitor GDC-0941 were provided by SU2C/PI3K Dream Team Mouse Pharmacy that obtains compounds from Shanghai Haoyuan Chemexpress Co. All compounds were dissolved in DMSO for in vitro experiments.

Determination of IC₅₀

To calculate IC₅₀, 5 × 10⁵ cells were seeded in 96-well plates and treated with escalating concentration of BYL719 (from 20 nM to 12.5 μM). After 3 days of treatment, cell proliferation was analyzed with CellTiter-Glo Luminescent Cell Viability Assay (Promega) as described by the manufacturer. IC₅₀ values were calculated with GraphPad Prism (GraphPad Software).

Determination of cell cycle, apoptosis, and proliferation

Cell cycle was quantified by flow cytometry as described (45). Briefly, cells were washed with phosphate-buffered saline (PBS), fixed in cold 70% ethanol, and then stained with propidium iodide (PI) while being treated with ribonuclease (Sigma-Aldrich).

Cell death was measured by annexin V–FITC (fluorescein isothiocyanate)/PI staining and evaluated by flow cytometry according to the manufacturer's protocol (BD Pharmingen). Cells (2 × 10⁵) were washed twice with PBS and stained with 5 μl of annexin V–FITC and 10 μl of PI (5 μg/ml) in 1× binding buffer (BD) for 15 min at room temperature in the dark. The apoptotic cells were detected with an LSR flow cytometer. Both apoptotic (annexin V–positive, PI-negative) and necrotic (annexin V–positive and PI-positive) cells were included in cell death determinations. Quantitative analyses were carried out by FlowJo software.

For proliferation analysis, 5 × 10⁵ cells were seeded in 24-well plates and treated with the appropriate drug(s) for 3 to 11 days. Cells were fixed and stained with crystal violet or Cyro60.

For IGF1/NRG1-induced proliferation experiments, 5 × 10⁵ cells were seeded in 24-well plates, treated with BYL719 (1 μM) and/or RAD001 (1 nM) in the presence of IGF1 (50 ng/ml) or NRG1 (0.8 ng/ml) for 6 days, and then stained with crystal violet.

Western blotting

Cells were washed with ice-cold PBS and scraped into ice-cold radio-immunoprecipitation assay lysis buffer (Cell Signaling Technology) supplemented with phosphatase inhibitor cocktails (Complete Mini and PhosphoStop, Roche). Lysates were cleared by centrifugation at 13,000 rpm for 10 min at 4°C, and supernatants were removed and assayed for protein concentration with the Pierce BCA Protein Assay Kit (Thermo Scientific). Thirty-five micrograms of total lysate was resolved on NuPAGE 4 to 12% bis-tris gels (Life Technologies) and electrophoretically transferred to Immobilon transfer membranes (Millipore). Membranes were blocked for 1 hour in 5% nonfat dry milk in tris-buffered saline (TBS)–Tween and then hybridized with the primary antibodies in 5% bovine serum albumin (BSA)/TBS-Tween. List of primary antibodies and their working concentrations can be found in table S2. β-Actin was used as a loading control (1:5000, Sigma), also in 5% BSA/TBS-Tween. Mouse and rabbit horseradish peroxidase–conjugated secondary antibodies (1:50,000, Amersham Biosciences) were diluted in 2% nonfat dry milk in TBS-Tween. Protein-antibody complexes were detected by chemiluminescence with SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific), and images were captured with a G-BOX camera system.

Establishment of tumor xenografts and studies in nude mice

Six-week-old female athymic NU/NU nude mice purchased from Charles River were injected with 2 × 10⁷ MCF7, 5 × 10⁶ JIMT-1, and 1 × 10⁷ CAL51 cells subcutaneously in a volume of 100 μl of culture medium/Matrigel (1:1) (BD Biosciences). For MCF7 xenografts, 1 μM 17β-estradiol was added to the drinking water as described (45).

For cell line–derived xenograft studies, animals were randomized at a tumor volume of 70 to 120 mm³, about 2 to 4 weeks after injection. Animals were randomized to four to six groups, with n = 8 to 10 tumors per group. Animals were treated daily with BYL719 [25 or 50 mg/kg in 0.5% carboxymethylcellulose sodium salt (CMC) (Sigma) in water] and/or RAD001 (0.5 or 0.1 mg/kg, freshly dissolved in water, and mixed in CMC or BYL719 in CMC solution). All agents were delivered by oral gavage. Tumor xenografts were measured with digital calipers, and tumor volumes were determined with the following formula: (length × width²) × (π/6). At the end of the experiment, animals were euthanized with CO₂ inhalation. Tumor volumes are plotted as means ± SEM.

Mice were maintained and treated in accordance with Institutional Guidelines of Massachusetts General Hospital. Mice were housed in air-filtered laminar flow cabinets with a 12-hour light cycle and food and water ad libitum.

Patients

Tumor response was assessed according to the RECIST criteria. All potential sites of tumor lesions were assessed at screening/baseline with thoracic, abdominal, and pelvic computerized tomography or magnetic resonance imaging, complemented with brain scan in case of clinical evidence of brain metastatic disease. In addition, a bone scan was performed for all patients with clinical evidence of bone metastases. Subsequent scans were done at the end of cycle 2, and every 8 weeks thereafter (that is, at the end of cycles 4, 6, 8, etc.). A complete treatment cycle is defined as 4 weeks of daily continuous treatment with oral BYL719. All measurable lesions up to a maximum of 5 lesions per organ and 10 lesions in total, representative of all involved organs, were identified as target lesions and recorded and measured at baseline, and then followed throughout the study. A sum of the longest diameter for all target lesions at baseline was used as a reference by which the objective tumor response was characterized. Pretreatment biopsies were obtained within 2 weeks of starting the study agent. On-treatment tumor biopsies were collected at the end of cycle 2 (cycle 2 day 28), and progression tumor biopsy was performed at the time of disease progression. Both on-treatment and progression biopsies were collected 4 to 6 hours after dose.

Immunohistochemistry

Secreted protein screen

Secreted protein library complementary DNAs were reverse-transfected into HEK293T cells with FuGENE HD (4:1, transfection reagent to DNA ratio) and incubated for 4 days to allow accumulation of secreted proteins in supernatant. The supernatant was then transferred to MCF7 and T47D cells, followed by addition of BYL719 to a final concentration of 1 μM. After 96 hours, growth was measured with CellTiter-Glo.

Statistical analysis

Two-way t test was done with GraphPad Prism (GraphPad Software). Error bars represent the SEM. All the in vitro experiments were repeated at least three times. All the in vivo experiments were run in duplicate with at least n = 6 for each treatment arm.

Quantitative in-cell western

Cells were plated in 96-well black flat-bottom plates (BD Falcon) at a density of 3 × 10⁵ cells per well. After treatment, the cells were immediately fixed with 3.7% paraformaldehyde in PBS for 20 min and then washed three times for 10 min each with 0.1% Triton X-100 in PBS with gentle shaking. Cells were blocked with blocking buffer (LI-COR) for 1 hour with gentle shaking. Cells were then hybridized with the following primary antibodies diluted in blocking buffer, with gentle shaking at 4°C overnight: pS6 (Ser^240/4) (1:2000) and pS6 (Ser^235/6) (1:100), all from Cell Signaling Technology. After incubation with primary antibody, cells were washed three times for 10 min each with PBS-Tween and incubated in goat anti-rabbit secondary IRDye (LI-COR, 1:1200), Sapphire 700 (LI-COR, 1:1000), and DRAQ5 (BioStatus, 1:2000) in blocking buffer for 1 hour with gentle shaking. Cells were washed again three times for 10 min each with PBS-Tween, and plates were dried and read with a LI-COR Odyssey Infrared Imager.

shRNA screen

Novartis Pharma generated the shRNA library. MCF7R, T47DR, and MDA453R cells were infected with shRNAs from the library, incubated for several days, selected with puromycin selection, and divided into two experiment arms (treatment and DMSO control, fig. S5). Because the total number of cells sometimes differed between the two arms, the number of shRNA reads for each arm was normalized to 5 × 10⁶ and log-transformed. Quantile normalization was applied across all the screens to allow for comparison of results between cell lines. A fold change was determined for each shRNA in each screen by subtracting the log₂ count in the control arm from the treatment arm.

A confidence value was calculated for each shRNA on the basis of percent knockdown of the gene target relative to other genes. For each gene K, we let {k₁, k₂, …, k_m} be the set of percent knockdowns for the shRNAs targeting K and let k_max denote the maximum percent knockdown in this set. The weights for the shRNAs against gene K were then given by $\{w_1,w_2,\dots w_m\}=\{\frac{k_1}{k_{max}},\frac{k_2}{k_{max}},\dots ,\frac{k_m}{k_{max}}\}$. Thus, the weight of an shRNA corresponded to its percent knockdown of the target gene, normalized to the maximum percent knockdown for that target gene by any of its shRNAs.

For each gene, a score was calculated on the basis of the following formula:

where A = {a₁, a₂, …, a_m} are the indices of shRNAs targeting the gene and B = {b₁, b₂,…,b_n} are the indices of all the shRNAs targeting other genes in the pool. FC_x and W_x denote the fold change (compound versus DMSO) and confidence weight of the xth shRNA, as described above.

Thus, the score for a gene is the difference in the (weighted) log fold change of shRNAs targeting that gene versus all other genes. It therefore measures the degree to which targeting a specific gene differentially sensitizes or rescues the cell line from the effects of treatment compared to the other genes.

A permutation test was used to estimate the significance of the gene-level scores. The mapping of shRNAs to their gene targets was randomly permuted 10,000 times, and in each permutation, a score was calculated for each gene as described above. For any particular gene, the distribution of its 10,000 permuted scores approximated a normal distribution. Consequently, a P value could be determined from a two-sided comparison of the actual gene score against the fitted normal distribution derived from the permuted gene scores. The P values of all genes were also corrected for multiple hypotheses testing with the false discovery rate (FDR) method of Benjamini-Hochberg. Genes with an FDR <0.25 were considered to be significantly sensitizing or rescuing the cell line from the effects of drug treatment when compared to the average gene screened in the shRNA pool.

shRNA knockdown

shControl and shTSC [previously described in (48)] were purchased from Open Biosystems. 293T cells were seeded into 10-cm plates at a density of 0.5 × 10⁵ cells per plate. Twenty-four hours later, cells were transfected with shRNA against TSC2 or Silencer negative control shRNA against green fluorescent protein with FuGENE (Roche) according to the manufacturer's instructions. Lentiviral shRNA was collected after 48 hours. Tumor cells were infected with lentiviral shRNA for 24 hours, and then cell lines were grown in medium supplemented with 10% fetal bovine serum in the presence of puromycin (2 μg/ml) for 5 to 8 days.

3D acinar morphogenesis assay and scoring of 3D structures

Cells were grown in DMEM/F12 or RPMI medium supplemented with 2% inactivated calf serum, which was replaced every 4 days for 2 to 3 weeks before treatment. Spheres were treated with DMSO, 1 μM BYL719, 10 nM RAD001, or combination for 48 hours and fixed. Phase-contrast microscopy was used to image 3D structures (10×) as described (49). The diameter of >50 spheroids in each condition was analyzed and measured by ImageJ.

IF and confocal microscopy

3D structures were fixed and stained as described (49). Secondary antibodies were as follows: Alexa Fluor 488 anti-rabbit immunoglobulin G (IgG) (1:200, Invitrogen) and Alexa Fluor 594 anti-mouse IgG (1:200, Invitrogen). For confocal imaging of 3D structures, we used Multispectral Multimode Laser Scanning Confocal Microscope: Nikon TE-200U inverted microscope with Nikon C1 point scanning confocal with spectral detection, wide range of Nikon 40×, 1.4 numerical aperture (NA) magnification/NA differential interference contrast (DIC) and phase optics, Nikon halogen trans-illuminator with 0.52 NA long working distance (LWD) and 0.85 NA Dry condenser, 405-, 488-, and 561-nm laser lines, Nikon EZ-C1 software, TMC vibration-isolation table. 3D images are shown as single sections from midstructure.

We thank M.-A. Bray for helping generating CellProfiler pipelines for IHC analysis, B. Jillian for the IHC staining, C. Benes for scientific discussion and providing cell lines, and E. Edelman and O. Litvin for statistical support.

Funding: This work was funded by a Stand Up To Cancer Dream Team Translational Cancer Research Grant, a program of the Entertainment Industry Foundation (SU2C-AACR-DT0209 to J.B.), the Breast Cancer Research Foundation (to J.B.), the European Research Council (AdG09250244 to J.B.), the Instituto de Salud Carlos III (Intrasalud PSO9/00623 to J.B.), and RO1CA137008 (to J.A.E.). M.E. is an International Sephardic Education Foundation postdoctoral fellow. A.B.C. holds a Translational Research Fellowship from the Spanish Society of Medical Oncology (SEOM). S.V. is supported by an ASCO YIA, American Association for Cancer Research–Genentech BioOncology Fellowship, and Terri Brodeur Breast Cancer Foundation research grant.