The anti-tumor efficacy of CDK4/6 inhibition is enhanced by the combination with PI3K/AKT/mTOR inhibitors through impairment of glucose metabolism in TNBC cells

Human BC cell lines MDA-MB-231, MDA-MB-468, HCC-38 (all triple negative) and MCF-7 (ERα-positive) were cultured in RPMI supplemented with 2 mM glutamine, 10% fetal bovine serum (FBS), and 100 U/ml penicillin/100 μg/ml streptomycin.

Cells were purchased from the American Type Culture Collection (Manassas, VA), which authenticates the phenotypes of these cell lines on a regular basis (http://​www.​lgcstandards-atcc.​org). Hypoxic conditions were established by placing the cells in a tissue culture incubator with controlled O₂ levels (Binder GmbH, Tuttlingen, Germany).

Cell proliferation was evaluated by counting the cells in a Bürker hemocytometer with trypan blue exclusion method and by Crystal Violet (CV) staining as previously described [15]. The nature of the interaction between palbociclib and PI3K inhibitors was calculated using the Bliss additivism model [16]. A theoretical dose-response curve was calculated for combined inhibition using the equation of Bliss = EA + EB-EA*EB, where EA and EB are the percent of inhibition versus control obtained by BYL719, BEZ235 or BKM120 (A) and palbociclib (B) alone and the E Bliss is the percent of inhibition that would be expected if the combination was exactly additive. If the combination effect is higher than the expected Bliss equation value, the interaction is synergistic, while if the effect is lower, the interaction is antagonistic. Otherwise, the effect is additive and there is no interaction between the drugs. Cell death was analyzed by fluorescence microscopy after staining with Hoechst 3342 and Propidium Iodide (PI) [17]. Distribution of the cells in the cell cycle was determined as previously described [18]. Briefly, 5 × 10⁵ cells were incubated overnight at 4 °C in 1 ml of hypotonic fluorochrome solution. Analysis was performed with Coulter EPICS XL-MCL cytometer (Coulter Co., Miami, FL, USA). Cell-cycle-phase distributions were analyzed by MultiCycle DNA Content and FCS Express Software (De Novo Software, Glendale, CA 91203).

Analyses of western blotting were performed as previously described [19].

Antibodies against p-Rb^Ser780 (#9307), Rb (#9309), cyclin D1 (#2926), CDK6 (#3136), c-myc (#9402), p-AKT^Ser473 (#9271), AKT (#9272), p-mTOR^Ser2448 (#2971), mTOR (#2972), p-ERK1/2^{Thr202/Tyr204} (#4370), ERK1/2 (#4695), were from CST (Danvers, MA); anti-p-CDK6^Tyr24 (sc-293,097) was from Santa Cruz Biotechnology, Incorporated (Dallas, TX). Anti CDKN2A/p16^INK4a (ab81278) and anti-GLUT-1 (ab40084) were from Abcam (Cambridge, UK). Antibody against HIF-1α (#610959) was from BD Biosciences (Franklin Lakes, NJ). Anti-β-actin (#3598) was from BioVision (Milpitas, CA). All the antibodies were used at the recommended dilution of 1:1000. Horseradish peroxidase-conjugated secondary antibodies (1:10,000) and chemiluminescence system were from Millipore (Millipore, MA). Reagents for electrophoresis and blotting analysis were from BIO-RAD Laboratories (Hercules, CA).

Total RNA was isolated by RNeasy Mini Kit (Qiagen, Venlo, Netherlands) and the quantitative real-time polymerase chain reaction (PCR) was performed using the StepOne system instrument (Applied Biosystems) as previously described [20]. Briefly, samples were amplified using the following thermal profile: 95 °C for 20 s and 40 cycles of denaturation at 95 °C for 3 s followed by annealing and extension at 60 °C for 30 s. The primer to specifically amplify GLUT-1 (QT00068957) was obtained from Qiagen. HPRT1 (QT00059066) and PGK1 (QT00013776) were used as housekeeping genes and were purchased from Qiagen. The fold change was calculated by the ΔΔCT method.

Glucose uptake was measured as described previously [21]. Briefly, cells were rinsed with Kreb’s Ringer HEPES buffer (KRH) and incubated in KRH containing 1 μCi/ml Deoxy-D-glucose-2-[1,2-3H(N)] (2DG, PerkinElmer, Waltham, MA) at 37 °C for 5 min. Then, the cells were quickly rinsed three times in fresh cold Earle’s solution containing 0.1 mM phloretin (Sigma-Aldrich). Ice-cold trichloroacetic acid (TCA, 5%) was added and radioactivity in the acid extracts was measured by liquid-scintillation. Cell proteins, precipitated by TCA, were dissolved in 0.5 N NaOH and their concentration determined by a dye-fixation method (Bio-Rad, Hercules,CA). Glucose uptake was calculated as pmol of 2DG/mg protein/5 min and expressed as percent vs control condition. Glucose levels in the media were determined using a Glucose (HK) Assay Kit (product code GAHK-20) (Sigma-Aldrich, St. Louis, MI), according to the manufacturer’s instruction. Glucose consumption was calculated subtracting the glucose amount in the spent media to glucose in cell-free media. Data were calculated as mg glucose/mg protein and expressed as percent vs control.

Statistical analyses were carried out using GraphPad Prism 6.00 software. Statistical significance of differences among data was estimated by two-tailed Student’s t test. Comparison among groups was made using analysis of variance (one-way ANOVA, repeated measures) followed by Tukey’s post-test.

We firstly evaluated the effect of palbociclib on cell proliferation in a panel of TNBC cell lines (MDA-MB-231, MDA-MB-468, HCC38) in comparison with a ER+ luminal-A BC cell line (MCF-7), that reflects the cancer histotype for which the drug is currently clinically used. Cell sensitivity to palbociclib can be predicted by the presence of detectable levels of p-Rb and cyclin D1, and by a contemporary reduced expression of the cell cycle inhibitor p16^INK4 [22]. As shown in Fig. 1a, MDA-MB-231, HCC38, and MCF-7 cell lines expressed both p-Rb as well as cyclin D1, whereas p16^INK4 was not detectable; accordingly, palbociclib inhibited cell proliferation in these cell models with EC₅₀ values ranging from 0.3 to 1.4 μM (Fig. 1b). In contrast, MDA-MB-468 cells, characterized by the loss of Rb and low expression of cyclin D1, while showing p16^INK4 expression, were less sensitive to palbociclib (Fig. 1a,b). In addition, palbociclib elicited a robust cell cycle blockade in G0/G1 phase only on sensitive cell models (80 and 70% of cells in G0/G1 phase for MDA-MB-231 and HCC38, respectively), whereas MDA-MB-468 cells did not show any variation in the distribution of cells within cell cycle phases (Fig. 1c).

Then, to investigate the reversibility of palbociclib on cell cycle, we treated MDA-MB-231 and HCC38 cells with palbociclib for 24 h, after which the compound was removed and cell distribution within cell cycle phases was analyzed at increasing time intervals. After drug removal, the initial blockade in G0/G1 phase was progressively lost and after 24 h the normal distribution of cells in each cell cycle phase was restored in both cell lines, thus confirming that the growth-inhibitory effect of palbociclib is reversible (Fig. 1d). We then evaluated the effect of palbociclib on the expression of cell cycle-related proteins and observed that the treatment induced a decrease of p-Rb, Rb and p-CDK6 levels and a concomitant increase of cyclin D1 levels in a dose- and time-dependent manner (Fig. 2a,b, respectively). Hypophosphorylation of Rb protein, due to CDK4/6 inhibition, is known to result in its activation; active Rb maintains E2F in a repressed state, with consequent inhibition of downstream gene transcription [8]. Indeed, palbociclib treatment of MDA-MB-231 and HCC38 cells down-regulated the expression of myc, which contains E2F-binding sites in its regulatory region (Fig. 2a,b).

Since it has been recently reported that palbociclib treatment increases the activation of AKT/mTOR signaling [13, 23], we evaluated the activation status of this pathway in MDA-MB-231 and HCC38 cells after dose- and time-dependent exposure to palbociclib (Fig. 3a,b). Palbociclib induced a dose-dependent up-regulation of the phosphorylation levels of mTOR and AKT proteins. On the contrary, no regulatory effects were observed for ERK-1/2 pathway. The activation of both AKT and mTOR increased also over time up to 24 h.

These findings prompted us to investigate the efficacy of combining palbociclib with three PI3K/mTOR inhibitors: BYL719, an inhibitor of the p110α catalytic subunit of PI3K, BEZ235, a dual PI3K and mTORC1–2 inhibitor, and BKM120, a pan-class I PI3K inhibitor. The exposure of TNBC cell lines to these compounds resulted in the inhibition of cell proliferation as shown in Additional file 1: Table S1. We initially evaluated the simultaneous combination of increasing concentrations of the PI3K/mTOR inhibitors with a fixed concentration of palbociclib (0.5 μM) on MDA-MB-231 (Fig. 4a-c) and HCC-38 cells (Fig. 4d-f). Such combination gave rise to an additive effect according to Bliss experimental model. Since AKT activation increased up to 24 h of treatment with palbociclib, we then evaluated the efficacy of a combinatory schedule involving a pre-incubation with palbociclib for 24 h followed by a simultaneous treatment with the PI3K inhibitors plus palbociclib for further 48 h (sequential combined treatment). As shown in Fig. 5a-f, this sequential combined approach resulted in a synergistic inhibition of cell proliferation in both MDA-MB-231 and HCC-38 cells. Of note, when palbociclib was removed during treatment with PI3K inhibitors the synergism was lost, resulting in an additive inhibition of cell proliferation (Additional file 2: Figure S1). The sequential combined treatment was associated with a greater increase of the percentage of cells accumulating in the G0/G1 cell cycle phase (Fig. 5g), with values of ~ 90% for all the PI3K inhibitors used. In addition, the sequential combined treatment induced a stronger down-regulation of the PI3K/AKT/mTOR pathway, as shown by the inactivation of mTOR (Fig. 5h), together with a complete dephosphoryation of p-Rb, in comparison with the single drug treatments. Moreover, in these conditions myc expression was strongly inhibited due to Rb inactivation. Directly comparing the growth-inhibitory effects induced by the three different schedules of treatment, we confirmed the superior efficacy of the sequential combined treatment over the others (23% of cell proliferation vs 43–44% for the combined and the sequential treatments); in addition, palbociclib alone did not induce cell death, as expected, mainly exerting a cytostatic effect; however, its combination with BEZ235 potentiate the pro-apoptotic effects associated with PI3K/AKT/mTOR inhibition, with the sequential combined treatment showing the stronger cell death induction (31% of cell death vs 15% and 21% for the combined and the sequential treatment, respectively) (Fig. 6a,b).

Taken together these results indicate that palbociclib may be used to potentiate the anti-tumor activity of PI3K inhibitors, and suggest the relevance of maintaining palbociclib during treatment with these inhibitors to provide the greatest benefit for TNBC.

A variety of evidence indicates that cell cycle-related proteins are directly involved in the regulation of cell energy metabolism [24]. Therefore, we evaluated the effect of palbociclib, alone or combined with BYL719, on cell glucose metabolism under both normoxic and hypoxic conditions (Fig. 7a-d). Palbociclib down-regulated glucose uptake and consumption, as well as the expression of GLUT-1 glucose transporter in MDA-MB-231 cells, and combination with BYL719 enhanced these inhibitory effects under normoxic conditions. In particular, palbociclib induced ~ 15% decrease in glucose uptake and consumption, the same produced by the treatment with BYL719 alone; a higher decrease (25%) was promoted by the drug combination. Exposure to hypoxia for 24 h induced the stabilization and accumulation of HIF-1α and up-regulated the expression of GLUT-1, resulting in a significant increase of glucose utilization. Again, the combination of palbociclib with BYL719 hindered hypoxia-mediated stimulation of glucose uptake more efficaciously than individual treatments (40% decrease vs 25% after palbociclib or BYL719 single treatment). Similar effects were observed for glucose consumption. The greater efficacy of such combination was presumably ascribed to the inhibition of both PI3K/mTOR signaling and c-myc expression (see Fig. 5h), whose involvement in the modulation of glucose metabolism is well-recognized [25]. Interestingly, the sequential combined treatment with palbociclib maintained during the incubation with BYL719 promoted a ~ 50% decrease of glucose uptake as compared to control (Fig. 7e), which was associated with a significant inhibition of GLUT-1 expression (Fig. 7f).

Altogether these results suggest that inhibition of PI3K/mTOR signaling may improve the efficacy of palbociclib also through a negative modulation of glucose metabolism.