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
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KeywordsPalbociclib Triple-negative breast cancer CDK4/6 inhibition PI3K/mTOR inhibitors Glucose metabolism
Triple-negative Breast Cancer
Cell cycle regulators have gain attention as potential targets for anticancer therapy. Palbociclib is a selective inhibitor of the cyclin-dependent kinases 4 and 6 (CDK4/6), which coordinate the G1-S transition. Palbociclib is currently approved for the treatment of hormone receptor positive, HER2-negative advanced breast cancer (BC) in association with letrozole or fulvestrant. In contrast, its efficacy in triple negative BC (TNBC), either alone or in combined therapies, has not been fully investigated to date.
Here we evaluated the potential of combining palbociclib with PI3K/mTOR inhibitors in Rb-proficient TNBC cells comparing different schedules of treatment: simultaneous, sequential, or sequential combined treatment (pre-incubation with palbociclib followed by exposure to both palbociclib and PI3K/mTOR inhibitors). We assessed the effects on cell proliferation, cell death, and cell cycle distribution, and looked at the impact of such treatments on glucose metabolism.
Palbociclib exerted cytostatic effects in Rb-positive TNBC cells, inducing a reversible blockade in G0/G1 cell cycle phase associated with down-regulation of CDK6, Rb, and c-myc expression and/or activity. Palbociclib treatment induced AKT signaling, providing a rationale for its combination with PI3K/mTOR inhibitors. The simultaneous or sequential treatment resulted in an additive inhibition of cell proliferation. On the other hand, the sequential combined treatment in which palbociclib was maintained also during exposure to PI3K/mTOR inhibitors gave rise to synergistic anti-proliferative and pro-apoptotic effects, by inhibiting both CDK4/6/Rb/myc and PI3K/mTOR signaling. Interestingly, the inhibition of the Rb/E2F/myc axis mediated by palbociclib resulted in a significant down-regulation of glucose metabolism; most importantly, these inhibitory effects were enhanced by the combination of palbociclib with BYL719 (specific inhibitor of the p110α PI3K-subunit), which promoted a stronger inhibition of GLUT-1 glucose transporter expression, glucose uptake and consumption in comparison with individual treatments, under both normoxic and hypoxic conditions.
Combination of palbociclib with PI3K/mTOR inhibitors may represent a promising therapeutic option for the treatment of Rb-proficient TNBC, with the sequential combined schedule showing a superior efficacy over the other schedules. In addition our results demonstrate that the impairment of glucose metabolism may contribute to the anti-tumor activity of such drug combinations.
In spite of the multitude of pharmacologic approaches which have become clinically available during the last decades and novel screening improvements, breast cancer (BC) remains the second leading cause of cancer-related death among women . BC subtypes are based on the expression of hormone receptors, i.e. estrogen receptor (ER) and/or progesterone receptor (PR) (∼75% of cases), and overexpression/amplification of the human epidermal growth factor receptor 2 (HER2) (∼20% of cases, half of which are also positive for hormone receptors). Tumors lacking the expression of such receptors are commonly referred to as Triple-negative BCs (TNBCs) (∼5%–10%) . In addition, the development of gene expression profiling using high-throughput analysis has provided a molecular classification of BC into luminal A, luminal B, HER2-enriched, basal-like, claudin-low, and normal-like subtypes . TNBCs are mostly basal-like and are associated with high aggressiveness and poor prognosis. Due to the lack of druggable targets, treatment of TNBC is based on chemotherapy and the identification of new targets is a high clinical priority.
p16INK4 is a cyclin-dependent kinase inhibitor (CDKI), that blocks the binding site of cyclin D1 on CDK4/6. Loss of functional p16INK4 gives rise to deregulated CDK4/6 activity, leading to persistent retinoblastoma protein (Rb) phosphorylation and increasing cell proliferation . The loss of p16INK4 has been reported to occur with higher frequency in TNBC in comparison with other BC histotypes and has been correlated with the poor prognosis of TNBC . In addition, the lack of p16INK4 expression has been associated with the acquisition of cancer stem cell-like properties and with a reduced response of TNBC to paclitaxel treatment . Also the inactivation of Rb, due to both mutation or homozygous loss of the gene, may be observed in all BC subtypes, with a higher frequency in TNBC (7–20%) [7, 8].
Palbociclib, an orally-available inhibitor of CDK4 and CDK6, represents the most widely studied compound among cell cycle inhibitors. Palbociclib is a cytostatic drug, which efficiently blocks cell cycle progression from G1 to S phase by preventing the CDK4/6-cyclin D1-mediated phosphorylation of Rb and the subsequent release of the transcription factor E2F .
Palbociclib granted accelerated approval in 2015 for the treatment of ER-positive, HER2-negative advanced BC in association with letrozole , and in combination with fulvestrant in patients with ER-positive/HER2-negative advanced BC with disease progression following endocrine therapy . Intriguingly, some early preclinical evidences have been documenting a possible efficacy of palbociclib in other BC molecular subvariants, including TNBC cell lines . Based on the aforementioned considerations, TNBC with a Rb-positive, p16INK4-negative profile might represent the subpopulation of TNBC suitable for treatment with palbociclib.
In addition to a direct effect of palbociclib on sensitive malignant cells, the mechanism of action of the drug also suggests a possible role for combinatory schedules of treatment. In particular, the recently described mechanism of palbocicilib-mediated activation of Protein Kinase B (AKT) signaling  provides a strong rationale for the combination with inhibitors of the phosphoinositide 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway. This pathway plays a critical role in the control of cell growth, proliferation, migration, and metabolism, and, being frequently deregulated in BC cells, PI3K/mTOR inhibitors are under evaluation in numerous clinical trials .
These preliminary observations represent the bases of the present work, designed to evaluate the activity of palbociclib on a panel of TNBC cell lines with regards to the possible utilization of the drug also in association with PI3K/mTOR inhibitors. Our findings demonstrated that the combination of palbociclib with PI3K/mTOR inhibitors enhances the growth inhibitory effects of the single agents, and impairs tumor cell metabolism, suggesting new therapeutic strategies to challenge the aggressive behavior of TNBC.
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).
Palbociclib (PD-0332991) was provided by Pfizer (New York City, NY); NVP-BEZ235, NVP-BYL719, and NVP-BKM120 (hereafter, referred to as BEZ235, BYL719, and BKM120) were provided by Novartis Institutes for BioMedical Research (Cambridge, MA). Drugs were prepared in DMSO, and DMSO concentration never exceeded 0.1% (v/v); equal amounts of the solvent were added to control cells.
Analysis of cell proliferation, cell death and cell cycle
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 . The nature of the interaction between palbociclib and PI3K inhibitors was calculated using the Bliss additivism model . 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) . Distribution of the cells in the cell cycle was determined as previously described . 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 .
Antibodies against p-RbSer780 (#9307), Rb (#9309), cyclin D1 (#2926), CDK6 (#3136), c-myc (#9402), p-AKTSer473 (#9271), AKT (#9272), p-mTORSer2448 (#2971), mTOR (#2972), p-ERK1/2Thr202/Tyr204 (#4370), ERK1/2 (#4695), were from CST (Danvers, MA); anti-p-CDK6Tyr24 (sc-293,097) was from Santa Cruz Biotechnology, Incorporated (Dallas, TX). Anti CDKN2A/p16INK4a (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).
Quantitative real-time PCR
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 . 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 and consumption
Glucose uptake was measured as described previously . 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.
Effects of palbociclib on cell proliferation and cell cycle distribution in TNBC cells
Effects of palbociclib in combination with PI3K/mTOR inhibitors
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.
Effect of palbociclib and PI3K/mTOR inhibition on glucose energy metabolism
Altogether these results suggest that inhibition of PI3K/mTOR signaling may improve the efficacy of palbociclib also through a negative modulation of glucose metabolism.
In the present study we demonstrate that CDK4/6 inhibition by palbociclib in combination with PI3K/mTOR inhibitors may be an effective strategy for treatment of Rb-proficient TNBC, affecting both cell proliferation/viability and energy metabolism.
A functional Rb pathway is more frequently found in luminal A hormone-positive BC, and preclinical data have indicated these tumors as exquisitely sensitive to CDK4/6 inhibition, providing the basis for the development of novel therapeutic strategies for this BC subtype . Actually, palbociclib has received accelerated approval for first-line treatment in combination with letrozole or fulvestrant in postmenopausal women with ERα+/HER2 − locally advanced or metastatic BC. In contrast, TNBC has been considered less likely to respond to CDK4/6 inhibitors , being frequently associated with Rb loss . However, a number of preclinical data have been suggesting that palbociclib treatment may provide benefit also in a proportion of TNBC [12, 27]. Here we show that palbociclib as a single-agent treatment reduced cell proliferation in TNBC cell models, by eliciting reversible blockade of the cell cycle in G1 phase. This effect was observed only in cell lines expressing the predictive markers of response to palbociclib, i.e. Rb, cyclin D1, and CDK6 proteins, associated with undetectable levels of p16INK4. Palbociclib treatment in these cells inhibited p-Rb, Rb, and p-CDK6 levels, and more interestingly down-regulated the expression of c-myc, a direct target of E2F transcription factor. Very recently, CDK4 has been found highly expressed in TNBC, especially in the basal-like subtype , and this feature may serve as an additional predictive marker of response to palbociclib in this type of BC. Blocking CDK4 expression or activity in TNBC cells has been shown to prevent BC stem cell self-renewal , thus providing a further rationale for exploiting palbociclib as a therapy for TNBC. In addition, it has been recently demonstrated, in BC cell lines including TNBC cells, that treatment with CDK4/6 inhibitors triggers anti-tumor immunity by enhancing tumor antigen presentation and suppressing regulatory T cells proliferation , thus opening new perspectives in the use of CDK4/6 inhibitors in association with immunotherapies.
In our study, palbociclib treatment gave the most promising results when combined with PI3K/mTOR inhibitors.
A relevant feature of palbociclib treatment is the induction of AKT activation, through the release of p-Rb-mediated suppression of mTORC2. Hyperphosphorylated Rb inhibits the activity of the mTORC2 complex, by directly binding to the complex component Sin1. Since AKT is a substrate of mTORC2, the inhibition of Rb phosphorylation, associated with CDK4/6 inhibition, results in mTORC2 activation, thus increasing AKT activation .
This provided a rationale for evaluating the efficacy of combining palbociclib with PI3K/mTOR inhibitors following different schedules of treatment: a simultaneous combination, a sequential treatment in which a pre-incubation with palbociclib was followed by treatment with PI3K/mTOR inhibitors alone, or a sequential combined treatment in which palbociclib, given as a pre-treatment, was also maintained during exposure to PI3K/mTOR inhibitors. The latter schedule produced a synergistic growth inhibitory effect as calculated by the Bliss experimental model, whereas an additive effect was observed with the other two schedules.
The synergistic effect of the association of palbociclib and PI3K inhibitors has been recently reported for luminal androgen receptor-positive TNBC cell lines and has been associated with low level of CDK2 activity, rather than with p16INK4 loss and cyclin D1 expression . Here we show that also other TNBC subtypes, i.e. mesenchymal and basal-like subgroups, represented by MDA-MB-231 and HCC38 cell models respectively , may benefit from a combined therapy with palbociclib and PI3K/mTOR inhibitors, provided that they express the predictive factors of response to palbociclib. Importantly, our results demonstrate that the sequential combined treatment of palbociclib and PI3K/mTOR inhibitors is more effective than simultaneous treatment and might therefore represent a novel therapeutic approach for the treatment of TNBC of different cell subtypes.
Aberrant energy metabolism is an important hallmark of cancer, that is being exploited as a therapeutic target . Here we provide evidence that inhibition of glucose metabolism may contribute to palbociclib anti-tumor activity in TNBC cells. The cyclin D1/CDK6/Rb/E2F pathway has been involved in the control of a variety of metabolic processes, such as glucose production and glycolytic metabolism, indicating a close relationship between metabolic responses and proliferative stimuli . There is evidence that E2F pathway is a negative regulator of energy expenditure, through repression of mitochondrial oxidative metabolism ; E2F is also able to stimulate the glycolytic flux through regulation of phosphofructokinase enzyme expression . In addition, downstream of E2F, c-myc transcription factor may affect cancer metabolic reprogramming through a variety of mechanisms, and in particular it has been shown to drive glucose metabolism in TNBC cells .
Here we demonstrate that palbociclib-mediated inhibition of E2F/c-myc hinders glucose metabolism in TNBC cells, by inhibiting GLUT-1 expression and glucose uptake under both normoxic and hypoxic conditions. c-Myccooperates with HIF-1 to induce the expression of glycolytic enzymes ; interestingly, palbociclib inhibited also hypoxia-induced HIF-1α accumulation, which might contribute to reduce glucose utilization under hypoxia. This result is in line with a recent study showing that palbociclib destabilizes HIF-1α in colon cancer cells under either normoxic or hypoxic conditions . In our study, the inhibitory effects of palbociclib on glucose metabolism were further enhanced by the combination with PI3K/mTOR inhibitors, being the AKT/mTOR signaling another key player in cancer metabolic reprogramming . Accordingly, palbociclib combined with the mTOR inhibitor everolimus has been recently demonstrated to inhibit aerobic glycolysis in glioblastoma cells . Importantly, we observed a strong impairment of glucose metabolism when the combination with palbociclib and PI3K/mTOR inhibitors was preceded by treatment with palbociclib alone, likely contributing to the synergistic anti-tumor effects associated with this schedule of treatment.
In conclusion, our study provides a pre-clinical rationale for the combination of palbociclib with PI3K/mTOR inhibitors as a therapeutic strategy for TNBC, highlighting the superior efficacy of the sequential combined schedule of treatment. A limitation of this study is that these results were obtained in in vitro experiments and would require confirmation in vivo in animal models, also to ascertain whether palbocilcib may be safely maintained during treatment with PI3K/mTOR inhibitors in order to produce synergistic anti-tumor effects without worsening toxicity.
This work was supported by Associazione per la Ricerca in Campo Oncologico – ARCO, Cremona; A.VO.PRO.RI.T., Parma; Di mano in mano Onlus, Parma; Fondazione Italiana Biologi – Ordine Nazionale dei Biologi, Roma. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Availability of data and materials
Data and materials will be shared.
DC, AR, conducted the cell biology and molecular biology experiments. AC and GD performed cytofluorimetric analysis. AB and DC performed glucose metabolism analysis. CF and SLM performed statistical analysis. MB, CF and PGP analyzed results. CF and MB wrote the paper. DG, AF and PGP revised the manuscript. All authors approved the final version for publication.
The authors declare that they have no competing interests.
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