Multi-targeted kinase inhibition alleviates mTOR inhibitor resistance in triple-negative breast cancer
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Owing to its genetic heterogeneity and acquired resistance, triple-negative breast cancer (TNBC) is not responsive to single-targeted therapy, causing disproportional cancer-related death worldwide. Combined targeted therapy strategies to block interactive oncogenic signaling networks are being explored for effective treatment of the refractory TNBC subtype.
A broad kinase inhibitor screen was applied to profile the proliferative responses of TNBC cells, revealing resistance of TNBC cells to inhibition of the mammalian target of rapamycin (mTOR). A systematic drug combination screen was subsequently performed to identify that AEE788, an inhibitor targeting multiple receptor tyrosine kinases (RTKs) EGFR/HER2 and VEGFR, synergizes with selective mTOR inhibitor rapamycin as well as its analogs (rapalogs) temsirolimus and everolimus to inhibit TNBC cell proliferation.
The combination treatment with AEE788 and rapalog effectively inhibits phosphorylation of mTOR and 4EBP1, relieves mTOR inhibition-mediated upregulation of cyclin D1, and maintains suppression of AKT and ERK signaling, thereby sensitizing TNBC cells to the rapalogs. siRNA validation of cheminformatics-based predicted AEE788 targets has further revealed the mTOR interactive RPS6K members (RPS6KA3, RPS6KA6, RPS6KB1, and RPS6KL1) as synthetic lethal targets for rapalog combination treatment.
mTOR signaling is highly activated in TNBC tumors. As single rapalog treatment is insufficient to block mTOR signaling in rapalog-resistant TNBC cells, our results thus provide a potential multi-kinase inhibitor combinatorial strategy to overcome mTOR-targeted therapy resistance in TNBC cells.
KeywordsMulti-kinase inhibitor mTOR-targeted therapy Drug resistance Triple-negative breast cancer (TNBC) Polypharmacology
Cyclin-dependent kinase 4
Human epidermal growth factor
Half-maximal inhibitory concentration
Luminal androgen receptor-like
The mammalian target of rapamycin
Receptor tyrosine kinase
Triple-negative breast cancer
Triple-negative breast cancer (TNBC) constitutes a small subtype (10–20%) of breast cancer, but causes the majority of breast cancer-related deaths [1, 2]. As defined by the absence of ER and PR expression and HER2 overexpression, TNBC is not curable by hormone receptor or HER2-targeted therapies . Furthermore, TNBC is highly heterogeneous. Gene expression profiling has further classified TNBC into six unique molecular subtypes, namely basal-like (BL1 and BL2), mesenchymal (M), mesenchymal stem-like (MSL), immunomodulatory (IM), and luminal androgen receptor-like (LAR) subtype . The TNBC molecular signatures have been explored for targeted therapies in clinical trials, including those targeting receptor tyrosine kinases (RTKs, e.g., EGFR, VEGFR, c-Met), PI3K/AKT, Ras/MAPK, JAK/STAT, cell cycle regulators [5, 6]. Yet, TNBC has not benefited from above mono-targeted therapies so far, due to intrinsic or acquired resistance .
The mammalian target of rapamycin (mTOR), a conserved serine/threonine protein kinase, is a central regulator of cell growth and proliferation, by sensing and integrating multiple signals from growth factors and nutrient signals [7, 8]. mTOR hyperactivity is frequently observed in TNBC compared to other breast cancer subtypes and is often correlated with poor prognosis, underpinning the potential of mTOR-targeted therapy for TNBC treatment [9, 10, 11]. Although mTOR-targeted interventions, such as rapamycin and its analogs (rapalogs) temsirolimus and everolimus, delay progression and extend survival, patients with TNBC eventually develop resistance to mTOR inhibitors with undesired outcome [9, 12]. Evidence has shown that rapalog treatment could release mTOR negative feedback on upstream kinases and activate compensatory pathways, for instance, PI3K/AKT and MAPK/ERK signaling pathways, thereby bypassing mTOR inhibition [13, 14, 15]. This observation underscores the need for alternative combinatorial therapeutic approaches for TNBC treatment.
Since oncogenic pathways incorporate multiple signaling components and axes to promote tumor malignancy, monotherapy may not be sufficient for long-term control of TNBC [9, 13, 16]. Hence, simultaneously targeting different signaling molecules represents a promising strategy to impede tumor growth and progression [8, 17]. Several reports have documented that co-targeting growth factor receptors and mTOR exerts cooperative anti-cancer effects in various cancer types, including TNBC [18, 19, 20, 21, 22]. However, these studies focus on a particular combination in the questioned cancer type. Little is known about the interactive kinases involved in rapalog resistance and the mechanisms of the combinatorial effect remain unclear. Here, we systematically screened a broad collection of kinase inhibitors across a large panel of TNBC lines treated with rapamycin. Our data demonstrated that multiple targeted kinase inhibition, for instance, by inhibitor AEE788, sensitizes TNBC cells to various mTOR inhibitors, rapamycin, temsirolimus, and everolimus. Integrated cheminformatics study and siRNA validation revealed additional putative targets of AEE788, which interact closely with mTOR signaling. Most importantly, our study provided an efficacious approach for exploring cancer combination treatment. Moreover, the combinatorial therapy is more effective than single drug application and thus demonstrates a therapeutic advantage over either agents as a monotherapy in TNBC treatment.
Kinase inhibitor library combination screen
One day post seeding into 96-well plates, cells were treated with individual kinase inhibitors alone or combined with rapamycin at 1 µM. After 4-day treatment, proliferation was evaluated by sulforhodamine B (SRB) colorimetric assay . Detailed information on materials and methods can be found in Supplementary file ESM_3.
TNBC cell lines are differentially responsive to mTOR inhibitor rapalogs
Rapalogs are highly selective allosteric inhibitors of mTOR, by binding to FKBP12/rapamycin-binding domain to block mTOR Ser2448 phosphorylation and function [24, 25]. mTOR Ser2448 is a predominant phosphorylation residue for mTOR kinase activity in response to mitogen-derived stimuli . Therefore, we examined the inhibitory effect of rapamycin (Rap), temsirolimus (Tem), and everolimus (Eve), on Ser2448-mTOR phosphorylation with a focus on rapalog-resistant TNBC cell lines HCC1806 and SUM149PT and rapalog-sensitive Hs578T TNBC cells. The rapalogs potently inhibited phosphorylation of mTOR in the sensitive Hs578T cells, but not or less effectively in the resistant HCC1806 and SUM149PT cells, respectively (Fig. 1c, d). These data suggest that mTOR kinase activity and its sustained phosphorylation render the TNBC cells resistant to rapalogs.
Combinatorial drug screen identifies kinase inhibitors sensitizing TNBC cells to mTOR inhibition
These data implicate that while the resistant SUM149PT cells poorly respond to inhibitors of EGFR or VEGFR and mTOR inhibitor rapamycin alone, concurrent blockage of upstream EGFR or VEGFR RTK activity or MEK signaling transduction, and downstream mTOR signaling could converge re-sensitization of TNBC cells.
Multi-targeted RTK inhibitor AEE788 enhances proliferative inhibition and cell death in rapalog-resistant TNBC cells
Co-treatment of rapamycin and AEE788 abolishes mTOR phosphorylation and sustains downregulation of ERK and AKT signaling in TNBC cells
As AEE788 has been described as an EGFR/VEGFR dual RTK inhibitor, we further evaluated the co-treatment effect of AEE788 and rapamycin on EGFR RTK signaling activity in both resistant SUM149PT and HCC1143 cells upon EGF stimulation (Fig. 4c). To reach maximal activation of the EGFR signaling pathway, cells were first serum starved followed by EGF treatment. EGF caused the activation of the EGF receptor as evidenced by increased p-EGFR and downstream p-ERK and p-AKT. AEE788 effectively blocked EGF-stimulated phosphorylation of these components, in both SUM149PT and HCC1143 cells. It has been reported that 4EBP1 has multiple phosphorylation sites and an increase in 4EBP1 phosphorylation is accompanied by a decrease in its electrophoretic mobility [26, 27, 28]. EGF also effectively caused enhanced p-mTOR and p-4EBP1. Rapamycin could inhibit 4EBP1 phosphorylation by EGF in both cell lines but with no (SUM149PT) or limited (HCC1143) effects on mTOR activation; and, reversely, AEE788 could inhibit mTOR activation but with marginal effects on 4EBP1 phosphorylation. However, co-treatment with AEE788 and rapamycin particularly could shut down the EGF-mediated phosphorylation of mTOR and 4EBP1 signaling.
Several cellular processes have been linked to the immunogenicity of cell death, including autophagy and ER stress [29, 30, 31, 32]. Given that mTOR is a key regulator of autophagy, we then tested the combination effect of autophagy in TNBC cells. Interestingly, the combination sustained the elevated LC3B level induced by monotherapy and demonstrated accumulated Beclin-1 expression 24 h post treatment in both SUM149PT and HCC1143 cells (Fig. 4d, e). The combination increased the expression level of BiP, a key regulator of ER stress, 24 h post treatment in SUM149PT cells, but not so much in HCC1143 cells (Fig. 4d), suggesting the involvement of immunogenic cell death-related events in TNBC cells by the combination treatment.
Silencing of AEE788 targets enhances mTOR inhibition in TNBC cells
AEE788 abolished rapalog-upregulated cyclin D1 expression in TNBC cells
mTOR acts as a central regulator of multiple signaling networks in control of cell growth, proliferation, and survival [7, 37]. mTOR signaling is frequently upregulated in malignant tumors, including TNBC, highlighting the potential of mTOR kinase targeted therapy in cancer modulation [7, 8, 9, 12]. However, patients with TNBC often experience mTOR targeting failure due to acquired resistance and activation of bypass surviving pathways [13, 15, 38]. Our drug combination screen revealed that co-treatment with AEE788, a multiple RTK-targeted inhibitor, restores the sensitivity of TNBC cells towards the clinically applied mTOR inhibitors (rapamycin, temsirolimus, and everolimus). The effect of AEE788 is likely due to polypharmacology to shut down the crosstalk among receptors as well as mTOR pathway within signaling networks in the resistant scenario. The combination of targeted agents profoundly improves therapeutic efficacy and overcomes resistance that might develop under single-agent therapy.
mTOR inhibition can relieve distinct negative feedback loops that normally serve to attenuate upstream RTKs, PI3K, and MAPK signaling, leading to rapalog resistance . As such, mTOR inhibition alone is not sufficient to overcome the entire oncogenic program propagated from the alternate proliferative signaling pathways. By exploiting a high-throughput kinase drug combination screen, our study has identified the effective kinase inhibitor, AEE788, that can block compensatory mechanisms conferring aberrant cell cycle progression upon rapalog treatment. The repression of EGFR/VEGFR- and mTOR-related pathways in concert seemingly reverts processes predominantly responsible for uncontrolled TNBC tumor proliferation. Our results are in line with the above observations that co-inhibition of upstream RTKs (such as EGFR, VEGFR, PDGFR, and IGF1R), PI3K and MAPK signaling transduction, and mTOR signaling elicited enhanced therapeutic efficacy in various cancer types.
Sustained mTOR signaling drives resistance to targeted therapeutics in cancer treatment . In TNBC tumor cells, mTOR signaling is frequently upregulated . We demonstrated that while rapalogs alone were insufficient to inhibit the sustained mTOR signaling in resistant TNBC cells, co-treatment of rapalogs with the multi-targeted RTK inhibitor AEE788 synergistically blocked mTOR phosphorylation in SUM149PT and HCC1143 cells. Interestingly, SUM149PT cells have been characterized for the constitutively activated EGFR via a self-sustaining amphiregulin autocrine loop, and subsequently, altered receptor signaling and gene expression [41, 42]. As a further validation, AEE788 and rapamycin treatment blocked EGF-mediated EGFR downstream signaling in both SUM149PT and HCC1143. We recognize that these experiments were performed under short-term EGF treatment conditions that may not fully reflect the normal TNBC cell signaling. Therefore, we cannot exclude that under more physiological conditions in the tumor microenvironment other distinct RTK signaling cascades are more prominent in the modulation of RTK signaling and cancer progression.
A mechanism of resistance to mTOR inhibition in cancer is the rapalog-mediated activation of upstream PI3K/AKT and MAPK/ERK signaling . Co-treatment with AEE788 and rapamycin maintained the inhibitory effect on AKT and ERK signaling in TNBC cells. These data suggest that AEE788 and rapamycin synergistically inhibit the sustained mTOR activity in TNBC cells, thus blocking mTOR’s potential feedback loop on activation of alternative ERK and AKT proliferative signaling pathways.
Polypharmacology, the action of drugs against multiple targets , is commonly observed in drug development including the effective marketed kinase inhibitors . Our ChEMBL-based cheminformatics analysis demonstrated that AEE788 is a kinase inhibitor that likely targets several kinases; this is consistent with other reports using protein kinase assays [19, 35]. Complementary to a recent kinobeads study on target landscape of clinical kinase drugs , our cheminformatics approach presented that the multi-targeted RTK inhibitor AEE788 likely interacts with EGFR, VEGFR, ABL2, PDGFRB, and several mTOR signaling pathway components, including AKT and S6K family members. Subsequent siRNA-based knockdown of these various kinases, e.g., RTKs (EGFR, VEGFR2/3, and PDGFRB), AKTs (AKT2 and AKT3), RPS6Ks (RPS6KA3, RPS6KA6, RPS6KB1, and RPS6KL1), MAPKAPK2, ABL2, and CDK7, sensitized rapalog-resistant TNBC cells to rapamycin. Several reports have demonstrated the synergistic effects of targeting EGFR or MEK on anti-mTOR therapies in TNBC [20, 44, 45, 46]. However, our study demonstrated that simultaneous use of EGFRi gefitinib or MEKi PD184352 only exerts additive effects on rapamycin-mediated proliferative inhibition, suggesting that AEE788-rapalog synergy results presumably from multi-targeted kinase inhibition. These data support the anticipated polypharmacology of AEE788 as the mode-of-action of the synergy with rapalogs. Further studies are required to determine the detailed kinome target landscape of AEE788 in TNBC.
mTOR pathway regulates cell growth through its downstream effectors, such as 4EBP1 and RPS6KB1 [7, 37]. Another primary way that mTOR confers its regulatory effects on cell proliferation is to upregulate expression of the cell cycle regulator cyclin D1 . CCND1, the cyclin D1 encoding gene, is frequently amplified in breast cancer, and depletion of cyclin D1 suppresses breast cancer progression [48, 49]. In response to mTOR inhibition, however, cyclin D1 is elevated by everolimus in various types of cancer [21, 22]. Consistently, we found that treatment with rapalogs (rapamycin, temsirolimus, and everolimus) commonly upregulated cyclin D1 in rapalog-resistant TNBC cells, indicating an alternative activation of cyclin D1 proliferative signaling pathway after mTOR inhibition. Considering that cyclin D1 was lost in the presence of the AEE788-rapalog combination, AEE788 seems to compensate the undesired effects of rapalog, further highlighting the therapeutic advantage of the drug combination. Interestingly, while we discovered the AEE788-rapamycin interaction through a wider screening effort in TNBC cells, our findings were further supported by the observations on the synergistic effects of AEE788-everolimus combination in prostate, germ, and renal tumor cell lines [19, 21, 22]. Moreover, a xenograft-bearing mice study also documented the beneficial action of AEE788-everolimus combination in glioblastoma tumor regression . However, these studies did not further the mode-of-action of AEE788. Since AEE788 is recognized as a multiple targeting kinase inhibitor, their observations were limited to EGFR/VEGFR, lacking the notion on other potentially targeted candidate kinases. Our study, for the first time, revealed the synergy on rapalogs treatment in TNBCs and its underlying polypharmacology by utilizing integrated systematic screen and cheminformatics approach. Moreover, either genetic or pharmacological ablation of cyclin D1 significantly enhanced mTOR-inhibition-mediated proliferative inhibition. This is concordant with the recent reports on the synergistic anti-cancer activity of combined CDK4/6 and mTOR targeting [51, 52, 53].
In conclusion, our work supports that polypharmacology to target multiple kinase targets in combination with rapalog treatment may offer a distinct combinatorial benefit to TNBC patients that are otherwise resistant to mTOR-targeted therapeutics.
JH was financially supported by the China Scholarship Council.
JH, YZ, and BvdW conceived and designed the experiments. YZ and BvdW supervised the research. JH, RM, VvdN, and YZ performed the experiments. JM and JF co-supervised the research. GvW performed ligand-based target prediction. JH, YZ, and BvdW wrote the manuscript. All authors read, reviewed, and approved the final manuscript.
This work was supported by the European Research Council Advanced grant Triple-BC (Grant No. 322737) and the Dutch Cancer Society project (Grant No. 2011-5124). GvW was supported by the Dutch Scientific Council (NWO domain AES Veni 14410).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
This article does not contain any studies with human participants or animals performed by any of the authors.
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