Preclinical evidence that MNK/eIF4E inhibition by cercosporamide enhances the response to antiangiogenic TKI and mTOR inhibitor in renal cell carcinoma

https://doi.org/10.1016/j.bbrc.2020.06.133Get rights and content

Highlights

  • Cercosporamide is active against RCC tumor and endothelia cells.

  • Cercosporamide acts synergistically with sunitinib and temsirolimus in RCC.

  • Cercosporamide suppressed pro-angiogenic, EMT and cell cycle proteins.

  • Cercosporamide targets MNK/e-IF4E axis in RCC.

Abstract

Eukaryotic translation initiation factor 4E (eIF4E) is deregulated in patients with renal cell carcinoma (RCC) and associated with poor prognosis, and is activated and regulated by Mnk kinases. In this study, we investigated the anti-RCC potential of a unique Mnk inhibitor cercosporamide. We showed that cercosporamide is active against RCC cells via suppressing growth, survival and migration. Combination indices value indicated that the combination of cercosporamide with sunitinib or temsirolimus are synergistic in RCC. In two independent RCC xenograft mouse models, complete tumor growth arrest or reverse was observed throughout the duration of drug treatment in the combination of cercosporamide with sunitinib or temsirolimus groups. Of note, cercosporamide inhibited RCC angiogenesis via negatively regulating a number of RCC endothelial cellular events including morphogenesis, migration, growth and survival. Mechanistically, we found that cercosporamide suppressed pro-angiogenic factors VEGF and HIFα, inhibited EMT and reduced pro-survival and cell cycle proteins; and furthermore this was attributed to cercosporamide’s ability in inhibiting eIF4E. This work demonstrates the anti-RCC activity of cercosporamide through targeting both RCC tumor cells and angiogenesis, and provides the first preclinical proof-of-concept of evidence of Mnk inhibition for RCC treatment.

Introduction

Renal cell carcinoma (RCC), most commonly clear-cell RCC, is an epithelial tumor derived from the proximal tubules of nephrons, and accounts for 90% of kidney cancers [1]. Approximately 35% of RCC patients are present with metastatic RCC (mRCC) at diagnosis and are refractory to cytotoxic chemotherapy and radiotherapy [2]. Clear-cell RCC is characterized by alterations to the VHL gene which activates angiogenesis factors, such as hypoxia-inducible factor (HIF) and vascular endothelial growth factor (VEGF). Current first-line treatments for mRCC are the anti-VEGFR tyrosine kinase inhibitors, such as sunitinib [3]. However, after initial response, the majority of mRCC patients will progress within 12 months of starting therapy [4]. The mammalian target of rapamycin (mTOR) inhibitor temsirolimus is another first-line treatment drug for mRCC but demonstrated only modest efficacy in patients with poor-risk clear-cell mRCC [5]. There is a need to identify more effective treatment strategies for mRCC patients [6].

Mitogen-activated protein kinase (MAPK) interacting kinases (MNKs), MNK1 and MNK2, play essential roles in human tumorigenesis and progression but are redundant for growth of non-transformed cells [7]. MNK modulates functions of eukaryotic translation initiation factor 4E (eIF4E) through phosphorylation of a conserved serine (Ser209) [8]. eIF4E is a well-known player in protein translation via preferentially binds to carcinogenesis associated mRNAs including VEGF and cyclin D that favor aberrant cancer cell proliferation and survival [[9], [10], [11]]. MNKs are therefore attractive targets for pharmacological inhibition with a broad therapeutic window for cancer treatment [12]. Cercosporamide is an orally bioavailable anti-fungal agent and was recently identified during a chemical screen as a potent and selective Mnk inhibitor [13]. Although unstudied in RCC, the anti-cancer activity of cercosporamide has been shown in melanoma, hepatocellular carcinoma, leukemia and glioblastoma [[13], [14], [15], [16]]. In this study, we investigated efficacy of cercosporamide alone with its combination with sunitinib and temsirolimus in various RCC models. We found that cercosporamide is active against RCC via targeting both RCC tumor cells and angiogenesis. Importantly, we found that combination of cercosporamide with sunitinib or temsirolimus achieved much greater efficacy in RCC, providing the evidence to support clinical trials of cercosporamide with other first-line drugs for the treatment of mRCC.

Section snippets

Cells and drugs

Two RCC cell lines 786–0 and Caki-1 were grown in Eagle’s Minimal Essential Media (MEM) supplemented with a final concentration of 10% fetal bovine serum (Hyclone) and 100 u/ml penicillin-streptomycin (Invitrogen). Primary human kidney tumor associated-endothelial cells (HKT-EC, CellBiologics) was cultured in Complete Human Endothelial Cell Medium (CellBiologics). HKT-EC at passages 2–5 were used for the experiments. Sunitinib and temsirolimus (LC Laboratories), and cercosporamide (CalBiochem)

Cercosporamide acts synergistically with sunitinib and temsirolimus in RCC in vitro

We examined the effects of cercosporamide alone and its combination with first-line treatment drugs for advanced RCC on the biological activities in two RCC cell lines: 786–0 is the primary and used most commonly in RCC-focused research and Caki-1 is a widespread model line of metastasis RCC [20]. Exposure to cercosporamide alone significantly inhibited proliferation and induced apoptosis in a dose-dependent manner in 786–0 and Caki-1 cells (Fig. 1A and B). Cercosporamide also dose-dependently

Discussion

This preclinical study is the first to show that Mnk-eIF4E inhibition by cercosporamide is a rational means to improve the response to sunitinib and temsirolimus in mRCC, a disease with exceedingly poor survival. Combination of first-line drugs with Mnk inhibitor for mRCC is an attractive therapeutic approach because Mnk-mediated eIF4E phosphorylation seems not to be a requirement for normal cellular development and growth [7] but is indispensable for malignant transformation and progression [23

Declaration of competing interest

All other authors declare no conflict of interest.

Acknowledgement

This work was supported by research grants provided by the Scientific and Technological Project of Shiyan City of Hubei Province (No. 18Y10) and Hubei University of Medicine (No. 2016QDJZR22).

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    These authors have contributed equally to this work.

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