Mammalian Target of Rapamycin Inhibitors Induce Tumor Cell ApoptosisIn VivoPrimarily by Inhibiting VEGF Expression and Angiogenesis

Patrick Frost,Alan Lichtenstein,Bao Hoang,Yijiang Shi,Eileen Berlanger,Joseph Gera,Veena Mysore

doi:10.1155/2013/897025

Patrick Frost, Alan Lichtenstein + Show 5 more

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https://doi.org/10.1155/2013/897025

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Abstract

We found that rapalog mTOR inhibitors induce G1 arrest in the PTEN-null HS Sultan B-cell lymphoma line in vitro, but that administration of rapalogs in a HS Sultan xenograft model resulted in significant apoptosis, and that this correlated with induction of hypoxia and inhibition of neoangiogenesis and VEGF expression. Mechanistically, rapalogs prevent cap-dependent translation, but studies have shown that cap-independent, internal ribosome entry site (IRES)-mediated translation of genes, such as c-myc and cyclin D, can provide a fail-safe mechanism that regulates tumor survival. Therefore, we tested if IRES-dependent expression of VEGF could likewise regulate sensitivity of tumor cells in vivo. To achieve this, we developed isogenic HS Sultan cell lines that ectopically express the VEGF ORF fused to the p27 IRES, an IRES sequence that is insensitive to AKT-mediated inhibition of IRES activity and effective in PTEN-null tumors. Mice challenged with p27-VEGF transfected tumor cells were more resistant to the antiangiogenic and apoptotic effects of the rapalog, temsirolimus, and active site mTOR inhibitor, pp242. Our results confirm the critical role of VEGF expression in tumors during treatment with mTOR inhibitors and underscore the importance of IRES activity as a resistance mechanism to such targeted therapy.

Highlights

Whilst inhibitors of the mTOR signaling pathways have been approved for use against advanced renal cell carcinoma [1, 2], their effectiveness against hematological malignancies remains unclear [3]
We demonstrated that 10 daily IP injections of temsirolimus had an antitumor effect in mice challenged with the human B-cell myeloma cell lines 8226, U226, and OPM-2 [6, 9, 20]
In order to determine if tumor regression was due to apoptosis, sections from tumor harvested after the last treatment with temsirolimus were stained for cleaved caspase-3 and demonstrated a dose-dependent induction of apoptosis (Figure 1(c))

Summary

Introduction

Whilst inhibitors of the mTOR signaling pathways have been approved for use against advanced renal cell carcinoma [1, 2], their effectiveness against hematological malignancies remains unclear [3]. Several rapalog mTOR inhibitors, including rapamycin [4, 5], temsirolimus (CCI-779) [6,7,8,9], and everolimus (RAD001) [10], have shown preclinical potential in hematological malignancies. One factor potentially limiting the effectiveness of rapalogs for treating hematological malignancies is the fact that in vitro exposure to mTOR inhibitors often only induces G1/S cell cycle arrest without apoptosis [11, 12]. Lack of in vitro tumor cell apoptosis may not accurately reflect the in vivo situation where tumor cell survival can be regulated by the microenvironment which itself may be impacted by mTOR inhibitors

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