Abstract
Pemetrexed (ALIMTA) is a folate anti-metabolite that has been approved for the treatment of non-small cell lung cancer, and has been shown to stimulate autophagy. In the present study, we sought to further understand the role of autophagy in the response to pemetrexed and to test if combination therapy could enhance the level of toxicity through altered autophagy in tumor cells. The multikinase inhibitor sorafenib (NEXAVAR), used in the treatment of renal and hepatocellular carcinoma, suppresses tumor angiogenesis and promotes autophagy in tumor cells. We found that sorafenib interacted in a greater than additive fashion with pemetrexed to increase autophagy and to kill a diverse array of tumor cell types. Tumor cell types that displayed high levels of cell killing after combination treatment showed elevated levels of AKT, p70 S6K and/or phosphorylated mTOR, in addition to class III RTKs such as PDGFRb and VEGFR1, known in vivo targets of sorafenib. In xenograft and in syngeneic animal models of mammary carcinoma and glioblastoma, the combination of sorafenib and pemetrexed suppressed tumor growth without deleterious effects on normal tissues or animal body mass. Taken together, the data suggest that premexetred and sorafenib act synergistically to enhance tumor killing via the promotion of a toxic form of autophagy that leads to activation of the intrinsic apoptosis pathway, and predict that combination treatment represents a future therapeutic option in the treatment of solid tumors.
Initial studies noted that the anti-folate pemetrexed (ALIMTA®) interacts with the multikinase inhibitor sorafenib (NEXAVAR®) in a greater than additive fashion to kill a wide variety of tumor cell types (breast, liver, lung, brain). Pemetrexed was originally developed as an inhibitor of thymidylate synthase; however, it became clear that the drug has at least one other target that became apparent from a continued anti-proliferative effect of drug treatment in cell cultures exposed to exogenous thymidine, which prevents the cytotoxic effects of thymidylate synthase inhibition. Subsequently, the secondary target was shown to be the folate-dependent enzyme, aminoimidazole-carboxamide ribonucleotide formyl-transferase (AICART). Pemetrexed inhibition of AICART elevates the levels of ZMP, a substrate of the AICART reaction. Accumulation of ZMP causes activation of AMP-activated protein kinase with subsequent inhibition of mTOR and the induction of autophagy. Sorafenib is a multikinase inhibitor whose biological actions have often been tied to inhibition of class III receptor tyrosine kinases, e.g., vascular endothelial growth factor receptors (VEGFRs) and platelet-derived growth factor receptor β (PDGFRβ).
We found that pemetrexed-induced autophagic vesicles are necessary for tumor cell killing in the majority of cell lines tested. Data from two colon cancer lines (HCT116, DLD1) show that pemetrexed-induced autophagy is a mildly protective effect. Prior studies from our laboratories have shown in a dose-dependent fashion that sorafenib can increase the levels of autophagy in tumor cells, and this autophagy can either act to promote survival or to cause tumor cell killing. Sorafenib interacts in a greater than additive fashion with pemetrexed to increase autophagy levels and to kill a diverse array of tumor cell types, including colon cancer cells. Based on our in vitro findings we moved our drug combination approach to the vivarium using a variety of animal models. We demonstrated that the sorafenib plus pemetrexed drug combination significantly reduces tumor growth and prolongs animal survival compared with either drug individually.
The development of estrogen independence can play a significant role in the recurrence of ER+ breast cancer, which in turn resulted in the development of anti-estrogen therapeutics such as Faslodex (Fulvestrant, ICI 182,780). We discovered that fulvestrant-resistant MCF7 cells (MCF7F) express higher levels of the autophagy regulatory protein Beclin 1 and the class III receptor tyrosine kinase and sorafenib target PDGFRβ. MCF7F cells are more sensitive to pemetrexed and sorafenib drug combination toxicity compared with their estrogen-dependent MCF7 counterparts.
It is well known that MCF7 cells, and for that matter many untreated breast cancers in situ, have a haplotype insufficiency in Beclin 1, and thus are presumed to be less effective at inducing autophagy than nontransformed mammary epithelial cells. These findings suggest that loss of autophagy in a premalignant mammary epithelial cell undergoing the process of transformation facilitates tumor formation. And, as we noted that MCF7F cells express higher basal levels of Beclin 1, it could also be argued that a portion of the process by which an MCF7 or any established estrogen-dependent breast cancer cell maintains its viability in the face of anti-estrogen therapy is to select for survival of clones that have an increased ability to induce a protective form of autophagy. In agreement with this finding, agents that act to suppress the later lysosomal fusion stage of autophagy, e.g., chloroquine, can facilitate the antitumor effects of the anti-estrogen tamoxifen. Thus, our data argue that the autophagic survival response of breast cancer cells following anti-estrogen therapy selects for tumor cell clones that may subsequently facilitate tumor cell killing by drug combinations that utilize toxic forms of autophagy to achieve their therapeutic effects.
As noted above, in several models of breast cancer we demonstrated that pemetrexed and sorafenib interact to suppress tumor growth that correlates with increased levels of apoptosis within the tumor. In vitro we also noted that lung, liver and brain cancers are killed by this drug combination. In the clinic, sorafenib is approved for treatment of renal and hepatocellular carcinomas and is believed to act by suppressing tumor angiogenesis. Pemetrexed is approved for treatment and maintenance therapy of non-small cell lung cancer. Based on the data presented in the present manuscript we are preparing to develop and implement a protocol for an open label phase I trial combining sorafenib and pemetrexed in patients with recurrent solid tumors.
Acknowledgements
Support for the present study was provided to P.D. from PHS grants (R01-DK52825; P01-CA104177; R01-CA108325; R01-CA141703; R01-CA150214), The Jim Valvano “V” foundation, and Department of Defense Award (W81XWH-10-1-0009); to S.G. from PHS grants (R01-CA63753; R01-CA77141) and a Leukemia Society of America grant 6405-97; to R.G.M. from PHS grants (R01-CA39687; R01-CA140416); and to K.P.N. (NCI U54CA113001).
Punctum to: Bareford MD, Park MA, Yacoub A, Hamed HA, Tang Y, Cruickshanks N, Eulitt P, Hubbard N, Tye G, Burow ME, Fisher PB, Moran RG, Nephew KP, Grant S, Dent P. Sorafenib enhances pemetrexed cytotoxicity through an autophagy-dependent mechanism in cancer cells. Cancer Res 2011; 71:4955 - 4967; PMID: 21622715; http://dx.doi.org/10.1158/0008-5472.CAN-11-0898