Establishing that autophagy, in the absence of apoptosis, was
partially responsible for the heightened radiosensitivity conferred
by mTOR inhibition lends further credence to the idea that
the traditional tenets of cancer treatment may exaggerate the role of
p53-dependent cell death. Brown and Wouters (41) have argued that
neither p53 status nor the ability to undergo apoptosis significantly
affects the sensitivity of solid tumor cells to DNA-damaging agents.
For example, many malignant cells have lost the capacity to undergo
normal apoptosis, so it is reasonable to expect alternative modes of
cell death to play a more prominent role in determining the
sensitivity of a particular tumor to genotoxic agents (21). With the
emergence of this new paradigm questioning the apoptotic
sensitivity of solid tumors, our results showing the autophagy
secondary to mTOR and apoptosis inhibition underscore the need
to further investigate how to augment alternative mechanisms of
cell death, such as autophagy, programmed necrosis that can
occur after prolonged or massive autophagy, or mitotic
catastrophe. The genetic constitution of a cancer cell may
determine which mode of death to pursue.
Although this study was conducted in vitro, there is evidence to
suggest that RAD001 efficacy may be improved under in vivo
conditions. For instance, rapamycin is known to impede angio-
genesis (42), and our group has previously shown decreased tumor
vascular density in murine models and sensitized vascular
endothelium when mTOR inhibition is coupled with radiation
(18). This raises the possibility of an additional antitumor effect in
the stroma, and suggests that a greater in vivo response to mTOR
inhibition may be observed than would be predicted by in vitro
In summary, we believe that this is the first article to show
that mTOR inhibition with RAD001 is capable of radiosensitiz-
ing these cells by amplifying the autophagic pathway of cell death.
This effect is more pronounced in the clinically relevant PTEN-
deficient cell line. As more in vitro studies show the feasibility
and efficacy of mTOR inhibition, methods of monitoring mTOR
levels in vivo will become increasingly important. The duration
of S6K1 inactivation in peripheral blood mononuclear cells cor-
relates with tumor response to mTOR inhibition in rats, and thus
potentially offers a valuable biomarker to assay RAD001 efficacy
in patients (43). Additionally, because mTOR induces GLUT1
expression, preclinical studies have shown the viability of assessing
reduced glucose uptake using
FDG-positron emission tomogra-
phy as a way of imaging areas of mTOR inhibition in vivo (44).
These preclinical results provide valuable guidance in designing
clinical trials using mTOR inhibitors.
Received 3/2/2006; revised 8/3/2006; accepted 8/10/2006.
Grant support: Vanderbilt Discovery Grant, Vanderbilt Physician Scientist Grant,
Mesothelioma Applied Research Foundation, and Department of Defense grants
PC031161 and BC030542.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1. Zelefsky MJ, Fuks Z, Hunt M, et al. High dose radiation
delivered by intensity modulated conformal radiother-
apy improves the outcome of localized prostate cancer.
J Urol 2001;166:876–81.
2. Pollack A, Hanlon AL, Horwitz EM, Feigenberg SJ,
Uzzo RG, Hanks GE. Prostate cancer radiotherapy dose
response: an update of the fox chase experience. J Urol
3. Cantley LC. The phosphoinositide 3-kinase pathway.
4. Nicholson KM, Anderson NG. The protein kinase B/
Akt signaling pathway in human malignancy. Cell Signal
5. Majumder P, Sellers W. Akt-regulated pathways in
prostate cancer. Oncogene 2005;24:7465–74.
6. McMenamin ME, Soung P, Perera S, Kaplan I, Loda M,
Sellers WR. Loss of PTEN expression in paraffin-
embedded primary prostate cancer correlates with high
Gleason score and advanced stage. Cancer Res 1999;59:
7. Nave BT, Ouwens M, Withers DJ, Alessi DR, Shepherd
PR. Mammalian target of rapamycin is a direct target for
protein kinase B: identification of a convergence point
for opposing effects of insulin and amino-acid deficien-
cy on protein translation. Biochem J 1999;344:427–31.
8. Brunn GJ, Hudson CC, Sekulic A, et al. Phosphoryla-
tion of the translational repressor PHAS-I by the
mammalian target of rapamycin. Science 1997;277:
9. Burnett PE, Barrow RK, Cohen NA, Snyder SH,
Sabatini DM. RAFT1 phosphorylation of the transla-
tional regulators p70 S6 kinase and 4E-BP1. Proc Natl
Acad Sci U S A 1998;95:1432–7.
10. Huang S, Houghton PJ. Targeting mTOR signaling for
cancer therapy. Curr Opin Pharmacol 2003;3:371–7.
11. Ruggero D, Pandolfi PP. Does the ribosome translate
cancer? Nat Rev Cancer 2003;3:179–92.
12. Hay N, Sonenberg N. Upstream and downstream of
mTOR. Genes Dev 2004;18:1926–45.
13. Chan S. Targeting the mammalian target of rapamy-
cin (mTOR): a new approach to treating cancer. Br J
14. Workman P. ‘‘Drugging the PI3K pathway’’ Program
and Proceedings of AACR-NCI-EORTC internal confer-
ence. Molecular targets and cancer therapeutic abstract
book. Boston: Hynes Center; 2003. p 257–8.
15. Vignot S, Faivre S, Aguirre D, Raymond E. mTOR-
targeted therapy of cancer with rapamycin derivatives.
Ann Oncol 2005;16:525–37.
16. Neshat MS, Mellinghoff IK, Tran C, et al. Enhanced
sensitivity of PTEN-deficient tumors to inhibition of
FRAP/mTOR. Proc Natl Acad Sci U S A 2001;98:10314–9.
17. Majumder PK, Febbo PG, Bikoff R, et al. mTOR
inhibition reverses Akt-dependent prostate intraepithe-
lial neoplasia through regulation of apoptotic and HIF-
1-dependent pathways. Nat Med 2004;10:594–601.
18. Shinohara ET, Cao C, Niermann K, et al. Enhanced
radiation damage of tumor vasculature by mTOR
inhibitors. Oncogene 2005;24:5414–22.
19. Paglin S, Lee N, Nakar C, et al. Rapamycin-sensitive
pathway regulates mitochondrial membrane potential,
autophagy, and survival in irradiated MCF-7 cells.
Cancer Res 2005;65:11061–70.
20. Albert JM, Kim KW, Cao C, et al. Targeting the Akt/
mammalian target of rapamycin pathway for radio-
sensitization of breast cancer. Mol Cancer Ther 2006;5:
21. Brown JM, Attardi LD. The role of apoptosis in
cancer development and treatment response. Nat Rev
22. Kabeya Y, Mizushima N, Ueno T, et al. LC3, a
mammalian homologue of yeast Apg8p, is localized in
autophagosome membranes after processing. EMBO J
23. Contessa JN, Hampton J, Lammering G, et al. Ionizing
radiation activates Erb-B receptor dependent Akt and
p70 S6 kinase signaling in carcinoma cells. Oncogene
24. Edwards E, Geng L, Tan J, Onishko H, Donnelly E,
Hallahan DE. Phosphatidylinositol 3-kinase/Akt signal-
ing in the response of vascular endothelium to ionizing
radiation. Cancer Res 2002;62:4671–7.
25. Sunavala-Dossabhoy G, Fowler M, De Benedetti A.
Translation of the radioresistance kinase TLK1B is
induced by g-irradiation through activation of mTOR
and phosphorylation of 4E-BP1. BMC Mol Biol 2004;5:1.
26. Maehama T, Dixon JE. The tumor suppressor, PTEN/
MMAC1, dephosphorylates the lipid second messenger,
phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem
27. Di Cristofano A, Pesce B, Cordon-Cardo C, Pandolfi
PP. Pten is essential for embryonic development and
tumour suppression. Nat Genet 1998;19:348–55.
Mutation of Pten/Mmac1 in mice causes neoplasia in
multiple organ systems. Proc Natl Acad Sci USA 1999;96:
29. Wang S, Gao J, Lei Q, et al. Prostate-specific dele-
tion of the murine Pten tumor suppressor gene leads
to metastatic prostate cancer. Cancer Cell 2003;4:
30. Ma X, Ziel-van der Made AC, Autar B, et al. Targeted
biallelic inactivation of Pten in the mouse prostate leads
to prostate cancer accompanied by increased epithelial
cell proliferation but not by reduced apoptosis. Cancer
31. Suzuki H, Freije D, Nusskern DR, et al. Interfocal
heterogeneity of PTEN/MMAC1 gene alterations in
multiple metastatic prostate cancer tissues. Cancer
32. Law M, Forrester E, Chytil A, et al. Rapamycin
disrupts cyclin/cyclin-dependent kinase/p21/proliferat-
ing cell nuclear antigen complexes and cyclin D1
reverses rapamycin action by stabilizing these com-
plexes. Cancer Res 2006;66:1070–80.
33. Semenza GL. Hypoxia, clonal selection, and the role
of HIF-1 in tumor progression. Crit Rev Biochem Mol
34. Arsham AM, Howell JJ, Simon MC. A novel hypoxia-
inducible factor-independent hypoxic response regulat-
ing mammalian target of rapamycin and its targets.
J Biol Chem 2003;278:29655–60.
35. Peng T, Golub TR, Sabatini DM. The immunosup-
pressant rapamycin mimics a starvation-like signal
Cancer Res 2006; 66: (20). October 15, 2006