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. 2009 Sep;83(17):8565-74.
doi: 10.1128/JVI.00603-09. Epub 2009 Jun 10.

Simian virus 40 small T antigen activates AMPK and triggers autophagy to protect cancer cells from nutrient deprivation

Sravanth Hindupur Kumar  1 , Annapoorni Rangarajan
Affiliations

Simian virus 40 small T antigen activates AMPK and triggers autophagy to protect cancer cells from nutrient deprivation

Sravanth Hindupur Kumar et al. J Virol. 2009 Sep.

Abstract

As tumors grow larger, they often experience an insufficient supply of oxygen and nutrients. Hence, cancer cells must develop mechanisms to overcome these stresses. Using an in vitro transformation model where the presence of the simian virus 40 (SV40) small T (ST) antigen has been shown to be critical for tumorigenic transformation, we investigated whether the ST antigen has a role to play in regulating the energy homeostasis of cancer cells. We find that cells expressing the SV40 ST antigen (+ST cells) are more resistant to glucose deprivation-induced cell death than cells lacking the SV40 ST antigen (-ST cells). Mechanistically, we find that the ST antigen mediates this effect by activating a nutrient-sensing kinase, AMP-activated protein kinase (AMPK). The basal level of active, phosphorylated AMPK was higher in +ST cells than in -ST cells, and these levels increased further in response to glucose deprivation. Additionally, inhibition of AMPK in +ST cells increased the rate of cell death, while activation of AMPK in -ST cells decreased the rate of cell death, under conditions of glucose deprivation. We further show that AMPK mediates its effects, at least in part, by inhibiting mTOR (mammalian target of rapamycin), thereby shutting down protein translation. Finally, we show that +ST cells exhibit a higher percentage of autophagy than -ST cells upon glucose deprivation. Thus, we demonstrate a novel role for the SV40 ST antigen in cancers, where it functions to maintain energy homeostasis during glucose deprivation by activating AMPK, inhibiting mTOR, and inducing autophagy as an alternate energy source.

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Figures

FIG. 1.
FIG. 1.
Effect of glucose deprivation on the survival of +ST and −ST cells. (a) Phase-contrast microscopic images of +ST and −ST cells in the presence and absence of glucose. (b) Graph representing quantification of trypan blue-positive (dead) cells upon glucose deprivation. (c) Photomicrographs showing fluorescent images of glucose-deprived cells stained with Hoechst 33342 to detect apoptosis. (d) Graph representing quantification of Hoechst 33342-positive (apoptotic) cells upon glucose deprivation. (e) Immunoblot analysis of whole-cell lysates of glucose-deprived +ST and −ST cells for cleaved PARP using an antibody that specifically recognizes the large fragment of cleaved PARP protein. Doxorubicin (Dox)-treated HeLa cells served as a positive control. (f) Phase-contrast microscopic images of HEK+ST and HEK−ST cells in the presence and absence of glucose. (g) Graph representing quantification of trypan blue-positive (dead) cells upon glucose deprivation (n = 6). ***, P < 0.001; **, P value between 0.001 and 0.01. In all experiments, statistical significance was determined using one-way analysis of variance. Error bars, standard errors of the means (n = 3).
FIG. 2.
FIG. 2.
OA mimics the effects of the ST antigen. (a) Phase-contrast microscopic images of +ST and −ST cells in the presence and absence of OA. (b) Quantification of trypan blue-positive cells in the presence and absence of OA. Dimethyl sulfoxide (DMSO) was used as a vehicle control (n = 3). (c) Reverse transcription-PCR analysis for ΔST 1-100 in HEK−ST and HEKΔST 1-100 cells. (d) Phase-contrast microscopic images of HEK+ST, HEK−ST, and HEKΔST 1-100 cells in the presence and absence of glucose. (e) Quantification of trypan blue-positive cells in the presence and absence of glucose. ***, P < 0.001; ns, nonsignificant.
FIG. 3.
FIG. 3.
Involvement of AMPK in ST antigen-mediated protection under glucose-deprived conditions. (a) Whole-cell extracts from +ST and −ST cells in the presence and absence of glucose were subjected to immunoblot analysis for pAMPK (Thr172), total AMPK, and pACC (Ser79). α-Tubulin was used as a loading control for all immunoblots. (b) Whole-cell extracts of HEK+ST and HEK−ST cells in the presence and absence of glucose were subjected to immunoblot analysis for pAMPK (Thr172) and total AMPK. (c) Whole-cell extracts from +ST cells deprived of glucose and treated with compound C (Comp C) were subjected to immunoblot analysis for pACC (Ser79). (d) Phase-contrast microscopic images of +ST and −ST cells in the presence and absence of compound C. (e) Quantification of trypan blue-positive cells in the presence and absence of compound C (n = 3). (f) Whole-cell extracts of +ST and −ST cells deprived of glucose in the presence and absence of AICAR were subjected to immunoblot analysis for pAMPK (Thr172), total AMPK, and pACC (Ser79). (g) Phase-contrast microscopic images of +ST and −ST cells in the presence and absence of AICAR. (h) Quantification of trypan blue-positive cells in the presence and absence of AICAR (n = 3). (i) Whole-cell extracts of −ST cells deprived of glucose and treated with OA were subjected to immunoblot analysis for pAMPK (Thr172) and pACC (Ser79). *, P value between 0.01 and 0.05; ***, P < 0.001.
FIG. 4.
FIG. 4.
Involvement of mTOR in ST antigen-AMPK-mediated rescue. (a) Immunoblot analysis of whole-cell extracts of glucose-deprived +ST and −ST cells for phospho-p4EBP1 (P-p4EBP1) (Ser65). (b) Immunoblot analysis of whole-cell extracts of rapamycin-treated −ST cells for P-p4EBP1 (Ser65). (c) Photomicrographs represent phase-contrast images of +ST and −ST cells treated with rapamycin. (d) Quantification of trypan blue-positive +ST and −ST cells upon glucose deprivation and rapamycin treatment (n = 3). ***, P < 0.001. (e) Whole-cell extracts of glucose-deprived +ST and −ST cells were subjected to immunoblot analysis for pAMPK (Thr172), total AMPK, pACC (Ser79), total ACC, phospho-raptor (pRaptor) (Ser792), and total raptor. (f) Whole-cell extracts of glucose-deprived +ST and −ST cells were subjected to immunoblot analysis for pAMPK (Thr172), pACC (Ser79), phosphorylated mTOR (pmTOR) (Ser2448), and total mTOR.
FIG. 5.
FIG. 5.
Autophagy in +ST and −ST cells upon glucose deprivation. (a) eGFP-LC3 localization in +ST and −ST cells upon glucose deprivation and rapamycin treatment. (b) Quantification of eGFP-LC3 punctate cells among +ST and −ST cells upon glucose deprivation and rapamycin treatment (n = 8). ***, P < 0.001. (c) Immunoblot analysis of whole-cell extracts for the cleaved LC3 fragment upon glucose deprivation.
FIG. 6.
FIG. 6.
Schematic of tumor development. As incipient tumors (or micrometastases) grow beyond the homeostatic limit (∼150 μM), they often experience insufficient supplies of oxygen and nutrients. Tumor cells must overcome this stress before replenishment arrives by way of neoangiogenesis. Our data show that ST antigen-mediated induction of autophagy can be one such mechanism that provides an alternate source of energy for tumor growth and the establishment of metastases. Thus, pathways activating autophagy can be targeted for cancer therapy.
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