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. 2011 Feb 18;144(4):499-512.
doi: 10.1016/j.cell.2011.01.017.

Identification of aneuploidy-selective antiproliferation compounds

Yun-Chi Tang  1 , Bret R Williams, Jake J Siegel, Angelika Amon
Affiliations

Identification of aneuploidy-selective antiproliferation compounds

Yun-Chi Tang et al. Cell. .

Abstract

Aneuploidy, an incorrect chromosome number, is a hallmark of cancer. Compounds that cause lethality in aneuploid, but not euploid, cells could therefore provide new cancer therapies. We have identified the energy stress-inducing agent AICAR, the protein folding inhibitor 17-AAG, and the autophagy inhibitor chloroquine as exhibiting this property. AICAR induces p53-mediated apoptosis in primary mouse embryonic fibroblasts (MEFs) trisomic for chromosome 1, 13, 16, or 19. AICAR and 17-AAG, especially when combined, also show efficacy against aneuploid human cancer cell lines. Our results suggest that compounds that interfere with pathways that are essential for the survival of aneuploid cells could serve as a new treatment strategy against a broad spectrum of human tumors.

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Figures

Figure 1
Figure 1. AICAR inhibits proliferation in trisomic MEFs
(A) Wild-type (filled symbols) and trisomic primary (open symbols) MEFs were grown for 72 hours either in the absence (circles) or presence (0.2 mM, triangles; 0.5 mM, squares) of AICAR and cell number was determined at the indicated times. (B) Cell number of wild-type (filled bars) and trisomic cells (open bars) was determined after 3 days and is shown as the percentage of the untreated control. The data in this and all subsequence figures are shown as the mean ± standard deviation. *P<0.05, **P<0.005, t test.
Figure 2
Figure 2. The proteotoxic compounds 17-AAG and chloroquine exaggerate the anti-proliferative effects of AICAR
(A, B) Wild-type (filled bars) and trisomic cells (open bars) were treated with the indicated concentrations of 17-AAG (A) or chloroquine (B) and cell number was determined after 3 days. (C, D) Cells were treated with 0.2 mM AICAR and the indicated concentrations of 17-AAG (C) or chloroquine (D). Cell number was determined after 3 days. *P<0.05, **P<0.005, t test.
Figure 3
Figure 3. AICAR, 17-AAG and chloroquine induce apoptosis in trisomic MEFs
(A) Wild-type (top) and trisomy 1 cells (bottom) were treated with AICAR for 24 hours and apoptosis was measured using annexin V-FITC/ PI staining. Early apoptotic cells are found in the bottom right quadrant. (B, C) Quantification of the percentage of annexin V-FITC positive, PI negative cells in wild-type, trisomy 1 and trisomy 13 cultures 24 hours after AICAR treatment (B) and in wild-type and trisomy 13 cultures 24 hours after 17-AAG or chloroquine treatment (C). (D) Wild-type and trisomy 13 cells were treated with 0.2 mM AICAR and p53 Serine 15 phosphorylation, and p53 and p21 protein levels were analyzed. Quantifications of the ratio of phosphorylated p53/actin protein are shown underneath the P-p53 blot. The ratios were normalized to untreated wild-type cells. Asterisk denotes S15 phosphorylated p53. (E) Wild-type and trisomy 13 cells were treated with 0.2 or 0.5 mM AICAR for 24 hours. Equal amounts of cytoplasmic or mitochondrial protein extracts were probed for the presence of Bax by immunoblotting. Mitochondrial Hsp60 served as loading control in mitochondrial extracts. Quantifications of the ratio of mitochondrial Bax/total Bax protein normalized to untreated wild-type cells are shown underneath the mitochondrial Bax blot. (F) p53 knockdown efficiency revealed by immunoblotting using an anti-p53 antibody. Actin serves as a loading control in Western blots. (G) Cells were transfected with a p53 knockdown shRNA and treated with AICAR for 24 hours at the indicated doses. *P<0.05, **P<0.005, t test.
Figure 4
Figure 4. AICAR antgonizes proliferation of trisomic MEFs in an AMPK-dependent manner
(A) AMPKα knock down efficiency revealed by immunoblotting using an anti-AMPK antibody. (B) Cells infected with either empty vector or an AMPKα knockdown construct were counted 24 hours after AICAR treatment. (C) Wild-type (filled bars) and trisomic (open bars) cells were treated with the indicated concentrations of compound C for 3 days. Even though the effects of compound C were less severe in trisomic cells than euploid controls, it is important to note that the treated trisomic cells grew poorly compared to euploid control cells. (D) Wild-type (filled bars) and trisomic cells (open bars) were treated with 0.5 mM AICAR and compound C at the indicated doses for 3 days and cell number was counted. (E, F) AMPK activity was analyzed by determining the extent of threonine172 phosphorylation on AMPK (E) or by in vitro kinase assays using the substrate peptide, IRS-1 S789 (F) in wild-type and trisomic cells after 24 hours of AICAR treatment. Quantifications of the ratio of phosphorylated AMPK/total AMPK protein normalized to untreated wild-type cells are shown underneath the P-AMPK blot. (G) AMPK activity was measured by in vitro kinase assays at the indicated time point following addition of 0.2 mM AICAR. *P<0.05, **P<0.005, t test.
Figure 5
Figure 5. AICAR exaggerates the stressed state of trisomic MEFs
(A) Lipidated LC3-II was analyzed by immunoblotting in wild-type and trisomy 13 and 16 cells after 24 hours of AICAR treatment. Quantifications of the ratio of lapidated LC3II /actin protein normalized to untreated wild-type cells are shown underneath the LC3-II blot. (B) Quantitative RT-PCR analysis of mRNA abundance of the autophagy genes ATG1, ATG4, Beclin1, LC3, Bnip3 and GAPRAPL1. mRNA levels were quantified in untreated wild-type (black bars) and trisomic (white bars) cells as well as wild-type (grey bars) and trisomic (blue bars) cells treated with 0.5 mM AICAR for 24 hours. RNA levels were normalized to those of the ribosomal RPL19 gene. (C) The extent of autophagy was quantified by determining the number of LC3-GFP puncta in cells. Typical images are shown as examples for LC3-GFP puncta formation in trisomy 13 and 16 and wild-type cells after AICAR treatment (left). Incubation in HBSS induces acute starvation and served as a positive control. 24 hours after AICAR treatment, the number of cells that harbor more than 4 LC3-GFP puncta was determined (right). *P<0.05, **P<0.005, t test. (D) Wild-type and trisomic MEFs were treated with AICAR at the indicated doses and levels of inducible Hsp72 were determined by immunoblotting. Quantifications of the ratio of inducible Hsp72/Hsp90 protein normalized to untreated wild-type cells are shown underneath the Hsp72 blot.
Figure 6
Figure 6. AICAR and 17-AAG inhibit the proliferation of MEFs with decreased chromosome segregation fidelity and of aneuploid human cancer cells
(A, B) Wild-type (filled bars) and Bub1bH/H cells (open bars; A), or wild-type (filled bars) and Cdc20AAA cells (open bars; B) were treated with the indicated concentrations of AICAR, 17-AAG or both, and cell number was determined after 3 days. *P<0.05, **P<0.005, t test. (C) Cells were treated with the indicated concentration of AICAR (top) or 17-AAG (center) or both compounds (bottom). Cell number was determined 3 days after the addition of compound and is shown as the percentage of the untreated control. Primary euploid cells (black symbol), MIN colon cancer cell lines (blue, green symbols) and aneuploid CIN colon cancer cells (red, purple symbols) were analyzed. (D) Cell number of euploid (black symbols) and aneuploid lung cancer cells (red, purple symbols) was determined after 3 days of treatment with the indicated compounds and is shown as the percentage of the untreated control. The data presented are the mean and the P value results of t test. NS, not significant.
Figure 7
Figure 7. AICAR and 17-AAG inhibit growth of human colon cancer cells in xenografts
(A) Mice were implanted with 4 ×106 MIN cells on the left flank and with the same number of CIN cells on the right flank. Seven days after injection (indicated by the arrow) mice were treated with daily i.p. injections of AICAR, 17-AAG, both or PBS. Tumor volume (mm3) was measured at the indicated time points and shown as mean tumor volumes. (B) Mice treated with PBS (left) or AICAR+17AAG (right) 25 days after transplantation. (C) Quantification of the percentage of annexin V-FITC positive, PI negative cells in wild-type, MIN and CIN cell cultures 24 hours after AICAR or AICAR+17-AAG treatment. *P<0.05, **P<0.005, t test.
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