Figure 5. FOXO3a mediated upregulation of PUMA changes the mode of action of an anti-cancer drug.

(A) Publicly available gene expression data set of cancer cell lines from multiple tumor types via Oncomine shows that cell lines sensitive to Nutlin (IC₅₀ < 5 μM) have increased expression of BBC3/PUMA. Values were Log2 transformed and the median values were scaled to zero according to Garnett et al. 2012. See also Figure S5.

(B) HCT116 cells were treated with vehicle, bafilomycin A1 10nM, chloroquine 40μM, Nutlin 20μM—an MDM2 inhibitor that activates p53, Nutlin + bafilomycin A1, or Nutlin + chloroquine for 24hrs and the resulting BBC3/PUMA mRNA levels were measured relative to 18s rRNA control.

(C) Clonogenic survival assay using HCT116 cells treated for 72hrs with vehicle, chloroquine 40μM, Nutlin 20μM, or Nutlin + chloroquine. Cells were then allowed to grow back for 7 days in full media.

(D) HCT116 cells were treated with vehicle, chloroquine 40μM, Nutlin 20μM, or Nutlin + chloroquine. Cell permeability by Sytox green (an indicator of cell death) was measured and normalized to cell number per mm².

(E) Representative images from panel D of cell permeability by Sytox green in HCT116 parental cells treated with vehicle, chloroquine 40μM, Nutlin 20μM, or Nutlin + chloroquine.

(F) HCT116 cells were treated with vehicle, chloroquine 40μM, Nutlin 20μM, or Nutlin + chloroquine. Caspase 3/7 activation (an indicator of apoptosis execution) was measured and normalized to cell number per mm². See also Figure S5.

(G) Representative images of caspase 3/7 activation from panel F in HCT116 parental cells treated with vehicle, chloroquine 40μM, Nutlin 20μM, or Nutlin + chloroquine.

(H) HCT116 parental cells or ΔFHRE cells lacking the endogenous Forkhead Response Element in the BBC3/PUMA locus were treated with Nutlin 20μM + chloroquine 40μM. Caspase 3/7 activation was measured and normalized to cell number per mm². See also Figure S5.

(I) Representative images of caspase 3/7 activation from panel H in HCT116 parental cells or ΔFHRE cells lacking the Forkhead Response Element in the endogenous BBC3/PUMA locus treated with Nutlin 20μM + chloroquine 40μM.

(J) Athymic nude mice given subcutaneous parental or ΔFHRE HCT116 tumors were treated with vehicle, Nutlin, chloroquine, or Nutlin + chloroquine and PUMA mRNA levels in the tumors were measured relative to 18s rRNA control.

(K) Kaplan-Meier survival curves based on tumor burden of athymic nude mice given subcutaneous parental or ΔFHRE HCT116 tumors and treated with vehicle, Nutlin, chloroquine, or Nutlin + chloroquine over indicated duration.

(L) Schematic representation of global model when autophagy is perturbed. Upon genetic or pharmacological basal autophagy inhibition, autophagy homeostasis is perturbed and FOXO3a becomes transcriptionally active. Both autophagy genes and pro-apoptotic proteins like PUMA are transcriptionally activated to compensate for the autophagy perturbation. If homeostasis is not re-established, cells undergo apoptosis. Statistical significance in A was determined by unpaired t-test, one-way ANOVA (Tukey post-hoc) in B, a repeated measures two-way ANOVA (Tukey post-hoc) in D, F, H, a two-way ANOVA in J, and five individual comparisons using Log-rank (Mantel-Cox) test in K. Due to five individual comparisons in K, p=0.05 ÷ 5= 0.01 is the corrected threshold for statistical significance. *P<0.01 for KM survival curves; n.s.=not significant. *P<0.05; **P<0.01; ***P<0.001 for all others. Data represented as mean ± SEM