Suppression of inhibitor of differentiation 2, a target of mutant p53, is required for gain-of-function mutations

Abstract

Overexpression of mutant p53 is a common theme in human tumors, suggesting a tumor-promoting gain-of-function for mutant p53. To elucidate whether and how mutant p53 acquires its gain-of-function, mutant p53 is inducibly knocked down in the SW480 colon cancer cell line, which contains mutant p53(R273H/P309S), and the MIA PaCa-2 pancreatic cancer cell line, which contains mutant p53(R248W). We found that knockdown of mutant p53 markedly inhibits cell proliferation. In addition, knockdown of mutant p53 sensitizes tumor cells to growth suppression by various chemotherapeutic drugs. To determine whether a gene involved in cell growth and survival is regulated by mutant p53, gene expression profiling analysis was performed and showed that the expression level of Id2, a member of the inhibitor of differentiation (Id) family, was markedly increased upon knockdown of mutant p53. To confirm this, Northern blot analysis was performed and showed that the expression level of Id2 was regulated by various mutant p53s in multiple cell lines. In addition, we found that the Id2 promoter is responsive to mutant but not wild-type p53, and mutant p53 binds to the Id2 promoter. Consistent with these observations, expression of endogenous Id2 was found to be inhibited by exogenous mutant p53 in p53-null HCT116 cells. Finally, we showed that knockdown of Id2 can restore the proliferative potential of tumor cells inhibited by withdrawal of mutant p53. Together, these findings suggest that one mechanism by which mutant p53 acquires its gain-of-function is through the inhibition of Id2 expression.

Figure 1. Knockdown of mutant p53 inhibits cell proliferation

(A) Generation of SW480 (left) and MIA-PaCa-2 (middle) cell lines in which mutant p53 can be inducibly knocked down. Western blots were prepared with extracts from SW480 and MIA-PaCa-2 cells, that were uninduced (−) or induced (+) to knock down mutant p53 for 3 days, and then probed with antibodies against p53 and actin, respectively. Right panel: scrambled shRNA and treatment with tetracycline have no effect on mutant p53 expression. Western blots were prepared with extracts from SW480 cells transfected with a scrambled shRNA, mock-treated (−), or treated with tetracycline for 3 d, and then probed with antibodies against p53 and actin, respectively. (B) Left and middle panels: knockdown of mutant p53 inhibits cell proliferation and sensitizes SW480 cells to camptothecin (CPT). Right panel: treatment with tetracycline has little if any effect on cell proliferation for parental SW480 cells regardless of CPT treatment. (C) Left panel: knockdown of mutant p53 in SW480 cells inhibits colony formation in the absence and presence of camptothecin treatment. Top right panel: knockdown of mutant p53 in SW480 cells leads to cell cycle arrest in G1. SW480 cells were uninduced or induced to knock down mutant p53 for 3 days, and then both floating dead cells in the medium and live cells on the plate were collected, and stained with propidium iodide for DNA histogram analysis. Bottom right panel: tetracycline treatment alone has no effect on the cell cycle in parental SW480 cells. (D) Knockdown of mutant p53 in MIA-PaCa-2 cells inhibits cell proliferation (left panel) and colony formation (right panel).

Figure 2. Knockdown of mutant p53 up-regulates Id2 expression

(A) Northern blots were prepared with total RNAs isolated from SW480 and MIA-PaCa-2 cells that were uninduced (−) or induced (+) to knock down mutant p53 for 3 days. The blots were probed with cDNAs derived from the Id2 and GAPDH genes, respectively. The level of GAPDH mRNA was measured as a loading control. (B) Western blots were prepared with extracts from MIA-PaCa-2 cells transfected with scrambled shRNA and from MIA-PaCa-2 and SW480 cells uninduced or induced to knock down mutant p53 for 3 days, and then probed with antibodies against p53, Id2, p21, and actin, respectively. (C) Id2 protein is induced and localized in nucleus. SW480 cells were uninduced or induced to knockdown mutant p53 for 3 days and immunofluorescent staining of SW480 cells with DAPI and anti-Id2 was performed as described in the Materials and Methods.

Figure 3. Over-expression of mutant p53 can inhibit expression of endogenous Id2 in p53-null HCT116 cells

(A) Western blots were prepared with extracts from p53-null HCT116 cells induced to express mutant R175H for 0, 2, 4, or 6 days and then probed with antibodies against p53 and actin, respectively. (B) Over-expression of mutant R175H inhibits endogenous Id2 expression. Left panel: Northern blots were prepared with total RNAs purified from p53-null HCT116 cells induced to express mutant p53 for 0–6 days and probed with cDNAs derived from the Id2 and GAPDH genes, respectively. Right panel: real-time PCR was performed to quantify the levels of Id2 transcripts in HCT116(p53−/−) cells inducibly expressing mutant p53 (R175H) at day 0, 4, and 6.

Figure 4. The Id2 promoter is responsive to mutant but not wild-type p53

(A) Schematic presentation of the Id2 (left) and p21 (right) promoters and luciferase reporter constructs. (B) The Id2 promoter is responsive to mutant p53. Left panel: the luciferase activity under the control of various Id2 promoter fragments was measured in the presence or absence of wild-type p53 or mutant R249S. Right panel: the Id2 promoter is responsive to mutant R175H and R273H in addition to R249S. The response of the p21 promoter to these p53 mutants was measured as a control. (C) Location of a potential mutant p53 binding site in the Id2 gene (left), the p53-REs in the p21 gene (middle), and the GAPDH promoter (right) along with the location of PCR primers used for ChIP assay. (D) Left panel: mutant p53 in SW480 cells binds to the Id2 promoter. Mutant p53-DNA complexes were captured with anti-p53 antibody along with rabbit IgG as a control. The binding of mutant p53 protein to the p21 and GAPDH promoters was measured as a control. Right panel: mutant p53 in MIA-PaCa-2 cells binds to the Id2 promoter.

Figure 5. Stable knockdown of Id2 restores the proliferative potential of SW480 cells upon inducible knockdown of mutant p53

(A) Identification of inducible mutant p53 knockdown cell lines. Western blots were prepared with extracts from SW480 cells uninduced (−) or induced (+) to knock down mutant p53 for 3 days, and then probed with antibodies against p53 and actin, respectively. (B) Identification of stable Id2 knockdown cell lines. Northern blots were prepared with total RNAs isolated from positive clones identified in (A) that were uninduced or induced to knock down mutant p53 and probed with cDNAs derived from the Id2 and GAPDH genes, respectively. (C) Stable knockdown of Id2 restores the proliferation potential of SW480 cells triggered by mutant p53 knockdown. The growth rate of SW480 cells with stable Id2 knockdown in the presence or absence of mutant p53 knockdown was measured over a 12-day testing period. (D) Left panel: stable knockdown of Id2 restores the colony-forming potential of SW480 cells upon knockdown of mutant p53. Colony formation assay was performed with stable Id2 knockdown SW480 cells uninduced or induced to knock down mutant p53. Right panel: stable knockdown of Id2 confers SW480 cells resistance to camptothecin treatment.