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Novel therapeutic interventions for p53-altered tumors through manipulation of its family members, p63 and p73 - PubMed

Novel therapeutic interventions for p53-altered tumors through manipulation of its family members, p63 and p73

Avinashnarayan Venkatanarayan et al. Cell Cycle. 2016.

Abstract

TP53 is highly mutated in human cancers, thus targeting this tumor suppressor pathway is highly desirable and will impact many cancer patients. (1,2) Therapeutic strategies to reactivate the p53-pathway have been challenging, (3,4) and no effective treatment exists. (5) We utilized the p53-family members, p63 and p73, which are not frequently mutated in cancer, to treat p53-defective cancers. The N-terminal splice variants of p63 and p73 are denoted as the TA and ΔN isoforms. We recently demonstrated that deletion of either ΔNp63 or ΔNp73 in p53-deficient mouse tumors results in tumor regression mediated by metabolic programming. Using this strategy, we identified pramlintide, a synthetic analog of amylin, as an effective treatment for p53 deficient and mutant tumors. Here, we show the utility of using pramlintide, as a potential cancer preventive option for p53-deficient tumors in mouse models. Additionally, we found that in vivo inhibition of both ΔNp63 and ΔNp73 in combination accelerates tumor regression and increases survival of p53-deficient mice. We report that inhibition of both ΔNp63 and ΔNp73 in combination results in upregulation of 3 key metabolic regulators, IAPP, GLS2, and TIGAR resulting in an increase in apoptosis and tumor regression in ΔNp63/ΔNp73/p53 deficient thymic lymphomas. These data highlight the value of generating inhibitors that will simultaneously target ΔNp63 and ΔNp73 to treat cancer patients with alterations in p53.

Keywords: p53 family; targeted therapy; tumor suppressors.

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Figures

Figure 1.
Figure 1.

Mutational status of p53, p63 and p73 in human cancers. Graphical representation of the frequency of p53 (red), p63 (blue) and p73 (red) mutations in human cancer. Mutation data was obtained from cbioportal, which represents multiple sequencing datasets from human cancers.

Figure 2.
Figure 2.

CALCR expression in patient cohort with p53-mutations. Graphical representation of expression of CALCR in patients with p53-mutations in lung squamous cell carcinoma (LUSC) (a) and head and neck squamous cell carcinoma HNSCC (b).

Figure 3.
Figure 3.

Combined deletion of ΔNp63 and ΔNp73 accelerated tumor regression in p53-deficient thymic lymphomas. Magnetic Resonance Imaging (MRI) of thymic lymphomas of mice of the indicated genotypes treated with Adenovirus (Ad)-mCherry (a-d) or Ad-Cre-mCherry (e-h, i-l and m-p). Age of mice is indicated across the top of each panel and the tumor volume is indicated (mm3) within each panel. UN-D indicates that the tumor was undetectable. The tumors are indicated by the dashed yellow line. Measurements of thymic lymphomas in mice of the indicated genotypes treated with Ad-mCherry (q) or Ad-Cre-mCherry (r-t), n = 3 to 5 mice per treatment followed over 8 weeks post infection. Kaplan Meier thymic lymphoma free survival curve of ΔNfl/fl;p53−/−, ΔNp63Δ/Δ;p53−/−, ΔNp73Δ/Δ;p53−/− orΔNp63Δ/Δ;ΔNp73Δ/Δ;p53−/− mice treated at 10 weeks with Ad-mCherry or Ad-Cre-mCherry to delete the ΔN isoforms of p63 and p73 (u), n = 3 – 5 mice, p<0.005. Boxed numbers on x-axis represent median time (weeks) of survival for the indicated genotypes.

Figure 4.
Figure 4.

ΔNp63 and ΔNp73 deletion mediates upregulation of metabolic regulators resulting in ROS accumulation and apoptosis in p53-deficient thymic lymphomas. Western blot analysis using the indicated antibodies and lysates of mice of the indicated genotypes 48 hours after treatment with Adenovirus (Ad)-mCherry or Ad-Cre-mCherry (a). Quantitative real time (qRT-PCR) of thymic lymphomas treated 48 hours after treatment with Ad-mCherry (ΔNfl/fl;p53−/−) or Ad-Cre-mCherry (ΔNp63Δ/Δ;p53−/− or ΔNp73Δ/Δ;p53−/− orΔNp63Δ/Δ;ΔNp73Δ/Δ;p53−/−) for the indicated target genes (b-d). n = 3, p<0.05. Immunohistochemistry for ROS, cleaved caspase 3 and PCNA in thymic lymphomas 48 hours after treatment with Ad-mCherry (ΔNfl/fl;p53−/−) or Ad-Cre-mCherry (ΔNp63Δ/Δ;p53−/− or ΔNp73Δ/Δ;p53−/− orΔNp63Δ/Δ;ΔNp73Δ/Δ;p53−/−) (e). Bar graph indicating quantification of cleaved caspase 3 (f) and PCNA (g) immunostaining.

Figure 5.
Figure 5.

Administration of pramlintide prior to thymic lymphoma formation in p53-deficient mice. Schedule of MRI imaging and injection (Inj.) of Pramlintide acetate in mice with p53 deficient thymus. MRI imaging at 10, 11, 12, and 13 weeks after treatment with placebo (b-h) or Pramlintide acetate (i-q'). Tumor volume is indicated (mm3) within each panel. The tumors are indicated by the dashed yellow line. Days of the week are shown as Monday (M), Wednesday (W), Friday (F). Blood glucose (B/G) levels were assessed every Wednesday at the time of MRI.

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