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IAPP-driven metabolic reprogramming induces regression of p53-deficient tumours in vivo

Abstract

TP53 is commonly altered in human cancer, and Tp53 reactivation suppresses tumours in vivo in mice1,2 (TP53 and Tp53 are also known as p53). This strategy has proven difficult to implement therapeutically, and here we examine an alternative strategy by manipulating the p53 family members, Tp63 and Tp73 (also known as p63 and p73, respectively). The acidic transactivation-domain-bearing (TA) isoforms of p63 and p73 structurally and functionally resemble p53, whereas the ΔN isoforms (lacking the acidic transactivation domain) of p63 and p73 are frequently overexpressed in cancer and act primarily in a dominant-negative fashion against p53, TAp63 and TAp73 to inhibit their tumour-suppressive functions3,4,5,6,7,8. The p53 family interacts extensively in cellular processes that promote tumour suppression, such as apoptosis and autophagy9,10,11,12,13,14, thus a clear understanding of this interplay in cancer is needed to treat tumours with alterations in the p53 pathway. Here we show that deletion of the ΔN isoforms of p63 or p73 leads to metabolic reprogramming and regression of p53-deficient tumours through upregulation of IAPP, the gene that encodes amylin, a 37-amino-acid peptide co-secreted with insulin by the β cells of the pancreas. We found that IAPP is causally involved in this tumour regression and that amylin functions through the calcitonin receptor (CalcR) and receptor activity modifying protein 3 (RAMP3) to inhibit glycolysis and induce reactive oxygen species and apoptosis. Pramlintide, a synthetic analogue of amylin that is currently used to treat type 1 and type 2 diabetes, caused rapid tumour regression in p53-deficient thymic lymphomas, representing a novel strategy to target p53-deficient cancers.

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Figure 1: In vivo deletion of ΔNp63 or ΔNp73 in p53-deficient mice suppresses lymphomagenesis.
Figure 2: IAPP is causally involved in tumorigenesis suppression in p53-deficient thymic lymphomas.
Figure 3: IAPP inhibits glycolysis and induces ROS and apoptosis in p53-deficient human cancer cell lines.
Figure 4: Calcitonin and RAMP3 receptors are required for secreted IAPP to suppress tumorigenesis.

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Gene Expression Omnibus

Data deposits

The RNA-Seq data has been deposited in the Gene Expression Omnibus (GEO) data repository and can be accessed using database accession number GSE60827.

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Acknowledgements

We thank A. Jain, V. Pant, J. Jackson, A. Marisetty, K. Michel and the Small Animal Imaging Facility (SAIF) for technical advice. This work was supported by grants to E.R.F. from NCI (R01CA160394) and (R01CA134796), CPRIT (RP120124), NCI-Cancer Center Core Grant (CA-16672) (University of Texas M.D. Anderson Cancer Center), a development award from the Lymphoma SPORE (P50CA136411), the Hildegardo E. and Olga M. Flores Foundation, and the Mel Klein Foundation and grant to J.A.B. from CPRIT (RP101243-P5). E.R.F. is a scholar of the Leukemia and Lymphoma Society, the Rita Allen Foundation and the V Foundation for Cancer Research. A.V. is a Schissler Scholar and D.C. is a CPRIT Scholar (RP101502).

Author information

Authors and Affiliations

Authors

Contributions

A.V. and E.R.F. conceived the study, designed experiments and analysed data. A.V., P.R., W.N., D.C., X.S., S.K.S., M.S.R., J.L., C.V.K., E.F.S., K.N., J.P.-T., J.A.B. and K.Y.T. designed and performed experiments. P.H.G., C.C. and K.R. performed bioinformatic analyses. E.R.F. and A.V. wrote the paper. All authors discussed the paper and commented on the manuscript.

Corresponding author

Correspondence to Elsa R. Flores.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Generation and characterization of ΔNp73 conditional knockout mice.

a, The ΔNp73 targeting vector was generated by inserting loxP sites (triangles) flanking exon 3′ and a neomycin cassette (neo) flanked by frt sites (squares). The location of PCR primers in each allele is shown by blue arrows. The targeted region of the floxed allele is depicted by yellow-dashed lines. b, Southern blot analysis using the 5′ probe shown in a and tail genomic DNA derived from mice of the indicated genotypes. c, PCR analysis using tail genomic DNA of the indicated genotypes. d, Western blot analysis using mouse embryo fibroblasts (MEFs) of the indicated genotypes. e, f, qRT–PCR in MEFs of the indicated genotypes, n = 4, P < 0.005. Statistical significance is indicated by black asterisks.

Source data

Extended Data Figure 2 Decreased thymic lymphomagenesis and increased survival in mice double deficient for ΔNp63 and p53 or ΔNp73 and p53.

a, Quantification of thymic lymphoma incidence (n = 30 mice). b, Table showing thymic lymphoma volumes. The difference in tumour volumes between p53−/− and ΔNp63+/−;p53−/− and p53−/− and ΔNp73−/−;p53−/− was statistically significant with P values < 0.03 and < 0.002, respectively. c, Kaplan–Meier survival in mice. Boxed numbers indicate median survival. d, e, Western blot analysis of thymic lymphomas of the indicated genotypes. Arrows indicate specific isoforms, and asterisks indicate non-specific bands. fh, qRT–PCR for PUMA (f), Noxa (g), and bax (h) in thymic lymphomas of the indicated genotypes, n = 4, P < 0.005. i, Immunohistochemistry (IHC) for cleaved caspase 3 in thymic lymphomas. j, Quantification of apoptosis as assessed by cleaved caspase 3 staining, n = 20 fields of 3 biological replicates, P < 0.005. km, qRT–PCR for PML (k), p16 (l), and p21 (m) in indicated thymic lymphomas, n = 4, P < 0.005. n, IHC for PCNA in indicated thymic lymphomas. o, Quantification of the percentage of proliferation as assessed by PCNA staining, n = 20 fields of 3 biological replicates, P < 0.005. Statistical significance indicated by black asterisks.

Source data

Extended Data Figure 3 Increased apoptosis and cell cycle arrest in ΔNp63+/−;p53−/− and ΔNp73−/−;p53−/− thymocytes after genotoxic stress.

a, Western blot analysis in thymocytes derived from mice 6 h after treatment with 0 Gy or 10 Gy gamma irradiation. bf, qRT–PCR for TAp63 (b), TAp73 (c), PUMA (d), Noxa (e), and bax (f) from samples shown in a, n = 4, P < 0.005. qRT–PCR normalized to samples treated with 0 Gy. g, Immunohistochemistry (IHC) for cleaved caspase 3 in samples from a. h, Quantification of the percentage of apoptosis as assessed by cleaved caspase 3 staining, n = 20 fields of 3 biological replicates, P < 0.005. ik, qRT–PCR for PML (i), p16 (j), and p21 (k) using total RNA from samples shown in a, n = 4, P < 0.005. l, IHC for PCNA in samples shown in a. m, Quantification of the percentage of proliferation as assessed by PCNA staining, n = 20 fields of 3 biological replicates, P < 0.005. Statistical significance is indicated by black asterisks.

Source data

Extended Data Figure 4 In vivo intra-thymic delivery of adenovirus-Cre-mCherry.

ac, IVIS Lumina imaging of thymic lymphomas of mice of the indicated genotypes infected with adenovirus (Ad)-mCherry (a) or Ad-Cre-mCherry (b, c) at 10 weeks of age and 48 h after adenoviral delivery. Red fluorescence indicates viral delivery to the thymus shown by the yellow dashed ovals. Red fluorescence near the mouth is due to auto-fluorescence of calcium and mineral deposits in the teeth. d, Western blot analysis using lysates from indicated thymic lymphomas 48 h after infection with adenovirus (Ad)-mCherry or Ad-Cre-mCherry. e, f, Quantitative real time (qRT–PCR) of thymic lymphomas 48 h after infection with Ad-mCherry (ΔNfl/fl;p53−/−) or Ad-Cre-mCherry (ΔNp63Δ/Δ;p53−/− or ΔNp73Δ/Δ;p53−/−), n = 4, P < 0.005. g, Immunohistochemistry (IHC) for cleaved caspase 3 in thymic lymphomas 48 h after infection with Ad-mCherry (ΔNfl/fl;p53−/−) or Ad-Cre-mCherry (ΔNp63Δ/Δ;p53−/− or ΔNp73Δ/Δ;p53−/−). h, Quantification of apoptosis as assessed by cleaved caspase 3 staining of the indicated thymic lymphomas, n = 20 fields of 3 biological replicates, P < 0.005. i, j, qRT–PCR of thymic lymphomas 48 h after treatment with Ad-mCherry (ΔNfl/fl;p53−/−) or Ad-Cre-mCherry (ΔNp63Δ/Δ;p53−/− or ΔNp73Δ/Δ;p53−/−), n = 4, P < 0.005. k, Senescence-associated β-galactosidase (SA-β-gal) staining (blue) of thymic lymphomas 48 h after treatment with Ad-mCherry (ΔNfl/fl;p53−/−) or Ad-Cre-mCherry (ΔNp63Δ/Δ;p53−/− or ΔNp73Δ/Δ;p53−/−). lo, Flow cytometry plots of the indicated thymocytes at 4-week of age. p, Bar graph showing quantification of CD4, CD8, and CD4/CD8 double-positive (DP) cells. n = 3 mice per genotype, P < 0.005. qs, Flow cytometry plots of thymic lymphoma cells 48 h after adenovirus-mCherry or adenovirus-CRE treatment for the indicated genotypes. t, Bar graph showing quantification of CD4, CD8, and CD4/CD8 double-positive (DP) cells in the indicated genotypes. n = 3 mice per genotype, P < 0.005. u, Cartoon representation of isolation of CD45-postive thymic lymphoma cells from 10-week-old mice of indicated genotypes. v, Western blot analysis of CD45-postive thymic lymphoma cells after treatment with Ad-mCherry (ΔNfl/fl;p53−/−) or Ad-CRE-mCherry (ΔNp63Δ/Δ;p53−/− and ΔNp73Δ/Δ;p53−/−). Statistical significance is indicated by black asterisks.

Source data

Extended Data Figure 5 Loss of ΔNp63/ΔNp73 induces TAp63 and TAp73 upregulation in the absence of p53.

a, Western blot analysis in ΔNp63fl/fl;p53−/− MEFs before (ΔNp63fl/fl;p53−/−) and after (ΔNp63Δ/Δ;p53−/−) Ad-Cre administration. b, c, qRT–PCR for ΔNp63 (b) and TAp63 (c) in indicated MEFs. d, Western blot analysis in ΔNp73fl/fl;p53−/− and ΔNp73Δ/Δ;p53−/− MEFs. e, f, qRT–PCR for ΔNp73 (e) and TAp73 (f) in indicated MEFs, n = 4, P < 0.005. g, Table showing ΔNp63 and ΔNp73 binding sites on the TAp63 and TAp73 promoter regions. h, i, qRT–PCR of chromatin immunoprecipitation using indicated MEFs and an antibody for p63 (h) or p73 (i) n = 3, P < 0.005. j, k, Western blot analysis in ΔNp63−/−;p53−/− (j) or ΔNp73−/−;p53−/− (k) MEFs treated with the indicated shRNAs; (shNT) indicates a non-targeting scramble shRNA. lq, qRT–PCR for PUMA (l), Noxa (m), bax (n), PML (o), p21 (p), and p16 (q) in the indicated MEFs expressing the indicated shRNAs, n = 5, P < 0.005. Statistical significance indicated by black asterisks.

Source data

Extended Data Figure 6 Metabolic genes including IAPP are upregulated in thymic lymphomas deficient for ΔNp63 or ΔNp73 and p53.

a, Supervised hierarchical clustering of RNA-sequencing data from thymic lymphomas 48 h after treatment with Ad-mCherry (ΔNfl/fl;p53−/−) or Ad-Cre-mCherry (ΔNp63Δ/Δ;p53−/− or ΔNp73Δ/Δ;p53−/−). b, c, qRT–PCR for GLS2 (b) and TIGAR (c) in the indicated thymic lymphomas, n = 4, P < 0.005. d, qRT–PCR for GLS2 in MEFs of the indicated genotypes expressing shRNAs for a non-targeting sequence (shNT), TAp63 (shTAp63) and TAp73 (shTAp73), n = 4, P < 0.005. e, Table showing the TAp63 and TAp73 binding sites on the IAPP promoter and intron 2. f, g, qRT–PCR of promoter site 1 using chromatin immunoprecipitation in MEFs of the indicated genotypes, n = 3, P < 0.005. hk, Dual luciferase reporter assay for pGL3-IAPP-promoter site 1 (h, i) and a mutant version of this reporter gene (pGL3-IAPP MUT) (j, k). Genotypes of MEFs and vectors used are shown. V represents pcDNA3 vector. l, m, Western blot analysis of the indicated MEFs expressing IAPP or siRNAs for a non targeting sequence (siNT) or IAPP (siIAPP). Statistical significance indicated by black asterisks.

Source data

Extended Data Figure 7 Systemic in vivo delivery of pramlintide results in tumour regression in p53-deficient thymic lymphomas.

a, Western blot analysis showing IAPP expression in the indicated thymic lymphomas, n = 5 mice. b, Kaplan–Meier survival indicating thymic lymphoma-free survival. n = 8 mice per group, P < 0.005. c, Cartoon indicating schedule of MRI imaging and injection (Inj.) of pramlintide in mice with p53-deficient thymic lymphomas. dq, MRI imaging at 10, 11, 12 and 13 weeks after treatment with placebo (d–g) or pramlintide (i–p); quantification of tumour volumes in placebo (n = 3) (h) and pramlintide-treated mice (n = 7) (q), P < 0.005. Statistical significance indicated by black asterisk.

Source data

Extended Data Figure 8 IAPP inhibits glycolysis by increasing intracellular G-6-P levels.

a, b, Quantification of apoptosis (a) and proliferation (b), n = 20 fields of 3 biological replicates, P < 0.005. c, qRT–PCR for the target genes indicated on the x-axis in the indicated H1299 cells expressing the indicated siRNAs, n = 4. Asterisks indicate statistical significance (P < 0.005) relative to siNT. d, Western blot analysis of H1299 cells treated with the indicated siRNAs. e, f, Bar graph indicating glucose-dependent proton secretion as a measure of glucose uptake and intracellular levels of glucose-6-phosphate in H1299 cells with the indicated siRNAs and treatments (f). g, Colour-coded legend for panels e, f and i. h, Western blot analysis of H1299 cells expressing the indicated siRNAs. i, Immunofluorescence analysis for ROS (red) or apoptosis (green or green/red) in H1299 cells expressing the indicated siRNAs and treated with 2DG and/or NAC.

Source data

Extended Data Figure 9 Treatment of p53-mutant human cancer cell lines with pramlintide inhibits glycolysis and induces ROS and apoptosis.

a, b, Western blot analysis of H1299 cells expressing the indicated siRNAs (a) or concentrated media derived from H1299 cells expressing siNT, siΔNp63, or siΔNp73 (b). c, Extracellular acidification rate (ECAR) using H1299 cells expressing the indicated siRNAs and treated with the indicated media containing secreted IAPP and treated with the indicated amylin inhibitor (AI). dg, Extracellular acidification rate (ECAR) as a measure of glycolysis in SW480 (d), MDA-MB-468 (e), SRB12 (f) and COLO16 (g) human cancer cell lines after treatment with placebo, pramlintide, or pramlintide and a calcitonin receptor inhibitor (CalR I.), n = 3, P < 0.005. Glucose, oligomycin, and 2-deoxy-d-glucose (2DG) were supplied to the media at the indicated time points shown on the x-axis. h, i, Immunofluorescence for ROS (red) (h) and apoptosis (green) (i) on the indicated cells, n = 3. j, k, Kaplan–Meier survival curves using data from patients with p53 mutant tumours with the indicated cancers and co-expression of IAPP, RAMP3 and CALCR. Boxed numbers represent median survival.

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Venkatanarayan, A., Raulji, P., Norton, W. et al. IAPP-driven metabolic reprogramming induces regression of p53-deficient tumours in vivo. Nature 517, 626–630 (2015). https://doi.org/10.1038/nature13910

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