IAPP-driven metabolic reprogramming induces regression of p53-deficient tumours in vivo
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doi:10.1038/nature13910 IAPP-driven metabolic reprogramming induces regression of p53-deficient tumours in vivo
Avinashnarayan Venkatanarayan1,2,3,4,Payal Raulji1,2,William Norton5,Deepavali Chakravarti1,2,3,4,Cristian Coarfa6,
Xiaohua Su1,2,4,Santosh K.Sandur1,2,4,7,Marc S.Ramirez8,Jaehuk Lee8,Charles V.Kingsley8,Eliot F.Sananikone1,2,3,4,
Kimal Rajapakshe6,Katherine Naff5,Jan Parker-Thornburg9,James A.Bankson8,Kenneth Y.Tsai2,10,Preethi H.Gunaratne11
&Elsa R.Flores1,2,3,4
TP53is commonly altered in human cancer,and Tp53reactivation suppresses tumours in vivo in mice1,2(TP53and Tp53are also known as p53).This strategy has proven difficult to implement therapeut-ically,and here we examine an alternative strategy by manipulating the p53family members,Tp63and Tp73(also known as p63and p73, respectively).The acidic transactivation-domain-bearing(TA)iso-forms of p63and p73structurally and functionally resemble p53, whereas the D N isoforms(lacking the acidic transactivation domain) of p63and p73are frequently overexpressed in cancer and act prim-arily in a dominant-negative fashion against p53,TAp63and TAp73 to inhibit their tumour-suppressive functions3–8.The p53family inter-acts extensively in cellular processes that promote tumour suppres-sion,such as apoptosis and autophagy9–14,thus a clear understanding of this interplay in cancer is needed to treat tumours with alterations in the p53pathway.Here we show that deletion of the D N isoforms of p63or p73leads to metabolic reprogramming and regression of p53-deficient tumours through upregulation of IAPP,the gene that encodes amylin,a37-amino-acid peptide co-secreted with insulin by the b 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 protein3 (RAMP3)to inhibit glycolysis and induce reactive oxygen species and apoptosis.Pramlintide,a synthetic analogue of amylin that is currently used to treat type1and type2diabetes,caused rapid tumour regression in p53-deficient thymic lymphomas,representing a novel strategy to target p53-deficient cancers.
Using D Np63(ref.15)and D Np73conditional knockout mice(Ex-tended Data Fig.1a,b),we generated D Np631/2and D Np732/2mice (Extended Data Fig.1c–f).To ask whether the D N isoforms of p63and p73act as oncogenes in vivo by interacting with p53,D Np631/2;p532/2 and D Np732/2;p532/2mice were aged for the development of thymic lymphomas,which form in nearly all p532/2mice16.We found a remark-able diminution in the number and size of thymic lymphomas in D Np631/2;p532/2and D Np732/2;p532/2mice,leading to an extended lifespan(Extended Data Fig.2a–c)and suggesting that the D N isoforms of p63and p73restrain a tumour suppressive program that can com-pensate for p53function.
We found that TAp63and TAp73were upregulated in thymic lym-phomas from D Np631/2;p532/2and D Np732/2;p532/2mice(Extended Data Fig.2d,e)along with an upregulation of apoptosis(Extended Data Fig.2f–j)and senescence(Extended Data Fig.2k–o).We also examined thymocytes from4-week-old mice after treatment with10Gy gamma irradiation,a dose that is known to elicit p53-dependent apoptosis9,17.Indeed,TAp63andTAp73arehigherin D Np631/2;p532/2and D Np732/2; p532/2thymocytes,which was further exacerbated after gamma irradi-ation(Extended Data Fig.3a–c)with an increase in apoptosis(Extended Data Fig.3d–h)and senescence(Extended Data Fig.3i–m).
To determine whether TAp63or TAp73compensate for p53function in tumours in vivo,we acutely removed D Np63or D Np73by intratumoral infection with adenovirus-Cre-mCherry(Extended Data Fig.4a–d and Fig.1a–f)in D Np63fl/fl;p532/2and D Np73fl/fl;p532/2at10weeks of age. Tumours were2.3–5.8mm3in size at the time of infection and moni-tored weekly by magnetic resonance imaging(MRI;Fig.1a–i).Mice defi-cient for either D Np63or D Np73and p53showed marked decreases in tumour burden(Fig.1h,i).The reduction of D Np63and D Np73expres-sion resulted in increased expression of TAp63and TAp73(Fig.1j–m and Extended Data4d)and increased apoptosis(Extended Data Fig. 4e–h)and senescence(Extended Data Fig.4i–k).D Np63D/D;p532/2and D Np73D/D;p532/2mice also had an increased lifespan(Fig.1n).We found differences in CD4/CD8-positive cells in young mice(4weeks)(Extended Data Fig.4l–p),indicating that changes in T-cell development may lead to a lower tumour incidence in double-mutant mice.Indeed,we found that p532/2thymic lymphomas are composed primarily of CD4/CD8 double-positive thymocytes while the D Np63D/D;p532/2and D Np73D/D; p532/2lymphomas contain very few CD4/CD8double-positive thy-mocytes(Extended Data Fig.4q–t).Lastly,we asked whether thymic stromal cells contribute to the apoptosis in the regressing lymphomas. We sorted CD45-positive cells to select for T lymphocytes in p532/2, D Np63fl/fl;p532/2and D Np73fl/fl;p532/2mice and infected them with adenovirus-Cre(Extended DataFig.4u).D Np63D/D;p532/2and D Np73D/D; p532/2thymocytes underwent apoptosis independent of the presence of the stromal cells(Extended Data Fig.4v).These data indicate that inhibition of the D N isoforms of p63and p73serves to upregulate TAp63and TAp73to compensate for loss of p53in tumour suppression. We found that the D N isoforms of p63and p73bind to the promo-ters of the TA isoforms of p63and p73,suggesting that the D N isoforms of p63and p73can transcriptionally repress TAp63and TAp73tran-scription(Extended Data Fig.5a–i).We also found that the increase in apoptosis and cellular senescence was dependent on TAp63and TAp73 (Extended Data Fig.5j–q).
We performed RNA sequencing of lymphomas after infection with Ad-mCherry(D Np63fl/fl;p532/2and D Np73fl/fl;p532/2)and Ad-Cre-mCherry(D Np63D/D;p532/2and D Np73D/D;p532/2)and found that thymic lymphomas from mice deficient for p53and D Np63clustered with those from mice deficient for p53and D Np73(Extended Data Fig.6a). Ingenuity pathway analysis(IPA)(Fig.1q)revealed genes involved in
1Department of Molecular and Cellular Oncology,The University of Texas M.D.Anderson Cancer Center,1515Holcombe Boulevard,Houston,Texas77030,USA.2Department of Translational Molecular Pathology,The University of Texas M.D.Anderson Cancer Center,1515Holcombe Boulevard,Houston,Texas77030,USA.3Graduate School of Biomedical Sciences,The University of Texas M.D.Anderson Cancer Center,1515Holcombe Boulevard,Houston,Texas77030,USA.4Metastasis Research Center,The University of Texas M.D.Anderson Cancer Center,1515Holcombe Boulevard,Houston,Texas 77030,USA.5Department of Veterinary Medicine and Surgery,The University of Texas M.D.Anderson Cancer Center,1515Holcombe Boulevard,Houston,Texas77030,USA.6Department of Molecular and Cellular Biology,Baylor College of Medicine,1Baylor Plaza,Houston,Texas77030,USA.7Radiation Biology&Health Sciences Division,Bhabha Atomic Research Center,Mumbai400085,India. 8Department of Imaging Physics,The University of Texas M.D.Anderson Cancer Center,1515Holcombe Boulevard,Houston,Texas77030,USA.9Department of Genetics,The University of Texas M.D. Anderson Cancer Center,1515Holcombe Boulevard,Houston,Texas77030,USA.10Department of Dermatology,The University of Texas M.D.Anderson Cancer Center,1515Holcombe Boulevard, Houston,Texas77030,USA.11Department of Biology and Biochemistry,University of Houston,Houston,Texas77204,USA.
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metabolism including TP53-inducible glycolysis and apoptosis regu-lator (TIGAR )18,and glutaminase 2(GLS2)19,20.While we found that TIGAR and GLS2were upregulated in either D Np63D /D ;p532/2or D Np73D /D ;p532/2thymic lymphomas,we identified a novel gene,islet amyloid polypeptide (IAPP )or amylin,which was upregulated by over fivefold in both double-mutant thymic lymphomas.IAPP limits glu-cose uptake,resulting in increased intracellular glucose-6-phosphate (G-6-P)21levels and decreased glycolysis 21.We validated IAPP ,TIGAR and GLS2expression in thymic lymphomas derived from D Np63D /D ;p532/2and D Np73D /D ;p532/2mice and found that IAPP is expressed at levels over twofold higher in double-mutant mice (Fig.1p and Extended Data Fig.6b–d).IAPP and GLS2expression depend on TAp63and TAp73(Fig.1q and Extended Data Fig.6d).To determine whether TAp63or TAp73transcriptionally regulate IAPP ,we performed chromatin immu-noprecipitation in mouse embryonic fibroblasts (MEFs;Extended Data Fig.6e–g)and thymocytes (Fig.1r,s).We found that TAp63and TAp73bind to sites located in the promoter (site 1),1,756nucleotides upstream of the transcriptional start site,and intron 2(site 2)of IAPP ,706nuc-leotides downstream of the transcriptional start site (Extended Data Fig.6e–g).Because a greater binding affinity of TAp63and TAp73was detected in the promoter region (site 1)of IAPP ,we cloned this site into a luciferase reporter gene and also mutated this site (Extended Data Fig.6h–k).Only the luciferase reporter gene containing wild-type IAPP promoter site 1was transactivated by TAp63and TAp73whereas the
mutant version was not.Taken together,these data indicate that IAPP is a transcriptional target gene of TAp63and TAp73(Fig.1t).
Expression of IAPP in p532/2MEFs resulted in lowlevels of glycolysis comparable to that in D Np632/2;p532/2and D Np732/2;p532/2MEFs (Extended Data Fig.6l–m and Fig.1u).Conversely,when we knocked down IAPP in D Np632/2;p532/2and D Np732/2;p532/2MEFs,the levels of glycolysis were similar to that of p532/2MEFs (Fig.1u)indicating that IAPP inhibits glycolysis.In vivo ,we detected massive tumour regression in D Np63fl/fl ;p532/2or D Np73fl/fl ;p532/2thymic lymphomas treated with IAPP (Extended Data Fig.7a and Fig.2a,b,h,i,o,p),P ,0.05.Conversely,in D Np63D /D ;p532/2and D Np73D /D ;p532/2thymic lym-phomas treated with Ad-shIAPP-mCherry the tumours continued to grow comparable to that of p532/2thymic lymphomas (Fig.2a–k,o–r),P .0.05at 13weeks.Additionally,p532/2mice treated with Ad-IAPP had an extended tumour-free survival period compared to p532/2mice or D Np63D /D ;p532/2and D Np73D /D ;p532/2mice treated intratumorally with Ad-shIAPP-mCherry (Extended Data Fig.7a,b),indicating that IAPP is a tumour suppressor gene and is causally involved in the in vivo effects seen upon inactivation of D Np63or D Np73.Given that pramlintide,a synthetic analogue of amylin,is used to treat type I and type II diabetes 22,we treated thymic lymphomas in D Np63fl/fl ;p532/2and D Np73fl/fl ;p532/2mice.Indeed,three-weekly intratumoral injections resulted in rapid tumour regression (Fig.2e,l,s),P ,0.005at 13weeks.This effect was exacerbated by systemic intravenous treatment
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Figure 1|In vivo deletion of D Np63or D Np73in p53-deficient mice
suppresses lymphomagenesis.a –f ,Magnetic resonance imaging (MRI)of thymic lymphomas of indicated mice.Tumour volume (mm 3)shown within each panel.UN-D,undetectable.Tumours indicated by the dashed yellow line.g –i ,Quantification of the indicated thymic lymphomas,n 54mice.
j –m ,Quantitative real time PCR (qRT–PCR),n 54,P ,0.005.n ,Kaplan–Meier curve,n 54,P ,0.005.Boxed numbers represent median survival.o ,Ingenuity pathway analysis (IPA)of RNA sequencing from thymic lymphomas 48h after infection with adenoviruses.Red oval indicates
significantly upregulated metabolic genes.p ,q ,qRT–PCR for IAPP in thymic lymphomas (p )or MEFs of the indicated genotypes using a non-targeting shRNA (shNT)or shRNAs for TAp63(shTAp63)or TAp73(shTAp73)(q ),n 54,P ,0.005.r ,s ,qRT–PCR of IAPP promoter site 1using chromatin immunoprecipitation,n 53,P ,0.005.t ,Cartoon showing transcriptional activation of IAPP by TAp63and TAp73.u ,Extracellular acidification rate (ECAR)as a measurement of glycolysis,P ,0.005.
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pramlintide(Fig.2f,m,t and ExtendedDataFig.7c–q),P ,0.005,similarto that seen in tumours treated with a known inhibitor of glycolysis,2-deoxy-D -glucose (2DG;Fig.2g,n,u).These data provide preclinical in vivo evidence that pramlintide can be used to effectively treat p53-deficient https://www.docsj.com/doc/9012618237.html,ing in vivo dynamic magnetic resonance spectroscopy to measure the conversion of hyperpolarized [1-13C]pyruvate to lactate as a proxy of glycolysis within the tumours,we found a marked reduction in glycolysis in D Np63/p53and D Np73/p53double-deficient mice and after introducing IAPP into p532/2thymic lymphomas similar to tumours treated with 2DG (Fig.2v).D Np63D /D ;p532/2and D Np73D /D ;p532/2thy-mic lymphomas infected with a short hairpin RNA for IAPP exhibited levels of glycolysis similar to those found in p532/2thymic lymphomas (Fig.2v).Pramlintide also inhibits glycolysis in tumours (Fig.2v).IAPP has been shown to induce reactive oxygen species (ROS)and activate apoptosis 23,24.We found a marked increase in the levels of ROS and apoptosis in thymic lymphomas expressing IAPP or treated with pramlintide or 2DG,whereas neither ROS nor apoptosis occurred upon inactivation of IAPP in thymic lymphomas from D Np63D /D ;p532/2and D Np73D /D ;p532/2mice (Fig.2w),indicating that upregulation of IAPP inhibits glycolysis similarly to 2DG and leads to oxidative stress that triggers apoptosis.While high levels of ROS are not commonly triggered by inhibition of glycolysis,nutrient deprivation or excess can result in the accumulation of ROS.Additionally,cancer cells tightly regulate ROS by acquiring additional mutations and compensatory mechanisms often ensue and may be at play in the thymic lymphoma cells that acutely downregulate glycolysis by IAPP 25.To extend our findings to human cancer where p53is altered in the majority of cases,we analysed human cancer cell lines containing p53deletions or mutations.We used short interfering RNA (siRNA)to knockdown D Np63or D Np73in cells derived from a lung adenocarci-noma (H1299)(Fig.3a).Downregulation of D Np63or D Np73resulted in upregulation of TAp63,TAp73and IAPP (Fig.3a)and an increase in apoptosis and decrease in cell proliferation (Fig.3b and Extended Data Fig.8a–d).To ask whether IAPP can also inhibit glycolysis in human cancer cell lines,we transfected H1299cells with si D Np63,si D Np73or IAPP (Fig.3a).Knockdown of D Np63or D Np73or expression of IAPP resulted in an inhibition of glycolysis (Fig.3c,d)and glucose uptake (Extended Data Fig.8e,g),accumulation of ROS (Fig.3d–f),and induc-tion of apoptosis (Fig.3d,g,h).We inhibited ROS in these cells using N -acetyl-L -cysteine (NAC)and observed no apoptosis (Fig.3d–h).Pre-vious studies have indicated that IAPP inhibits glycolysis by increasing intracellular G-6-P in turn leading to an inhibition of hexokinase 21,26.We measured the levels of intracellular G-6-P in H1299cells and found that cells expressing high levels of IAPP (H1299-si D Np63,H1299-si D Np73,or H12991IAPP)also had high levels of G-6-P while knock-down of IAPP resulted in a diminution in G-6-P (Extended Data Fig.8f,g).Overexpression of glucose hexokinase II (HKII)led to a rescue of the glycolytic capacity of H1299cells expressing si D Np63or si D Np73to levels similar to those in parental H1299cells (Fig.3c–g).These results indicate that IAPP inhibits glycolysis through the inhibition of HKII.We found that treatment of H1299cells with pramlintide led to similar effects on glycolysis and apoptosis (Fig.3g–n).Taken together,these
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Δ/Δ;p53–/–Δ/Δ;p53–/–Figure 2|IAPP is causally involved in tumorigenesis suppression in p53-deficient thymic lymphomas.a –n ,Thymic lymphomas were infected with adenovirus (Ad)-mCherry (a ,h ),Ad-IAPP-mCherry (1IAPP)(b ,i ),Ad-shIAPP-mCherry (c ,d ,j ,k ),or treated with pramlintide intratumorally (IT)(e ,l )or intravenously (IV)(f ,m ),or with 2DG (g ,n ).Yellow dashed lines indicate tumour.Volume of tumour shown.UN-D,undetectable.
o –u ,Quantification of the indicated thymic lymphomas,n 55mice per group.Significance indicated by the asterisks,P ,0.005.v ,Quantification of in vivo pyruvate to lactate conversion using dynamic magnetic resonance spectroscopy as a measurement of glycolysis,n 53mice,P ,0.005.w ,Immunohistochemistry for reactive oxygen species (ROS)or cleaved caspase 3.Positive nuclei are brown.
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