The execution of the transcriptional axis mutant p53, E2F1 ... - Nature

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The execution of the transcriptional axis mutant p53, E2F1 and ID4 promotes tumor neo-angiogenesis

© 2009 Nature America, Inc. All rights reserved.

Giulia Fontemaggi1–3,10, Stefania Dell’Orso1,2,10, Daniela Trisciuoglio4, Tal Shay5, Elisa Melucci6, Francesco Fazi7, Irene Terrenato8, Marcella Mottolese6, Paola Muti8, Eytan Domany5, Donatella Del Bufalo4, Sabrina Strano1,9 & Giovanni Blandino1,2 ID4 (inhibitor of DNA binding 4) is a member of a family of proteins that function as dominant-negative regulators of basic helix-loop-helix transcription factors. Growing evidence links ID proteins to cell proliferation, differentiation and tumorigenesis. Here we identify ID4 as a transcriptional target of gain-of-function p53 mutants R175H, R273H and R280K. Depletion of mutant p53 protein severely impairs ID4 expression in proliferating tumor cells. The protein complex mutant p53–E2F1 assembles on specific regions of the ID4 promoter and positively controls ID4 expression. The ID4 protein binds to and stabilizes mRNAs encoding pro-angiogenic factors IL8 and GRO-. This results in the increase of the angiogenic potential of cancer cells expressing mutant p53. These findings highlight the transcriptional axis mutant p53, E2F1 and ID4 as a still undefined molecular mechanism contributing to tumor neo-angiogenesis. The ID family consists of four proteins (ID1–ID4) that belong to helixloop-helix transcription factors and have been recognized as regulators of crucial cellular processes ranging from cell-fate determination and differentiation to proliferation and cell death. ID proteins, which lack a DNA-binding domain, associate with HLH transcription factors and prevent them from binding DNA or forming active heterodimers1–3. Biochemical and genetic evidence describes ID proteins as inhibitors of cellular differentiation and positive regulators of proliferation. Increased expression of ID proteins has been reported in many types of human tumors and has been frequently associated with loss of differentiation (anaplasia), enhanced malignancy and aggressive clinical behavior4–6. Loss of ID4 expression leads to the expression of BRCA1 (breast cancer 1), thereby indicating a role for the ID4 gene in mammary tumorigenesis7. BRCA1 upregulates the ID4 gene8, establishing a regulatory loop that maintains the expression of both genes during normal mammary cell division. ID4 mRNA and protein levels have been found to be upregulated in rat mammary carcinomas compared with adenomas and normal mammary glands9. Recently, an inverse correlation between ID4 mRNA and estrogen receptor expression was reported in human normal breast epithelium and carcinoma10. Here we report that ID4 is a transcriptional target gene positively regulated by gain-of-function p53 mutants. These mutants are the gene products of p53 missense mutations, the vast majority of which occur in the DNA-binding core domain of the protein; these mutants cannot bind to the wild-type p53 consensus sequence. Mutant p53 proteins are mostly full-length proteins whose half-life is largely prolonged compared

to that of wild-type p53. A combination of in vitro and in vivo studies have clearly shown that some of p53 mutant proteins gain oncogenic functions, conferring transformed properties to cell cultures and accelerated tumorigenicity in mice11,12. Tumor-associated forms of mutant p53 can contribute to genomic instability by abrogating the mitotic spindle checkpoint and, consequently, facilitating the generation of aneuploid cells13,14. Tumors bearing p53 mutations are less sensitive to the killing action of commonly used anticancer agents than are tumors carrying wild-type p53 protein. Whereas overexpression of human tumor-derived p53 mutants increases the resistance of cultured cells to anticancer agents, depletion of mutant p53 protein facilitates DNA damage–induced cell death15–19. The molecular mechanisms underlying the gain of function of mutant p53 proteins are not well understood. To date, three molecular scenarios have been described. (i) Mutant p53 binds to and sequesters proteins whose function is required for antitumor effects such as apoptosis or growth inhibition. Among these proteins, p63 and p73 interact with human tumor-derived p53 mutants, and the outcome of such interaction results in the severe impairment of p73 and p63 transcriptional activity and apoptosis mediated by these proteins20,21. (ii) Mutant p53 binds to DNA and contributes to the transcriptional control of putative target genes through its intact N-terminal transactivation domain18,22. (iii) Mutant p53 takes part in the formation of large transcriptional competent complexes through which the expression of its target genes is regulated23–26. By exploring the transcriptional activity of mutant p53-R175H through a microarray approach, we identify ID4 as an in vivo transcriptional

1Translational

Oncogenomics Unit, Regina Elena Cancer Institute, Rome, Italy. 2Rome Oncogenomic Center (ROC), Regina Elena Cancer Institute, Rome, Italy. Pathology Section, Department of Clinical and Experimental Medicine, Perugia University, Perugia, Italy. 4Experimental Chemotherapy Laboratory, Regina Elena Cancer Institute, Rome, Italy. 5Department of Physics of Complex Systems, Weizmann Institute of Science, Rehovot, Israel. 6Department of Pathology, Regina Elena Cancer Institute, Rome, Italy. 7Department of Histology and Medical Embryology, University “La Sapienza” & San Raffaele Bio-Medical Park Foundation, Rome, Italy. 8Department of Epidemiology, Regina Elena Cancer Institute, Rome, Italy. 9Molecular Chemoprevention Group, Scientific Direction, Regina Elena Cancer Institute, Rome, Italy. 10These two authors contributed equally to the work. Correspondence should be addressed to G.B. ([email protected]). 3General

Received 3 April; accepted 11 August; published online 27 September 2009; doi:10.1038/nsmb.1669

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target gene of human gain-of-function mutant p53 proteins. Indeed, we show that ID4 expression of diverse cancer cell lines is specifically driven by mutant p53 that is also recruited in vivo to ID4 gene promoter. We also find that mutant p53 protein is concomitantly recruited with the transcription factors p65 (Rel A) and E2F1 to ID4 regulatory regions. A tight cooperation between E2F1 and mutant p53 occurs in the transcriptional control of ID4 gene expression. This effect results in the recruitment of the acetyltransferase p300, thereby indicating an active chromatin status. We have observed elevated ID4 expression in the presence of p53 mutations both through analysis of public microarray data from breast cancer cell lines and by staining of 186 breast cancer specimens. The net biological output of the transcriptional activation of the ID4 gene by mutant p53 is the increase in the angiogenic potential of mutant p53–carrying tumor cells. Binding of ID4 protein to the mRNAs of pro-angiogenic factors, such as IL8 and GRO-α, which results in the increased stability of these transcripts, explains the pro-angiogenic effects of ID4 transactivation. RESULTS Mutant p53-R175H specifically induces ID4 transcript To evaluate whether mutant p53 proteins exert gain-of-function activity through the modulation of specific sets of genes, we used DNA micro arrays to analyze the gene expression profile of p53-null human non–small cell lung carcinoma cells expressing the ponasterone-inducible mutant p53-R175H (H1299 clone 41). Mutant p53-R175H protein upregulated the expression of 38 probe sets and down regulated the expression of of 326 probe sets (Supplementary Fig. 1 and Supplementary Tables 1a,b). Among the upregulated probe sets, the ID4 gene showed a substantial induction by mutant p53 (Fig. 1a, left, and Supplementary Fig. 1b), whereas ponasterone-inducible wild-type p53 (H1299 clone Figure 2 Mutant p53 drives the expression of ID4 and associates with its promoter. (a) qRT-PCR analysis of ID4 expression in SKBr3, MDA-MB-231 and MCF7 cells whose p53 expression was depleted (pRS-p53), compared with control cells (pRS-scr) with wild-type p53 expression. Relative ID4 mRNA levels were normalized to the amount of GAPDH transcript. p53 protein levels in SKBr3 and MDA-MB-231 cells, and mRNA levels in MCF7 cells, are shown below. Error bars represent s.d. (b) Analysis of ID4 protein expression by immunocytochemistry in SKBr3 cells depleted of mutant p53 expression (pRS-p53) and in control cells (pRS-scr). Mutant p53 protein levels following interference in these cells are shown at left.

23) had no effect on ID4 expression (Fig. 1a, middle), indicating that ID4 transcript is specifically regulated by mutant p53 expression. ID4 gene expression was also unchanged in H1299 cells that were transduced with the vectors of the ponasterone-inducible system (H1299-pIND) (Fig. 1a, right). We also followed ID4 expression upon infection with a retroviral vector expressing mutant p53-R175H. ID4 transcript was greatly induced following expression of mutant p53-R175H (Supplementary Fig. 1c). To a lesser extent, this effect was also seen upon ectopic expression of mutant p53-R273H (Fig. 1b). Taken together, these findings indicate that ID4 expression is specifically modulated by mutant p53 protein. To probe in more detail the induction of ID4 expression by mutant p53, we isolated ribosome- and polysome-associated fractions from H1299 clone 41 cells and followed the distribution of the ID4 transcript. The polysomal, highly translating fractions were strongly enriched for ID4 transcript (up to ten-fold) upon mutant p53 expression (induced with ponasterone (Pon+)) (Fig. 1c), indicating that the ID4 transcript is translated. In fact, we verified ID4 protein expression following mutant p53 induction by western blotting (Fig. 1d) and by immunocytochemistry (Fig. 1e). Mutant p53 proteins transactivate the ID4 promoter To analyze whether mutant p53 regulates ID4 promoter activity, we performed transactivation assays. Increasing amounts of both mutant p53-R175H, to a larger extent, and mutant p53-R273H, to a lesser

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Figure 1 ID4 expression is transcriptionally induced by mutant p53. (a) Quantitative RT-PCR (qRT-PCR) analysis of ID4 mRNA expression on clone 41 (expressing inducible p53-R175H), clone 23 (expressing inducible wild-type p53) and clone pIND (empty vector) cells in the presence or absence of ponasterone A and/or cisplatin (CDDP). Relative ID4 mRNA levels were calculated by normalization to the amount of GAPDH transcript. p53 protein induction was assessed by western blotting (below). (b) qRT-PCR analysis of ID4 expression in H1299 cells stably transfected with mutant p53-R273H or empty vector (EV). Western blot analysis for p53 expression is shown below. (c) qRT-PCR analysis of ID4 mRNA in ribosomal and polysomal cell fractions. Relative ID4 mRNA levels were calculated as in a. Pon+, ponasterone-treated cells; Pon−, untreated cells. (d) Western blot analysis of ID4 expression upon induction of p53-R175H in H1299 clone 41. (e) Analysis of ID4 protein expression by immunocytochemistry upon induction of p53-R175H in H1299 clone 41. (f,g) Relative luciferase activity (RLU) of ID4-luciferase vector transfected in H1299 cells in absence or in presence of increasing amounts of mutant p53-R175H (f) or p53-R273H (g) expression vectors. The data represent the mean of duplicate determinations for three separate experiments. Error bars represent s.d.

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extent, transcriptionally activate ID4 promoter (Fig. 1f,g). We asked whether mutant p53 directly binds to ID4 promoter. To this end, we performed chromatin immunoprecipitation (ChIP) experiments using H1299 clone 41 cells. We found that mutant p53 was recruited to the ID4 promoter only in cells whose mutant p53 expression was induced by the addition of ponasterone (Supplementary Fig. 2a). Endogenous mutant p53 proteins control ID4 gene expression To verify whether endogenous mutant p53 also controlled the expression of ID4, we evaluated the impact of mutant p53 silencing in breast cancer cell lines. Depletion of mutant p53-R175H in the SKBr3 breast cancer cell line strongly affected the expression of ID4 transcript and protein (Fig. 2a,b). Similarly, partial depletion of endogenous mutant p53-R280K from MDA-MB-231 cells led to reduced ID4 mRNA levels (Fig. 2a, middle). Knockdown of wild-type p53 expression in MCF7 breast cancer cells did not affect ID4 transcript levels (Fig. 2a, right). These findings further confirm the specificity of the effect of mutant p53 on ID4 mRNA levels. To evaluate whether the ID4 gene is differentially expressed in breast cancer cell lines carrying wild-type or mutant p53 protein, we queried public gene expression data repositories27 (http://www.oncomine.org/). Analysis of the data set from Neve and colleagues28 revealed that ID4 transcript is substantially upregulated in breast cancer cell lines carrying mutant p53, compared to those with wild-type p53 protein (Supplementary Fig. 2b). Mutant p53 proteins assemble in vivo on ID4 promoter To characterize ID4 promoter occupancy in cells bearing endogenous mutant p53, we performed ChIP experiments in SKBr3 cells (p53R175H). We evaluated occupancy of regions B, C and D (Fig. 3a) by real-time PCR and carried out semiquantitative PCR on region A, as its nucleotide composition does not allow real-time PCR analysis. Mutant p53-R175H bound to regions A, C and D of the ID4 promoter (Fig. 3b, i,ii), as assessed using two different antibodies (anti-p53 Ab1 and anti-p53 1088

Figure 3 Transcriptionally competent protein complexes containing mutant p53 assemble in vivo on ID4 promoter. (a) Schematic representation of ID4 promoter. (b) ChIP analysis using the indicated antibodies (‘Ab1’ and ‘Ab2’ represent two different antibodies to p53; ‘No Ab’ represents the no antibody control) on cross-linked extracts from proliferating SKBr3 cells. The occupancy of region A of the ID4 promoter was analyzed by PCR, whereas regions B, C and D were examined by qPCR. (c) Four additional aliquots of chromatin immunoprecipitated with anti-p53 antibody were eluted and re-immunoprecipitated with antibodies directed against p65, E2F1 and p300, or without antibody (No Ab). PCR analysis was performed on ID4 promoter using four pairs of primers spanning the four major clusters of consensus sequences on ID4 promoter (A, B, C and D), as indicated in a. Relative enrichments (Rel. enrich.) are presented as fold change over No Antibody. Error bars represent s.d.

Ab2) (Fig. 3b). Next, we investigated the recruitment of the transcription factors p65 and E2F1 to the ID4 promoter. p65 bound to region A, which contains a canonical consensus for NF-kB (Fig. 3b, iii), whereas E2F1 was recruited onto regions A to D (Fig. 3b, iv). We also found that the histone acetyltransferase p300 bound to ID4 promoter with increasing strength approaching to the transcription start site (Fig. 3b, v). We obtained similar results with cell line MDA-MB-231, which expresses mutant p53-R280K (Supplementary Fig. 2c). To investigate whether there is concomitant presence of mutant p53, p65, E2F1 and p300 on the ID4 promoter, indicating the formation of transcriptionally competent complexes, we performed sequential ChIP (Re-ChIP) analyses (Fig. 3c). We found that mutant p53 and p65 were present simultaneously on region A (Fig. 3c, i), whereas mutant p53 was bound to regions C and D, which contain the CDE consensus element, together with E2F1 and p300 (Fig. 3c, ii,iii). We obtained similar data for MDA-MB-231 (Supplementary Fig. 2c). On the other hand, ChIP analysis of ID4 promoter in MCF7 cells showed that wild-type p53 was not recruited to the ID4 promoter (Supplementary Fig. 2d). Mutant p53-R175H and E2F1 cooperate in ID4 transcription To further dissect whether the concomitant recruitment of mutant p53 and E2F1 results in the cooperative transcriptional control of ID4 gene expression, we analyzed the impact of E2F1 depletion on mutant p53 activity. Knockdown of E2F1 in SKBr3 cells abrogated the recruitment of mutant p53-R175H to region C of the ID4 promoter (Fig. 4a). This region contains a CDE sequence that is reported to interact with diverse transcription factors, including E2F1 (ref. 29). E2F1 silencing did not affect binding of mutant p53 to region A of the ID4 promoter (Fig. 4a). To further define the binding of the E2F1–mutant p53 complex to the CDE region of the ID4 promoter, we used the electrophoretic mobility shift assay (EMSA) with nuclear extracts from SKBr3 cells incubated with a 30-base pair (bp) probe that encompassed the CDE-binding site. We observed two specific complexes, and addition of an anti-E2F1 antibody reduced the amount of both forms (lanes 7 and 9) (Fig. 4b, left). The presence of an anti-p53 antibody led to a decrease in the amount of complex 1 and to the appearance of a supershifted band (lanes 8 and 9). The effect of the antibodies against E2F1 and p53 indicate that both complexes 1 and 2 contain E2F1 and p53. Thus, the two complexes observed by EMSA might represent different multiprotein complexes containing mutant p53, E2F1 and other cofactors. Next, we analyzed the effect of E2F1 depletion on ID4 transcript in H1299 clone 41 cells. ID4 transcript induction upon inducible mutant p53 expression was abolished by knockdown of E2F1 (Fig. 4c). We observed no change in E2F1 mRNA and protein levels upon induction of mutant p53 protein (Fig. 4d). Depletion of E2F1 with two different small interfering RNAs (siRNAs) also reduced ID4 expression in SKBr3 cells (Fig. 4e,f).


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To verify whether the cooperation of mutant p53 and E2F1 relies on their physical interaction, we performed coimmunoprecipitation analysis. Previous reports showed that E2F1 interacts with wild-type p53 (refs. 30,31), induces its nuclear retention and stimulates p53’s transactivation and apoptotic functions30. We show here that mutant p53 and E2F1associate in H1299 cells (Supplementary Fig. 3). Altogether, our data indicate that mutant p53-R157H cooperates with E2F1 in the transcriptional control of ID4 expression. Gain-of-function mutant p53 promotes neo-angiogenesis The findings above indicate that ID4 expression is constitutively driven by the gain-of-function mutant p53 protein, suggesting a potential role for the control of ID4 expression by mutant p53 in the maintenance or spreading of tumor cells. Thus, we asked whether the transcriptional activation of ID4 by mutant p53 has an impact on angiogenesis. We found that the conditioned medium derived from induced H1299 clone 41 cells promoted proliferation of endothelial cells (Fig. 5a). Indeed, we observed a time-dependent increase in endothelial cell proliferation when we incubated HUVEC cells with the conditioned medium derived from cells treated with ponasterone (pon+) for 36 h or 48 h when compared to medium derived from cells cultured in the absence of ponasterone (pon−) (36 h, P = 0.025; 48 h, P = 0.001) (Fig. 5a). We also evaluated the different conditioned media samples for their potential to induce endothelial cell migration. Conditioned medium from ponasteronetreated cells significantly increased HUVEC cell migration when compared to the activity of medium derived from untreated cells (optical density = 1.3 ± 0.26 versus 0.55 ± 0.22; P = 0.00025) (Fig. 5b). Finally, we carried out in vivo matrigel assays (Fig. 5c). Briefly, matrigel plugs containing conditioned medium from H1299 clone 41 cells, cultured in the absence or presence of ponasterone, were inoculated subcutaneously in mice and recovered after 5 d. Matrigel plugs containing the conditioned medium derived from cells expressing mutant p53 (pon+) showed an approximately three-fold increase in hemoglobin content when compared to the corresponding untreated counterparts (optical density = 0.4 ± 0.09 versus 0.12 ± 0.2; P = 0.026).

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Figure 4 E2F1 is required for mutant Region C 2 WB: E2F1 p53-mediated ID4 transcription. (a) ChIP CDE 1 analysis using antibodies directed against WB: GAPDH 0 Pon A – + Pon A – + p53 and E2F1 in SKBr3 cells following E2F1 interference (48 h). Regions A (above) Region A 60 and C (below) of the ID4 promoter were 50 Region C 40 amplified by PCR. (b) EMSA was performed 30 20 4 with a probe containing the CDE consensus 10 3 0 present in the region C of ID4 promoter. 2 1 Nuclear extracts from proliferating SKBr3 1 1 cells were incubated with the CDE probe * 0 WB: ID4 2 alone (lane 2) or in the presence of excess 2 – + Pon A – + siScr siE2F1 unlabeled CDE probe (lanes 3 and 4) or WB: p53 Pon A – + – + an unrelated probe containing a CCAAT WB: E2F1 WB: p53 consensus (lane 5); anti-E2F1 antibody (lane 7); WB: HSP70 anti-p53 antibody (lane 8); both anti-p53 and WB: HSP70 siScr siE2F1 anti-E2F1 antibodies (lane 9); anti-NF-YB 12 34 5 67 8 9 10 11 12 13 antibody as control (lane 13). Asterisk indicates the supershift band obtained in the presence of anti-p53 antibody. NE, nuclear extract; auto, self competition. (c) qRT-PCR analysis of ID4 expression was performed in H1299 cells expressing ponasterone A (Pon A)-inducible p53-R175H (clone 41), treated with E2F1 or control (scramble (Scr)) siRNAs. p53-R175H protein expression is shown below. (d) qRT-PCR and western blot (WB) analyses of E2F1 following p53-R175H expression in H1299 clone 41 cells. (e) qRT-PCR analysis of ID4 expression in SKBr3 cells following interference of E2F1 with two different siRNAs (indicated as siE2F1-1 and siE2F1-2). Relative ID4 mRNA levels were calculated by normalization for the amount of GAPDH transcript present in the RNA preparations. (f) ID4 protein expression was evaluated by western blot analysis following E2F1 interference. Error bars represent s.d.

ID4 contributes to mutant p53-R175H–mediated neo-angiogenesis We asked whether ID4 upregulation has a role in mutant p53-mediated neo-angiogenesis. First, we showed that ID4 ectopic expression is sufficient to generate conditioned medium that can induce endothelial cell proliferation: conditioned medium from H1299 cells overexpressing ID4 increased endothelial cell proliferation when compared with medium from cells transfected with the control vector (P = 0.0004; Fig. 5d). Second, we found that mutant p53-mediated neo-angiogenesis was dependent on ID4: depletion of ID4 from cells expressing mutant p53-R157H (Fig. 5e) severely impaired the endothelial cell proliferation (Fig. 5e,f; P = 0.01) and migration (Fig. 5g; P = 0.0009) induced by the conditioned medium. We obtained similar results with overexpression of either mutant p53 or ID4 proteins or ID4 depletion in immortalized EAHy926 endothelial cells (Supplementary Fig. 4). We also examined the contribution of ID4 to angiogenesis in SKBr3 cells expressing endogenous mutant p53-R157H: depletion of ID4 expression significantly reduced the proliferation rate of HUVEC cells that was induced by conditioned medium (Fig. 5h,i; P = 0.001 for siID4/1; P = 0.0001 for siID4/1+2). These findings strongly support the idea that ID4 contributes to the angiogenic process promoted by mutant p53. ID4 induces the expression of pro-angiogenic cytokines To verify whether the enhanced neovascularization promoted by mutant p53 and ID4 is mediated by the secretion of pro-angiogenesis factors, we used a cytokine antibody array system to screen for the presence of 60 human cytokines on the ponasterone-inducible mutant p53-H1299 clone 41 cell line and on ID4-overexpressing H1299 cells. Expression of mutant p53 and ID4 in these cells induced 10 and 9 cytokines, respectively; conversely, none of the cytokines was downregulated (Supplementary Table 2a,b). Of note, SGP130, IL-2R-α, GRO-α, sTNF-RII, IL-6R and IL8 showed increased levels in both systems upon ponasterone treatment, compared to untreated cells. The effect of mutant p53 on GRO-α secretion was also confirmed by enzyme-linked immunosorbent assay (ELISA) on conditioned medium derived from ponasterone-treated and untreated H1299 clone 41 cells (Fig. 6a). Two of the identified cytokines, IL8 (also known as CXCL-8) and GRO-α (also known as CXCL-1), have pro-angiogenic activity32,33, and we investigated them further. Quantitative RT-PCR analysis showed IL8


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Figure 5 Mutant p53 and ID4 expression promote angiogenesis. (a,b) Cell proliferation and migration of HUVEC cells incubated with conditioned medium collected from H1299 clone 41 cells grown in the absence (Pon−) or presence (Pon+) of ponasterone. (a) Conditioned media samples were collected after exposure of the cells to ponasterone for 36 h or 48 h. (b) Quantification and representative microscopic pictures of HUVEC cell migration in response to conditioned medium from H1299 clone 41 cells in the absence or presence of ponasterone for 48 h. Neg indicates negative control, represented by serum-free medium. (c) Hemoglobin (Hb) content of matrigel plugs containing conditioned medium from H1299 clone 41 cells expressing ponasterone-inducible mutant p53-R175H, inoculated subcutaneously into mice and recovered 5 d later (see Supplementary Methods for further details). (d) Cell proliferation of HUVEC cells exposed to conditioned medium from control (empty vector, EV) or ID4-overexpressing (ID4) H1299 cells. Western blots (WB) showing ID4 expression are shown below. (e) ID4 mRNA and protein levels following ID4 interference in H1299 clone 41 cells. (f,g) Cell proliferation (f) and migration (g) of HUVEC exposed to conditioned medium from H1299 clone 41 cells, in the absence or presence of ponasterone. Conditioned medium from ponasterone-treated cells with knocked-down ID4 expression was also used (pon+/pLL-GFP-ID4). (h,i) Cell proliferation of HUVEC exposed to conditioned medium from control (siCont) or ID4-interfered (siID4/1, siID4/1+2) SKBr3 cells (*P = 0.005; **P = 0.001) (h). ID4 mRNA levels in SKBr3 following ID4 interference are shown in i. Results represent the mean ± s.d. of three independent experiments performed in sextuplicate (proliferation) or triplicate (migration).

and GRO-α upregulation in H1299 clone 41 cells following induction of mutant p53 (Supplementary Fig. 5a). We analyzed IL8 and GRO-α mRNA levels following ID4 overexpression and observed increased IL8 and GRO-α mRNA levels in both H1299 and SKBr3 cells, whereas expression of vascular endothelial growth factor B (VEGFB) and thrombospondin-1 (THBS-1), two regulators of angiogenesis, remained unaffected (Fig. 6b–e). We also observed an induction of IL8 and GRO-α mRNAs and proteins after transfection of increasing amounts of ID4 expression plasmid in H1299 cells (Supplementary Fig. 5b,c).

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Endothelial cells migration


a

ActD 5 µg ml–1 IL8 GRO-α VEGFB GAPDH

1090

ID4 stabilizes IL8 and GRO- transcripts Since most cytokine-encoding mRNAs are tightly controlled at the posttranscriptional level34, we asked whether ID4 was involved in the posttranscriptional regulation of IL8 and GRO-α expression. We treated SKBr3 cells overexpressing ID4 with Actinomycin D, an inhibitor of transcription, and harvested the cells at different time points for RTPCR analysis. We found that the stability of endogenous IL8 and GRO-α mRNAs was strongly increased upon ID4 overexpression (Fig. 6f). We tested whether ID4 interacts physically with IL8 and GRO-α transcripts by performing ribonucleoprotein immunoprecipitation on fixed extracts from MDA-MB-231, MDA-MB-468 and SKBr3 cells (which express mutants p53-R280K, p53-R273H and p53-R175H, respectively) using an anti-ID4 antibody. Using RT-PCR, we detected IL8 and GRO-α mRNAs bound to ID4 protein, whereas no mRNA for ribosomal protein RPL19 was detected (Fig. 7a). We used antibodies to p53 and to MyoD, a muscle-specific transcription factor, as negative controls. Many RNA-binding proteins involved in post-transcriptional regulation act by binding the 3′ untranslated (UTR) regions of Figure 6 ID4 promotes IL8 and GRO-α expression. (a) GRO-α protein levels in conditioned medium from H1299 clone 41, in absence (Pon−) or presence (Pon+) of ponasterone for 48 h. Error bars represent s.d. (b,c) GRO-α, IL8, VEGFB and THSB-1 mRNA levels in SKBr3 (b) and H1299 (c) cells overexpressing ID4 or the control vector (EV), as evaluated by RT-PCR. (d,e) ID4 overexpression in SKBr3 and H1299 cells, as assessed by western blotting (WB) for the hemagglutinin (HA) tag. (f) GRO-α, IL-8 and VEGFB mRNA levels evaluated by RT-PCR in SKBr3 cells overexpressing ID4 (SKBr3-ID4) or the control vector (SKBr3-control), treated with actinomycin D (5 µg ml−1) to block transcription, and harvested at the indicated time points (min).


RPL19 MDA-MB-231

MDA-MB-468


SKBr3

f Pon– Pon+

80S

Polysomes

IL8-3′ UTR SV40 (511 bp) polyA

HSV-TK

g 10 8 6 4 2 0

Pon– Pon+

80S

RPL19 mRNA

12 10 8 6 4 2 0

VEGFB mRNA

IL8 mRNA

e

HSV-TK

–

ID4

Renilla Luc

Polysomes

d 4 3 2 1 0

ID4

–

GRO-α mRNA

GRO-α

c 6 5 4 3 2 1 0

RLU GRO-α 3′ UTR

ID4 IL8

RLU IL8-3′ UTR

b

MyoD

p53

ID4

MyoD

p53

ID4

p53

Input

a

MyoD

articles

5 4 3 2 1 0

Renilla Luc GRO-α-3′ UTR SV40 polyA (524 bp)

Pon– Pon+

80S

Polysomes

h 6 5 4 3 2 1 0

Pon– Pon+

80S

Polysomes

MDA-MB-468

SKBr3

MDA-MB-231

Figure 7 ID4 binds and stabilizes IL-8 and GRO-α mRNAs. (a) Ribonucleoprotein immunoprecipitation (RIP) assay was peformed on MDA-MB-231, MDA-MB-468 and SKBr3 cells using the indicated antibodies to analyze RNA-protein interactions. GRO-α, IL8 and RPL19 (as a negative control) mRNA levels were evaluated by RT-PCR. (b,c) 500 ng of pRL-TK, pRL-TK-IL8 or pRL-TK-GRO-α vectors (carrying a Renilla luciferase reporter) were transiently transfected into H1299 cells, with 500 ng of pGL2 control plasmid (carrying a firefly luciferase reporter), in the absence or in presence of increasing amounts of ID4 expression vector (0.5 µg, 1.0 µg and 1.5 µg). The cells were assayed for luciferase activities 48 h after transfection, with Renilla luciferase activity normalized over firefly luciferase activity. Relative luciferase units (RLUs) represent the fold induction over the activity of the pRL-TK control plasmid. (d–g) qRT-PCR analysis of GRO-α, IL8, VEGFB and RPL19 mRNAs in ribosomal and polysomal cell fractions. Relative mRNA levels were calculated by normalization for the amount of GAPDH transcript present in the RNA preparations. (h) Analysis of ID4 protein expression by immunocytochemistry in MDA-MB-231, MDA-MB-468 and SKBr3 cells. Error bars represent s.d.

their target transcripts34–38. Both IL8 and GRO-α 3′ UTRs present AU-rich elements (ARE), which have been shown to be involved in the control of stability and expression of mRNAs. Our query of the ARE database (http://brp.kfshrc.edu.sa/ARED/) revealed the presence of a specific type of ARE (class II, cluster 2) in both the IL8 and GRO-α 3′ UTRs. We generated two reporter constructs containing Renilla luciferase cDNA followed by 511 bp or 524 bp from the IL8 or GRO-α 3′ UTRs, respectively (Fig. 7b,c). We individually transfected these constructs or the control vector into H1299 cells along with increasing amounts of ID4 expression plasmid. ID4 expression caused a dose-dependent increase in the luciferase activity of both constructs (Fig. 7b,c), indicating that the IL8 and GRO-α 3′ UTRs are regulated by the ID4 protein. We obtained similar results with the IL8 3′ UTR in MDA-MB-468 cells (Supplementary Fig. 5d). ARE sequences have been implicated in the translational efficiency of some transcripts36–38. To evaluate whether the increased levels of IL8 and GRO-α following mutant p53 and ID4 expression could also rely on enhanced translational efficiency, were isolated ribosome- and polysome-associated cell fractions to evaluate the distribution of IL8 and GRO-α mRNAs in fractions presenting different translational rates in mutant p53-inducible H1299 clone 41 cells. We observed a reproducible increase of GRO-α and IL8 mRNA levels in the polysomal, actively translating fractions upon induction of mutant p53 (Pon+), compared to the non-induced counterparts (Pon−) (Fig. 7d,e). The transcript for the VEGFB cytokine, whose expression is not affected by mutant p53 or ID4 induction, and the transcript encoding the ribosomal protein RPL19 did not show any change in distribution (Fig. 7f,g), thereby highlighting the specificity of the effect obtained for GRO-α and IL8 mRNAs. It has been previously reported that ID4 protein localizes preferentially in the nucleus. By staining SKBr3, MDA-MB-231 and MDA-MB-468 cells for ID4 protein, we found that ID4 was present mainly in the cytoplasm in SKBr3 and MDA-MB-231 cells, whereas MDA-MB-468 cells show homogeneous nuclear and cytoplasmic staining of ID4 (Fig. 7h). Both of these conditions are compatible with the ability of ID4 to bind and stabilize mRNAs.

These findings indicate that the production of pro-angiogenic factors, mainly caused by an increase in the stability of their transcripts and in translation efficiency, could be the key molecular event underlying the neo-angiogenesis that is promoted by ID4 protein. ID4 correlates with p53 and high MVD in breast cancer To further evaluate the relevance of the findings discussed above in tumor biology, we evaluated ID4 protein expression by immuno histochemistry on a tissue microarray (TMA) containing 186 breast cancer specimens (102 luminal, 54 Her-2 and 30 basal-like subtypes, as described in Supplementary Table 3a). We found a higher percentage of ID4-expressing tissues in the Her-2 group compared to the luminal and basal-like subtypes (Supplementary Table 3b). We also analyzed these breast cancer specimens for p53 staining, as p53 overexpression is frequently the result of a mutation that confers high stability to the protein. We found a higher percentage of ID4-positive specimens in the p53-overexpressing group (45 cases) compared to the p53-negative group (141 cases) (Fig. 8 and Supplementary Table 3c). This result was much stronger when the correlation analysis was performed on breast cancer Her-2 subtype specimens separately (Fig. 8 and Supplementary Table 3d). We investigated whether ID4 expression correlates with increased vascularization. We stained CD31, a well-known marker for blood vessels, on 110 of the 186 breast cancer specimens, to quantify microvessel density (MVD). We observed a significant increase in the percentage of patients with high MVD (≥15) in the population that was positive for ID4 staining, compared to the ID4-negative population (Fig. 8 and Supplementary Table 3e), suggesting that ID4 expression affects angiogenesis in vivo. DISCUSSION This study reveals the existence and depicts the execution of a previously uncharacterized transcriptional axis comprising mutant p53, the transcription factor E2F1 and the ID4 gene, whose activation promotes neo-angiogenesis of tumor cells. By analyzing cancer cell transcriptomes, we found that the transcript of the ID4 gene was upregulated by mutant


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BC specimens (%)

a

BC specimens (%) BC specimens (%)


p53 positive

e

45 30 15 0

ID4– ID4+ p53 pos

ID4– ID4+ p53 neg

P value: 0.021

f

Cytoplasmic ID4

g

Nuclear ID4

CD31 (ID4 neg)

i

CD31 (ID4 pos)

80 60 40 20 0

ID4– ID4+ p53 neg

90

ID4– ID4+ p53 pos

P value < 0.0001

75

h

60 45 30 15 0

MVD