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HMGA2 functions as a competing endogenous RNA to promote lung cancer progression

A Retraction to this article was published on 10 June 2015

Abstract

Non-small-cell lung cancer (NSCLC) is the most prevalent histological cancer subtype worldwide1. As the majority of patients present with invasive, metastatic disease2, it is vital to understand the basis for lung cancer progression. Hmga2 is highly expressed in metastatic lung adenocarcinoma, in which it contributes to cancer progression and metastasis3,4,5,6. Here we show that Hmga2 promotes lung cancer progression in mouse and human cells by operating as a competing endogenous RNA (ceRNA)7,8,9,10,11 for the let-7 microRNA (miRNA) family. Hmga2 can promote the transformation of lung cancer cells independent of protein-coding function but dependent upon the presence of let-7 sites; this occurs without changes in the levels of let-7 isoforms, suggesting that Hmga2 affects let-7 activity by altering miRNA targeting. These effects are also observed in vivo, where Hmga2 ceRNA activity drives lung cancer growth, invasion and dissemination. Integrated analysis of miRNA target prediction algorithms and metastatic lung cancer gene expression data reveals the TGF-β co-receptor Tgfbr3 (ref. 12) as a putative target of Hmga2 ceRNA function. Tgfbr3 expression is regulated by the Hmga2 ceRNA through differential recruitment to Argonaute 2 (Ago2), and TGF-β signalling driven by Tgfbr3 is important for Hmga2 to promote lung cancer progression. Finally, analysis of NSCLC-patient gene-expression data reveals that HMGA2 and TGFBR3 are coordinately regulated in NSCLC-patient material, a vital corollary to ceRNA function. Taken together, these results suggest that Hmga2 promotes lung carcinogenesis both as a protein-coding gene and as a non-coding RNA; such dual-function regulation of gene-expression networks reflects a novel means by which oncogenes promote disease progression.

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Figure 1: Hmga2 promotes lung cancer cell transformation in a protein-coding-independent but let-7-site-dependent manner.
Figure 2: Hmga2 ceRNA activity enhances lung cancer progression in vivo.
Figure 3: Hmga2 ceRNA activity enhances TGF-β signalling through overexpression of Tgfbr3.
Figure 4: HMGA2 and TGFBR3 are reciprocally and coordinately upregulated in NSCLC patients.

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Sequence data have been uploaded to the Gene Expression Omnibus under accession number GSE50932.

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Acknowledgements

We thank the laboratory of T. Jacks for providing the 482N1 and 368T1 lung cancer cell lines and the laboratories of P.A. Sharp and P. Chambon for providing the KPD sarcoma cell lines. We thank the LRI FACS Facility for the BrdU analyses and the LRI Biological Resources Unit for assistance with the animal studies. We thank M.S. Ebert and E. De Bruin for critical review of the manuscript. M.S.K. is a Long-Term Fellow of the Human Frontier Science Program and the European Molecular Biology Organization. This work was funded by Cancer Research UK and by the European Commission’s Seventh Framework Programme (FP7/2007-2013) under the grant agreement Lungtarget (project no. 259770).

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Contributions

M.S.K. and J.D. designed the study. M.S.K. and E.A.-M. performed the experiments described. M.S.K., P.E. and P.C. conducted bioinformatics analyses. N.M. performed the next-generation sequencing studies. M.M.W. provided necessary reagents and conceptual advice. M.S.K. and J.D. wrote the manuscript.

Corresponding author

Correspondence to Julian Downward.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Hmga2 functions as a ceRNA in human cancer cells without changes in let-7 expression.

a, RNA was purified from 368T1 and 482N1 cells expressing the Hmga2 allelic series as in Fig. 1c and qRT–PCR was performed for a control miRNA (miR-10a) and various members of the let-7 family (let-7a, let-7c, let-7e, let-7f and let-7i). Expression of let-7 members is normalized to miR-10a. 368T1 values are normalized to empty and 482N1 values are normalized to shluc empty. Values are technical triplicates, have been performed independently three times, and represent mean ± standard deviation (s.d.) with propagated error. b, H1299 cells infected with control shRNA (shluc) or two different shRNAs targeting human HMGA2 (shHMGA2.1 and shHMGA2.2). Cells were then stably transfected with the Hmga2 allelic series as in Fig. 1a, b. Lysates were then prepared and Western analysis was performed for HMGA2, Rho, Tgfbr3, phosphorylated Smad2 (pSmad2) and Smad2 as in Figs 1b and 3a. c, H1299 cells with the Hmga2 allelic series described in b were plated in soft agar as in Fig. 1d. Values are technical triplicates, have been performed independently twice, and represent mean ± s.d. d, SK-MES-1 cells were infected with shRNAs and stably transfected with the Hmga2 allelic series and Western blot analysis was performed as in b. e, SK-MES-1 cells with the Hmga2 allelic series described in d were plated in soft agar as above. Values are technical triplicates, have been performed independently twice, and represent mean ± s.d. ***P < 0.0005; **P < 0.005; *P < 0.05.

Extended Data Figure 2 Let-7 regulates Hmga2 ceRNA activity, which functions through the 3′ UTR and controls Nkx2.1-mediated anchorage-independent growth.

a, Lysates were prepared from the 368T1 Hmga2 WT and Hmga2 ATG WT; 482N1 shluc empty, shHmga2 WT and shHmga2 ATG WT cells reverse transfected with either control miRNA (miR-10a) or let-7e. Western blot analysis was subsequently performed for pSmad2, Smad2, Tgfbr3 and Rho. b, Cells were transfected as in a and soft-agar analysis was performed as in Fig. 1d. Values are technical triplicates, have been performed independently three times, and represent mean ± s.d. c, 368T1 cells were transfected with empty vector, Hmga2 WT (as in Fig. 1b), the wild-type 3′ UTR of Hmga2 (WT 3′ UTR) or the let-7-site-mutated 3′ UTR (m7 3′ UTR). Soft-agar analysis was performed as in Fig. 1d. Values are technical triplicates, have been performed independently twice, and represent mean ± s.d. d, 482N1 cells were infected with empty retrovirus or a retrovirus overexpressing Nkx2.1. The Hmga2 allelic series was then introduced into Nkx2.1 cells (with just empty vector in the empty retrovirus cells) and soft-agar analysis was performed as in Fig. 1d. Values are technical triplicates, have been performed independently three times, and represent mean ± s.d. ***P < 0.0005; **P < 0.005; *P < 0.05.

Extended Data Figure 3 Hmga2 ceRNA activity affects proliferation in suspension without altering K-Ras signalling.

a, 368T1 and 482N1 cells with the Hmga2 allelic series from Fig. 1 were treated with 10 μM BrdU for 1 h. Cells were subsequently analysed for percentage BrdU positive cells. Values are mean ± s.d (n = 3). b, 482N1 cells with the Hmga2 allelic series from Fig. 1 were plated under normal adherent conditions (adherent) or on plates coated with polyHEMA (suspension) overnight. Cells were then treated with BrdU as in a and subsequently analysed for percentage BrdU-positive cells. Values are technical triplicates, have been performed independently three times, and represent mean ± s.d (n = 3). ***P < 0.0005; **P < 0.005; *P < 0.05. c, Lysates were prepared from the 368T1 and 482N1 Hmga2 allelic series and from 482N1 cells treated with either DMSO, 1 μM AZD6244 or 1 μM PIK-75 for 2 h. Western blot analysis was subsequently performed for phosphorylated Akt (pAkt), Akt, phosphorylated ERK1 and ERK2 (pERK1/2), ERK1/2, phosphorylated ribosomal protein S6 (pS6), ribosomal protein S6 (S6), K-Ras and Rab11.

Extended Data Figure 4 Hmga2 ceRNA function specifically upregulates let-7 target transcripts.

RNA-seq was performed for cells from the 482N1 Hmga2 allelic series. a, The set of transcripts expressed in shluc empty and shHmga2 empty cells were ranked in order of differential expression and Sylamer analysis was performed. Sylamer plots were then generated across the transcript set for all octomer miRNA strings statistically enriched (P < 0.001 by hypergeometric testing). The Sylamer plot for mmu.let-7a.8(A1) is delineated in red. b, The set of transcripts expressed in shHmga2 WT and shHmga2 empty cells were ranked in order and Sylamer analysis and plots were generated as in a. c, The set of transcripts expressed in shHmga2 ATG WT and shHmga2 empty cells were ranked in order and Sylamer analysis and plots were generated as in a. d, The set of transcripts expressed in shHmga2 m7 and shHmga2 empty cells were ranked in order and Sylamer analysis and plots were generated as in a. e, The set of transcripts expressed in shHmga2 ATG m7 and shHmga2 empty cells were ranked in order and Sylamer analysis and plots were generated as in a. f, Copies-per-cell analysis of Hmga2, Tgfbr3, and let-7 family members in 482N1 cells. Expression levels of transcripts in 482N1 cells were compared to a dilution series of in vitro transcribed mRNA (Hmga2 and Tgfbr3) or chemically synthesized microRNA (let-7a, let-7c, let-7e, let-7f and let-7i). Comparison of expression in 482N1 cells to the respective standard curves allowed calculation of copies per cell. Values are technical triplicates, have been performed independently three times, and represent mean with the 99% confidence interval. Confidence intervals were determined using a t-statistic.

Extended Data Figure 5 Hmga2 regulates target transcripts through both protein and ceRNA function to promote TGF-β signalling.

a, Effect of Hmga2 depletion on potential Hmga2 target genes from Supplementary Table 5. RNA was purified from 482N1 shluc and shHmga2 cells and qRT–PCR was performed for Gapdh and the overlapping set of transcripts upregulated in metastasis-generating lung cancer cells (Tmet) and predicted let-7 target transcripts described in Supplementary Table 5. Values are normalized to shluc. Values are technical triplicates, have been performed independently three times, and represent mean ± standard deviation (s.d.) with propagated error. b, Effect of re-expression of either Hmga2 WT or ATG WT on target transcripts in knockdown cells. RNA was purified from 482N1 shluc empty, shHmga2 empty, shHmga2 WT and shHmga2 ATG WT cells and qRT–PCR was performed for Gapdh and the Hmga2 regulated transcripts from a. Values are normalized to shluc empty. Values are technical triplicates, have been performed independently three times, and represent mean ± standard deviation (s.d.) with propagated error. c, Induction of target transcripts by let-7 loss-of-function. RNA was purified from 482N1 shHmga2 cells expressing tough decoys targeting miR-122 or let-7 and qRT–PCR was performed on the transcripts from a. Values are normalized to the miR-122 tough decoy. Values are technical triplicates, have been performed independently three times, and represent mean ± standard deviation (s.d.) with propagated error. d, Suppression of target transcripts by let-7 overexpression. RNA was purified from 482N1 cells transfected with either miR-10a or with let-7e and qRT–PCR was performed on the transcripts from a. Values are normalized to the miR-10a expressing cells. Values are technical triplicates, have been performed independently three times, and represent mean ± standard deviation (s.d.) with propagated error. e, Hmga2 WT and ATG WT specifically induce expression of a TGF-β luciferase reporter (CAGA12) but not a control luciferase reporter (GL3) in both 368T1 and 482N1 cells. Values are normalized to co-transfected pRL-CXCR4 plasmid30. 368T1 values are normalized to empty and 482N1 values are normalized to shluc empty. Values are technical triplicates, have been performed independently three times, and represent mean ± s.d. with propagated error. f, RNA was purified from 368T1 and 482N1 cells expressing the Hmga2 allelic series as in Fig. 1c and qRT–PCR was performed for various TGF-β target genes. Values are normalized to Gapdh. 368T1 values are normalized to empty and 482N1 values are normalized to shluc empty. Values are technical triplicates, have been performed independently three times, and represent mean ± standard deviation (s.d.) with propagated error. ***P < 0.0005; **P < 0.005.

Extended Data Figure 6 Hmga2 ceRNA activity does not induce an epithelial–mesenchymal transition and regulates the Tgfbr3 3′ UTR in a miRNA-dependent manner.

a, Lysates were prepared from the 368T1 and 482N1 Hmga2 allelic series and from Dicer1 expressing and Dicer1-null sarcoma cells (KPD F/- and -/-). Western blot analysis was subsequently performed for mesenchymal markers N-cadherin and vimentin, epithelial marker E-cadherin, and loading control Rho. b, Hmga2 WT and ATG WT specifically induce expression of a luciferase Tgfbr3 3′ UTR reporter in a let-7-site-dependent manner in KPD F/- cells but not KPD -/- cells. Cells were transfected with Renilla constructs of the control siCXCR4 multimer_ENREF_3030 and either the Tgfbr3 wild-type or let-7-mutant 3′ UTR reporter. Values are normalized to co-transfected pGL3 plasmid. KPD F/- and -/- values are each normalized to empty. Values are technical triplicates, have been performed independently three times, and represent mean ± s.d. with propagated error. ***P < 0.0005.

Extended Data Figure 7 Hmga2 ceRNA function does not alter Ago2 occupancy of let-7 family members.

Lysates from 368T1 and 482N1 cells of the Hmga2 allelic series underwent either control immunoprecipitation (IgG) or immunoprecipitation for Ago2 as in Fig. 3d. RNA was purified and qRT–PCR was performed for miR-10a and let-7 family members on both the immunoprecipitated and input RNAs. Values are depicted as the percentage of input RNA, are technical triplicates, have been performed independently three times, and represent mean ± s.d.

Extended Data Figure 8 Depletion of Tgfbr3 specifically represses TGF-β signalling and anchorage-independent growth without affecting general proliferation.

a, Tgfbr3 depletion reduces TGF-β reporter activity in 368T1 WT and ATG WT and 482N1 cells. Cells were infected with the listed shRNAs and transfected with either GL3 or CAGA12 luciferase reporters as in Extended Data Fig. 5e. Values are normalized to co-transfected pRL-CXCR4 plasmid. Values are normalized to shluc. Values are technical triplicates, have been performed independently three times, and represent mean ± s.d. with propagated error. b, Tgfbr3 knockdown suppresses TGF-β target gene expression. RNA was purified from 368T1 WT and ATG WT and 482N1 cells expressing the listed shRNAs and qRT–PCR was performed for various TGF-β target genes as in Extended Data Fig. 5f. Values are normalized to Gapdh and shluc. Values are technical triplicates, have been performed independently three times, and represent mean ± standard deviation (s.d.) with propagated error. c, Tgfbr3 knockdown does not generally affect proliferation. Cells were infected with the listed shRNAs and BrdU analysis was performed as in Extended Data Fig. 3a. Values are technical triplicates, have been performed independently three times, and represent mean ± s.d (n = 3). d, 482N1 cells were transfected with a scrambled siRNA (siscr) or siRNAs targeting Hmga2, Angptl2, Col1a2, Fndc3, Hmga1, Skil or Tgfbr3. RNA was purified and qRT–PCR was performed for Gapdh and the relevant target transcripts. Values are normalized to both Gapdh and specific transcript expression in siscr cells. Values are technical triplicates, have been performed independently twice, and represent mean ± standard deviation (s.d.) with propagated error. e, 482N1 cells transfected with the siRNAs described in d and were plated in soft agar, and the number of colonies was analysed as in Fig. 1d. Values are technical triplicates, have been performed independently twice, and represent mean ± s.d. ***P < 0.0005; **P < 0.005; *P < 0.05.

Extended Data Figure 9 Inhibition of TGF-β signalling suppresses Hmga2 ceRNA-driven lung cancer cell transformation without altering general proliferation.

a, SB-431542 (‘SB’) suppresses TGF-β pathway activation. 368T1 cells were treated with or without TGF-β ± SB, and 482N1 cells were treated with or without SB. Twenty-four hours later, lysates were prepared and Western blot analysis was performed for pSmad2 and total Smad2. b, SB suppresses TGF-β reporter activity. 368T1 WT and ATG WT and 482N1 cells were treated with or without SB for 24 hours and transfected with either GL3 or CAGA12 luciferase reporters as in Extended Data Fig. 5e. Values are normalized to co-transfected pRL-CXCR4 plasmid. Values are normalized to vehicle treatment (−SB). Values are technical triplicates, have been performed independently three times, and represent mean ± s.d. with propagated error. c, SB suppresses TGF-β target gene expression. 368T1 WT and ATG WT and 482N1 cells were treated with or without SB for 24 hours. RNA was purified and qRT–PCR was performed for various TGF-β target genes as in Extended Data Fig. 5f. Values are normalized to Gapdh and to vehicle treatment (−SB). Values are technical triplicates, have been performed independently three times, and represent mean ± standard deviation (s.d.) with propagated error. d, SB suppresses anchorage-independent growth driven by Hmga2 ceRNA activity. 368T1 WT and ATG WT and 482N1 cells were plated for soft-agar colony growth in the continual absence or presence of SB. Soft-agar colony analysis was performed as in Fig. 1d. Values are technical triplicates, have been performed independently three times, and represent mean ± s.d. e, SB does not generally affect proliferation. 368T1 WT and ATG WT and 482N1 cells were treated with or without SB for 24 hours and BrdU analysis was performed as in Extended Data Fig. 3a. Values are technical triplicates, have been performed independently three times, mean ± s.d (n = 3). f, SB treatment suppresses multiple Hmga2 ceRNA target genes. 482N1 cells were treated with or without SB for 24 hours. RNA was purified and qRT–PCR was performed for Hmga2 and Hmga2 ceRNA targets. Values are normalized to vehicle treatment (−SB). Values are technical triplicates, have been performed independently twice, and represent mean ± standard deviation (s.d.) with propagated error. g, TGF-β treatment induces multiple Hmga2 ceRNA target genes. 368T1 cells were treated with vehicle (DMSO) or TGF-β for 24 hours. RNA was purified and qRT–PCR was performed for Hmga2 and Hmga2 ceRNA targets. Values are normalized to Gapdh and vehicle treatment (DMSO). Values are technical triplicates, have been performed independently twice, and represent mean ± standard deviation (s.d.) with propagated error. ***P < 0.0005; **P < 0.005; *P < 0.05.

Extended Data Figure 10 HMGA2 and TGFBR3 are reciprocally and coordinately upregulated in an independent NSCLC-patient gene-expression data set.

a, The Director’s Challenge lung adenocarcinoma gene expression data set was sorted on HMGA2 expression. The top and bottom quartiles (HMGA2 low and high, respectively) were selected (117 patients per group) and HMGA2 expression was compared using box-and-whisker plots. b, The Director’s Challenge data set was sorted into top and bottom quartiles of HMGA2 expression as in a, and TGFBR3 expression was compared using box-and-whisker plots. c, The Director’s Challenge data set was sorted into top and bottom quartiles of TGFBR3 expression (TGFBR3 low and high, respectively) as in a, and TGFBR3 expression was compared using box-and-whisker plots. d, The Director’s Challenge data set was sorted into top and bottom quartiles of TGFBR3 expression as in c, and HMGA2 expression was compared by box-and-whisker plots. In all box-and-whisker plots, values are presented on a log2 scale. Significance was assessed by the Mann–Whitney test with a Bonferroni correction for multiple-hypothesis testing. e, Correlation analysis of HMGA2 and TGFBR3 expression was performed on all samples in the TCGA data set. All values are presented on a log2 scale. Significance was assessed by Spearman correlation analysis. f, Correlation analysis of HMGA2 and TGFBR3 and Spearman analysis were performed on all samples in the Director’s Challenge data set as in e. ***P < 0.0005; **P < 0.005.

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Kumar, M., Armenteros-Monterroso, E., East, P. et al. HMGA2 functions as a competing endogenous RNA to promote lung cancer progression. Nature 505, 212–217 (2014). https://doi.org/10.1038/nature12785

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