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LETTER Doi:10.1038/Nature14418 LETTER doi:10.1038/nature14418 TP53 loss creates therapeutic vulnerability in colorectal cancer Yunhua Liu1, Xinna Zhang2,3, Cecil Han1, Guohui Wan1, Xingxu Huang4, Cristina Ivan2,3, Dahai Jiang2,3, Cristian Rodriguez-Aguayo3,5, Gabriel Lopez-Berestein3,5, Pulivarthi H. Rao6, Dipen M. Maru7, Andreas Pahl8, Xiaoming He9, Anil K. Sood1,2,3, Lee M. Ellis10, Jan Anderl8 & Xiongbin Lu1,3 TP53, a well-known tumour suppressor gene that encodes p53, is POLR2A (Fig. 1c). However, no homozygous deletion of POLR2A was frequently inactivated by mutation or deletion in most human observed, consistent with the notion that POLR2A is essential for cell tumours1,2. A tremendous effort has been made to restore p53 survival. Analysis of TCGA and CCLE databases revealed that express- activity in cancer therapies3–7. However, no effective p53-based ion of POLR2A is tightly correlated with its gene copy number therapy has been successfully translated into clinical cancer treat- (Fig. 1d). This positive correlation was also validated in 20 pairs of ment owing to the complexity of p53 signalling. Here we dem- matched normal and CRC tissue samples and human CRC tissue onstrate that genomic deletion of TP53 frequently encompasses microarray (Fig. 1e and Extended Data Fig. 1). POLR2Aloss (hemizy- essential neighbouring genes, rendering cancer cells with hemizy- gous loss) cell lines expressed POLR2A proteins at significantly lower gous TP53 deletion vulnerable to further suppression of such levels than POLR2Aneutral cell lines (Fig. 1f–h). Unlike POLR2A, p53 genes. POLR2A is identified as such a gene that is almost always levels are determined by post-transcriptional and post-translational co-deleted with TP53 in human cancers. It encodes the largest and events16. Despite a correlation between TP53 copy number and catalytic subunit of the RNA polymerase II complex, which is spe- mRNA expression, p53 protein levels are not associated with its gene cifically inhibited by a-amanitin8,9. Our analysis of The Cancer copy numbers in human CRC (Extended Data Fig. 2 and Fig. 1h). Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia To assess the sensitivity of cells to POLR2A inhibition, a panel of (CCLE) databases reveals that POLR2A expression levels are POLR2Aneutral (HCT116, SW480) and POLR2Aloss (SW837, SNU283) tightly correlated with its gene copy numbers in human colorectal cells were treated with a-amanitin. Treatment of a-amanitin at high cancer. Suppression of POLR2A with a-amanitin or small inter- concentrations ($1 mgml21) caused complete cell death in all four cell fering RNAs selectively inhibits the proliferation, survival and lines. However, at concentrations ranging from 0 to 1.0 mgml21, tumorigenic potential of colorectal cancer cells with hemizygous a-amanitin inhibition had significantly higher levels of cell-killing TP53 loss in a p53-independent manner. Previous clinical applica- effect on the POLR2Aloss cells than on the POLR2Aneutral cells 10 tions of a-amanitin have been limited owing to its liver toxicity . (Fig. 2a, b). The half-maximum inhibitory concentration (IC50)was However, we found that a-amanitin-based antibody–drug conju- ,1.0 mgml21 for the POLR2Aneutral cells, which was tenfold greater gates are highly effective therapeutic agents with reduced toxicity11. than that of the POLR2Aloss cells. By contrast, the POLR2Aloss cells did Here we show that low doses of a-amanitin-conjugated anti-epi- not show any greater sensitivity to the treatment of actinomycin D, a thelial cell adhesion molecule (EpCAM) antibody lead to complete nonspecific transcription inhibitor (Extended Data Fig. 3a). In direct tumour regression in mouse models of human colorectal cancer competition assays, the POLR2Aneutral cells (HCT116, SW480) stably with hemizygous deletion of POLR2A. We anticipate that inhib- expressing POLR2A short hairpin RNAs (shRNAs) only had modestly iting POLR2A will be a new therapeutic approach for human can- reduced proliferation, in comparison with that of the corresponding cers containing such common genomic alterations. cells expressing control shRNAs (Extended Data Fig. 3b, c). However, Genomic deletion of a tumour suppressor gene often encompasses silencing POLR2A in the POLR2Aloss cells (SNU283, SW837) led to several neighbouring genes that may not contribute to cancer develop- markedly reduced proliferation. We generated HCT116 and SNU283 ment, but are essential for cell proliferation and survival12. This partial cell lines stably expressing doxycycline (Dox)-inducible POLR2A loss of essential genes has been postulated to render cancer cells highly shRNAs (Extended Data Fig. 3d). Despite significant knockdown of vulnerable to further inhibition of those genes13,14. Analysis of TCGA POLR2A, HCT116 cells continued to proliferate, whereas SNU283 revealed that hemizygous deletion of the TP53 gene occurs frequently cells exhibited severe G1 cell cycle arrest and apoptosis (Fig. 2c, d in human cancers (Fig. 1a). We identified POLR2A as an essential gene and Extended Data Fig. 3e–g). Approximately 50% of the decrease in the proximity of TP53 (Fig. 1b). Concomitant deletion of POLR2A in POLR2A expression (30–100 ng ml21 of Dox) remarkably reduced occurs in virtually all the human colorectal tumours containing hemi- the proliferation of SNU283 cells, but only had a modest effect on zygous TP53 deletion (Fig. 1c). HCT116 cells (Fig. 2d). Results of rescue experiments demonstrated Among the 12 subunits in the human RNA polymerase II complex, that gradual re-expression of exogenous POLR2A in SNU283 and POLR2A encodes the largest subunit that is indispensable for the poly- SW837 cells restored their resistance to a-amanitin up to a level com- merase activity in messenger RNA synthesis. Inhibiting POLR2A with parable to that of the POLR2Aneutral cells (Fig. 2e, f and Extended a specific inhibitor, a-amanitin, causes extensive cell death, and homo- Data Fig. 4). zygous deletion of POLR2A is lethal in human cells9,15. We found that To exclude genetic difference across cell lines, we used the CRISPR 104 (53%) out of 195 colorectal cancer (CRC) cases bear hemizygous (clustered regularly interspaced short palindromic repeat)/Cas9 sys- loss of the 17p13 region, resulting in concomitant deletion of TP53 and tem to generate an isogenic HCT116 cell line with hemizygous loss of 1Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA. 2Department of Gynaecologic Oncology and Reproductive Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA. 3Center for RNA Interference and Non-coding RNAs, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA. 4School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China. 5Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA. 6Department of Paediatrics, Baylor College of Medicine, Houston, Texas 77030, USA. 7Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA. 8Heidelberg Pharma GmbH, Ladenburg 68526, Germany. 9Department of Biomedical Engineering, The Ohio State University, Columbus, Ohio 43210, USA. 10Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA. 30 APRIL 2015 | VOL 520 | NATURE | 697 G2015 Macmillan Publishers Limited. All rights reserved RESEARCH LETTER a 70 b 60 Chr 17 50 40 SENP3 30 20 POLR2A TNFSF12 SHBG TP53 10 Alteration frequency (%) 0 200 kb c TCGA colon (n = 195) AML GBM Liver CCLE DLBCOvary POLR2A 53% GliomaBreast Cervix Uterine Bladder Prostate Pancreas Sarcoma Stomach TP53 53% Colorectal Renal pap. Melanoma Renal clear Lung adeno. Hemizygous deletion Head &Renal neck chrom. Lung squamous TCGA colon (n = 195) CCLE (n = 1,043) P < 0.005 de2.0 r 6.0 r 6.5 = 0.542 = 0.365 P P (ratio)) < 0.0001 (ratio)) < 0.001 2 5.5 2 5.5 1.5 4.5 5.0 1.0 3.5 4.5 0.5 2.5 (tumour/normal) 1.5 4.0 RNA expression (log RNA expression (log 0.0 –1.2 –0.8-0.4 0.0 0.4 –2.0 –1.5 –1.0 –0.5 0.0 0.5 1.0 Relative POLR2A expression Neutral Loss DNA copy number (log2(ratio)) DNA copy number (log2(ratio)) fhg 2.5 TP53neutral TP53loss TP53 1.5 POLR2A 2.0 POLR2A 1.5 1.0 HT29 HCT116DLD1SW480SW620SW837SNU283SNU1197 1.0 0.5 POLR2A 0.5 p53 0.0 Relative mRNA level 0.0 Genomic copy number β-actin HT29 DLD1 HT29 DLD1 SW480 HCT116 SW480SW837SNU283 HCT116 SW837SNU283 SNU1197 SNU1197 Figure 1 | Expression of POLR2A, but not TP53, is correlated with the gene mRNA expression in TCGA and CCLE databases. Pearson correlation copy number. a, Frequencies of hemizygous deletion of TP53 in human coefficients (r) and P values are shown. e, POLR2A protein levels in matched cancers. AML, acute myeloid leukaemia; DLBC, diffuse large B-cell lymphoma; normal and CRC tissue samples. Error bars denote s.d. f, g, Copy numbers GBM, glioblastoma. b, Schematic diagram of genes adjacent to TP53 in human (f) and relative mRNA expression levels (g)ofPOLR2A in human CRC cell genome. c, Concomitant deletion of POLR2A in human CRCs containing lines. Data are mean and s.d. of three independent experiments. h, Protein hemizygous loss of TP53. d, Scatterplots of POLR2A copy number versus levels of POLR2A and p53 in human CRC cell lines. POLR2A (refs 17, 18) (Extended Data Fig. 5a–c). POLR2A expression deletion of TP53. By contrast, hemizygous loss of POLR2A markedly was significantly reduced in the POLR2Aloss HCT116 cells (Extended sensitized these cells to a-amanitin treatment regardless of their TP53 Data Fig. 5d). The POLR2Aloss and parental HCT116 cells exhibited status (Fig. 3b and Extended Data Fig. 6d–g). The mRNA synthesis similar proliferation rates (Extended Data Fig. 5e), indicating that one activity of the RNA polymerase II is essential to any type of cells allele of POLR2A is sufficient to maintain cell proliferation. However, including therapy-resistant tumour cells.
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