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TP53 loss creates therapeutic vulnerability in colorectal cancer.

Liu Y, Zhang X, Han C, Wan G, Huang X, Ivan C, Jiang D, Rodriguez-Aguayo C, Lopez-Berestein G, Rao PH, Maru DM, Pahl A, He X, Sood AK, Ellis LM, Anderl J, Lu X - Nature (2015)

Bottom Line: Previous clinical applications of α-amanitin have been limited owing to its liver toxicity.However, we found that α-amanitin-based antibody-drug conjugates are highly effective therapeutic agents with reduced toxicity.Here we show that low doses of α-amanitin-conjugated anti-epithelial cell adhesion molecule (EpCAM) antibody lead to complete tumour regression in mouse models of human colorectal cancer with hemizygous deletion of POLR2A.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA.

ABSTRACT
TP53, a well-known tumour suppressor gene that encodes p53, is frequently inactivated by mutation or deletion in most human tumours. A tremendous effort has been made to restore p53 activity in cancer therapies. However, no effective p53-based therapy has been successfully translated into clinical cancer treatment owing to the complexity of p53 signalling. Here we demonstrate that genomic deletion of TP53 frequently encompasses essential neighbouring genes, rendering cancer cells with hemizygous TP53 deletion vulnerable to further suppression of such genes. POLR2A is identified as such a gene that is almost always co-deleted with TP53 in human cancers. It encodes the largest and catalytic subunit of the RNA polymerase II complex, which is specifically inhibited by α-amanitin. Our analysis of The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) databases reveals that POLR2A expression levels are tightly correlated with its gene copy numbers in human colorectal cancer. Suppression of POLR2A with α-amanitin or small interfering RNAs selectively inhibits the proliferation, survival and tumorigenic potential of colorectal cancer cells with hemizygous TP53 loss in a p53-independent manner. Previous clinical applications of α-amanitin have been limited owing to its liver toxicity. However, we found that α-amanitin-based antibody-drug conjugates are highly effective therapeutic agents with reduced toxicity. Here we show that low doses of α-amanitin-conjugated anti-epithelial cell adhesion molecule (EpCAM) antibody lead to complete tumour regression in mouse models of human colorectal cancer with hemizygous deletion of POLR2A. We anticipate that inhibiting POLR2A will be a new therapeutic approach for human cancers containing such common genomic alterations.

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POLR2Aloss cells are highly sensitive to POLR2A inhibitiona, Cell proliferation of POLR2Aneutral and POLR2Aloss cells treated with actinomycin D. b, Knockdown efficiency of POLR2A-specific shRNAs in HCT116, SW480, SW837 and SNU283 cells. c, Effect of POLR2A knockdown on the proliferation of four colorectal cancer cell lines. Cells expressing GFP and control or POLR2A-specific shRNAs were sorted and mixed with control GFP-negative cells (1:1) and the GFP positive cells were quantified at passage 2, 4 and 6. **p < 0.01, ns: not significant. d, Protein levels of POLR2A in HCT116 and SNU283 cells expressing Dox-inducible POLR2A shRNAs (1.0 μg ml−1 Dox). e, Cell proliferation of HCT116 and SNU283 cells expressing Dox-inducible POLR2A shRNA in the presence of 300 ng ml−1 Dox. **p < 0.01. f, g, Cell cycle profiles (f) and apoptosis (g) of control or POLR2A shRNA-expressing HCT116 and SNU283 cells. ** p < 0.01. Data are mean and s.d. of three independent experiments in the figure.
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Figure 7: POLR2Aloss cells are highly sensitive to POLR2A inhibitiona, Cell proliferation of POLR2Aneutral and POLR2Aloss cells treated with actinomycin D. b, Knockdown efficiency of POLR2A-specific shRNAs in HCT116, SW480, SW837 and SNU283 cells. c, Effect of POLR2A knockdown on the proliferation of four colorectal cancer cell lines. Cells expressing GFP and control or POLR2A-specific shRNAs were sorted and mixed with control GFP-negative cells (1:1) and the GFP positive cells were quantified at passage 2, 4 and 6. **p < 0.01, ns: not significant. d, Protein levels of POLR2A in HCT116 and SNU283 cells expressing Dox-inducible POLR2A shRNAs (1.0 μg ml−1 Dox). e, Cell proliferation of HCT116 and SNU283 cells expressing Dox-inducible POLR2A shRNA in the presence of 300 ng ml−1 Dox. **p < 0.01. f, g, Cell cycle profiles (f) and apoptosis (g) of control or POLR2A shRNA-expressing HCT116 and SNU283 cells. ** p < 0.01. Data are mean and s.d. of three independent experiments in the figure.

Mentions: To assess the sensitivity of cells to POLR2A inhibition, a panel of POLR2Aneutral (HCT116, SW480) and POLR2Aloss (SW837, SNU283) cells were treated with α-Amanitin. Treatment of α-Amanitin at high concentrations (≥ 1μg ml−1) caused complete cell death in all four cell lines. However, at concentrations from 0 to 1.0 μg ml−1, α-Amanitin inhibition had significantly higher levels of cell-killing effect on the POLR2Aloss cells than on the POLR2Aneutral cells (Fig. 2a, b). The half-maximum inhibitory concentration (IC50) was ~1.0 μg ml−1 for the POLR2Aneutral cells, which was ~10-fold greater than that of the POLR2Aloss cells. By contrast, the POLR2Aloss cells did not show any greater sensitivity to the treatment of actinomycin D, a nonspecific transcription inhibitor (Extended Data Fig. 3a). In direct competition assays, the POLR2Aneutral cells (HCT116, SW480) stably expressing POLR2A shRNAs only had modestly reduced proliferation, in comparison with that of the corresponding cells expressing control shRNAs (Extended Data Fig. 3b, c). However, silencing POLR2A in the POLR2Aloss cells (SNU283, SW837) led to markedly reduced proliferation. We generated HCT116 and SNU283 cell lines stably expressing doxycycline (Dox)-inducible POLR2A shRNAs (Extended Data Fig. 3d). Despite significant knockdown of POLR2A, HCT116 cells continued to proliferate, whereas SNU283 cells exhibited severe G1 cell cycle arrest and apoptosis (Fig. 2c, d and Extended Data Fig. 3e–g). Approximately 50% of decrease in POLR2A expression (30–100 ng ml−1 of Dox) remarkably reduced the proliferation of SNU283 cells, but only had a modest effect on HCT116 cells (Fig. 2d). Results of rescue experiments demonstrated that gradual re-expression of exogenous POLR2A in SNU283 and SW837 cells restored their resistance to α-Amanitin up to a level comparable to that of the POLR2Aneutral cells (Fig. 2e, f and Extended Data Fig. 4).


TP53 loss creates therapeutic vulnerability in colorectal cancer.

Liu Y, Zhang X, Han C, Wan G, Huang X, Ivan C, Jiang D, Rodriguez-Aguayo C, Lopez-Berestein G, Rao PH, Maru DM, Pahl A, He X, Sood AK, Ellis LM, Anderl J, Lu X - Nature (2015)

POLR2Aloss cells are highly sensitive to POLR2A inhibitiona, Cell proliferation of POLR2Aneutral and POLR2Aloss cells treated with actinomycin D. b, Knockdown efficiency of POLR2A-specific shRNAs in HCT116, SW480, SW837 and SNU283 cells. c, Effect of POLR2A knockdown on the proliferation of four colorectal cancer cell lines. Cells expressing GFP and control or POLR2A-specific shRNAs were sorted and mixed with control GFP-negative cells (1:1) and the GFP positive cells were quantified at passage 2, 4 and 6. **p < 0.01, ns: not significant. d, Protein levels of POLR2A in HCT116 and SNU283 cells expressing Dox-inducible POLR2A shRNAs (1.0 μg ml−1 Dox). e, Cell proliferation of HCT116 and SNU283 cells expressing Dox-inducible POLR2A shRNA in the presence of 300 ng ml−1 Dox. **p < 0.01. f, g, Cell cycle profiles (f) and apoptosis (g) of control or POLR2A shRNA-expressing HCT116 and SNU283 cells. ** p < 0.01. Data are mean and s.d. of three independent experiments in the figure.
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Figure 7: POLR2Aloss cells are highly sensitive to POLR2A inhibitiona, Cell proliferation of POLR2Aneutral and POLR2Aloss cells treated with actinomycin D. b, Knockdown efficiency of POLR2A-specific shRNAs in HCT116, SW480, SW837 and SNU283 cells. c, Effect of POLR2A knockdown on the proliferation of four colorectal cancer cell lines. Cells expressing GFP and control or POLR2A-specific shRNAs were sorted and mixed with control GFP-negative cells (1:1) and the GFP positive cells were quantified at passage 2, 4 and 6. **p < 0.01, ns: not significant. d, Protein levels of POLR2A in HCT116 and SNU283 cells expressing Dox-inducible POLR2A shRNAs (1.0 μg ml−1 Dox). e, Cell proliferation of HCT116 and SNU283 cells expressing Dox-inducible POLR2A shRNA in the presence of 300 ng ml−1 Dox. **p < 0.01. f, g, Cell cycle profiles (f) and apoptosis (g) of control or POLR2A shRNA-expressing HCT116 and SNU283 cells. ** p < 0.01. Data are mean and s.d. of three independent experiments in the figure.
Mentions: To assess the sensitivity of cells to POLR2A inhibition, a panel of POLR2Aneutral (HCT116, SW480) and POLR2Aloss (SW837, SNU283) cells were treated with α-Amanitin. Treatment of α-Amanitin at high concentrations (≥ 1μg ml−1) caused complete cell death in all four cell lines. However, at concentrations from 0 to 1.0 μg ml−1, α-Amanitin inhibition had significantly higher levels of cell-killing effect on the POLR2Aloss cells than on the POLR2Aneutral cells (Fig. 2a, b). The half-maximum inhibitory concentration (IC50) was ~1.0 μg ml−1 for the POLR2Aneutral cells, which was ~10-fold greater than that of the POLR2Aloss cells. By contrast, the POLR2Aloss cells did not show any greater sensitivity to the treatment of actinomycin D, a nonspecific transcription inhibitor (Extended Data Fig. 3a). In direct competition assays, the POLR2Aneutral cells (HCT116, SW480) stably expressing POLR2A shRNAs only had modestly reduced proliferation, in comparison with that of the corresponding cells expressing control shRNAs (Extended Data Fig. 3b, c). However, silencing POLR2A in the POLR2Aloss cells (SNU283, SW837) led to markedly reduced proliferation. We generated HCT116 and SNU283 cell lines stably expressing doxycycline (Dox)-inducible POLR2A shRNAs (Extended Data Fig. 3d). Despite significant knockdown of POLR2A, HCT116 cells continued to proliferate, whereas SNU283 cells exhibited severe G1 cell cycle arrest and apoptosis (Fig. 2c, d and Extended Data Fig. 3e–g). Approximately 50% of decrease in POLR2A expression (30–100 ng ml−1 of Dox) remarkably reduced the proliferation of SNU283 cells, but only had a modest effect on HCT116 cells (Fig. 2d). Results of rescue experiments demonstrated that gradual re-expression of exogenous POLR2A in SNU283 and SW837 cells restored their resistance to α-Amanitin up to a level comparable to that of the POLR2Aneutral cells (Fig. 2e, f and Extended Data Fig. 4).

Bottom Line: Previous clinical applications of α-amanitin have been limited owing to its liver toxicity.However, we found that α-amanitin-based antibody-drug conjugates are highly effective therapeutic agents with reduced toxicity.Here we show that low doses of α-amanitin-conjugated anti-epithelial cell adhesion molecule (EpCAM) antibody lead to complete tumour regression in mouse models of human colorectal cancer with hemizygous deletion of POLR2A.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA.

ABSTRACT
TP53, a well-known tumour suppressor gene that encodes p53, is frequently inactivated by mutation or deletion in most human tumours. A tremendous effort has been made to restore p53 activity in cancer therapies. However, no effective p53-based therapy has been successfully translated into clinical cancer treatment owing to the complexity of p53 signalling. Here we demonstrate that genomic deletion of TP53 frequently encompasses essential neighbouring genes, rendering cancer cells with hemizygous TP53 deletion vulnerable to further suppression of such genes. POLR2A is identified as such a gene that is almost always co-deleted with TP53 in human cancers. It encodes the largest and catalytic subunit of the RNA polymerase II complex, which is specifically inhibited by α-amanitin. Our analysis of The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) databases reveals that POLR2A expression levels are tightly correlated with its gene copy numbers in human colorectal cancer. Suppression of POLR2A with α-amanitin or small interfering RNAs selectively inhibits the proliferation, survival and tumorigenic potential of colorectal cancer cells with hemizygous TP53 loss in a p53-independent manner. Previous clinical applications of α-amanitin have been limited owing to its liver toxicity. However, we found that α-amanitin-based antibody-drug conjugates are highly effective therapeutic agents with reduced toxicity. Here we show that low doses of α-amanitin-conjugated anti-epithelial cell adhesion molecule (EpCAM) antibody lead to complete tumour regression in mouse models of human colorectal cancer with hemizygous deletion of POLR2A. We anticipate that inhibiting POLR2A will be a new therapeutic approach for human cancers containing such common genomic alterations.

Show MeSH
Related in: MedlinePlus