The p63/p73 network mediates chemosensitivity to cisplatin in a biologically defined subset of primary breast cancers - PubMed (original) (raw)

. 2007 May;117(5):1370-80.

doi: 10.1172/JCI30866. Epub 2007 Apr 19.

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The p63/p73 network mediates chemosensitivity to cisplatin in a biologically defined subset of primary breast cancers

Chee-Onn Leong et al. J Clin Invest. 2007 May.

Abstract

Breast cancers lacking estrogen and progesterone receptor expression and Her2 amplification exhibit distinct gene expression profiles and clinical features, and they comprise the majority of BRCA1-associated tumors. Here we demonstrated that the p53 family member p63 controls a pathway for p73-dependent cisplatin sensitivity specific to these "triple-negative" tumors. In vivo, DeltaNp63 and TAp73 isoforms were coexpressed exclusively within a subset of triple-negative primary breast cancers that commonly exhibited mutational inactivation of p53. The DeltaNp63alpha isoform promoted survival of breast cancer cells by binding TAp73 and thereby inhibiting its proapoptotic activity. Consequently, inhibition of p63 by RNA interference led to TAp73-dependent induction of proapoptotic Bcl-2 family members and apoptosis. Breast cancer cells expressing DeltaNp63alpha and TAp73 exhibited cisplatin sensitivity that was uniquely dependent on TAp73. Thus, in response to treatment with cisplatin, but not other chemotherapeutic agents, TAp73 underwent c-Abl-dependent phosphorylation, which promoted dissociation of the DeltaNp63alpha/TAp73 protein complex, TAp73-dependent transcription of proapoptotic Bcl-2 family members, and apoptosis. These findings define p63 as a survival factor in a subset of breast cancers; furthermore, they provide what we believe to be a novel mechanism for cisplatin sensitivity in these triple-negative cancers, and they suggest that such cancers may share the cisplatin sensitivity of BRCA1-associated tumors.

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Figures

Figure 1

Figure 1. Coexpression of ΔNp63 and TAp73 in triple-negative primary breast tumors.

(A) Overexpression of p63 in primary microdissected invasive breast carcinomas relative to specimen-matched normal luminal epithelia. The ratio of tumor/normal p63 mRNA was determined by QRT-PCR. (B) Nuclear p63 protein correlated with p63 mRNA expression, as assessed by immunohistochemistry of representative tumors from A. Photomicrographs demonstrate low and high expression of p63 mRNA. Original magnification, ×100. (C) TAp73 was overexpressed in ER/PR-negative tumors. Shown is QRT-PCR for TAp73 in 14 ER-positive and 23 ER/PR-negative primary breast carcinomas. Statistical significance was analyzed using both a mean-value approach (ER-positive, 0.373 ± 0.126; ER/PR-negative, 1.381 ± 0.303; P = 0.0172, 2-way Student’s t test) and a binning approach whereby tumors exhibiting p73 expression more than 2-fold the mean of the sample set were categorized as high and the rest as low (P = 0.0309, Fisher’s exact test). (D) Expression of TAp73 correlated with ΔNp63 overexpression (P = 0.0107, Fisher’s exact test) and with p53 mutation (P = 0.0257, Fisher’s exact test) in ER/PR-negative primary breast carcinomas. Levels of ΔNp63 were determined by QRT-PCR, and p53 mutation was determined by cDNA sequencing. Note that TAp73/ΔNp63 coexpression was not observed in _Her2_-overexpressing tumors as assessed by QRT-PCR.

Figure 2

Figure 2. Endogenous p63 is required for survival in breast cancer cells.

(A) Knockdown of endogenous p63–induced Puma, Noxa, and PARP-1 cleavage, as assessed by immunoblot of the indicated cells 72 hours after infection with lentiviral shRNA vectors targeting 2 distinct p63 sequences (p63si-1 and p63si-2) or a nonspecific sequence (si-NS). None of these effects were observed in MCF-7 cells, which do not express abundant p63. (B) Apoptotic morphology following lentiviral p63 knockdown in HCC-1937 cells. Photomicrographs were taken 72 hours after lentiviral shRNA infection. Original magnification, ×100. (C) Apoptosis was observed following endogenous p63 knockdown, as assessed by annexin V/PI staining of unfixed cells 72 hours following infection with the indicated lentiviral shRNA vectors. Percentages indicate apoptotic cells (annexin V–positive and/or PI-positive). (D) Apoptosis following p63 inhibition was specific to breast cancer cells expressing abundant p63. Shown are results of annexin V/PI staining for 3 independent experiments. Error bars represent SD. (E) Puma and Noxa were induced following p63 knockdown. RNA was prepared 72 hours following lentiviral infection and assayed by QRT-PCR for the indicated genes. Error bars represent SD for 2 independent experiments, each performed in duplicate.

Figure 3

Figure 3. Survival of breast cancer cells is promoted by p63 through repression of TAp73-dependent apoptosis.

(A) PARP-1 cleavage, Puma and Noxa induction, and apoptosis induced by p63 knockdown were TAp73 dependent. Pools of cells expressing a TAp73-targeted shRNA (TAp73si) or the control vector were then infected with a p63-directed lentiviral shRNA or control, and lysates were harvested at 72 hours for immunoblot and IP/immunoblot (for p73). (B) Morphologic features of apoptosis were TAp73 dependent. Shown are photomicrographs of representative fields of cells treated as in A and harvested 72 hours following p63 knockdown. Original magnification, ×100. (C) Rescue from apoptosis following ablation of TAp73 but not TAp63. Quantitation of apoptosis by annexin V/PI staining of cells treated as in A and harvested 72 hours following p63 knockdown. Error bars represent SD for 3 independent experiments.

Figure 4

Figure 4. TAp73 mediates cisplatin sensitivity in breast cancer cells expressing TAp73 and ΔNp63.

(A) Inhibition of TAp73 induced resistance specifically to cisplatin. Dose-response curves (MTT cell viability assay) of cells expressing the control vector or a TAp73-directed lentiviral shRNA 5 days following treatment with cisplatin (Cis), doxorubicin (Dox), or paclitaxel (Tax). Little or no effect of TAp73 knockdown was observed in MCF-7 cells. Error bars show SD for 3 independent experiments. (B) TAp73 mediated selective proapoptotic target gene induction in response to cisplatin. QRT-PCR analysis of the indicated genes in HCC-1937 cells as in A 6 hours after cisplatin treatment (at IC70, 6.6 μM). (C) TAp73 expression conveyed specific cisplatin sensitivity to normal basal mammary epithelial cells. MCF-10A cells were infected with a retrovirus encoding TAp73β or the control vector, followed by quantitative dose-response analysis as shown in A (P < 0.01, 1-tailed Student’s t test). TAp73β increased sensitivity (i.e., decreased the IC50) only for cisplatin. Error bars show SD for 3 independent experiments.

Figure 5

Figure 5. Cisplatin induces dissociation of the ΔNp63α/TAp73 complex.

(A) Quantitative binding of TAp73 to ΔNp63α in HCC-1937 cells and dissociation following cisplatin treatment. Left, IPs of control or cisplatin-treated cultures (IC70 for 6 hours); right, corresponding post-IP supernatants (Sup). IP for either p63 or p73 resulted in complete immunodepletion of TAp73 (lanes 11 and 12). Following cisplatin treatment, less TAp73 was associated with ΔNp63α (lanes 3 and 7), resulting in detectable “free” TAp73 in the depleted post-IP supernatant (lanes 11 and 15). Note the absence of change in ΔNp63α or TAp73 protein levels following cisplatin treatment (lanes 2, 6, 10, and 14). Controls demonstrated these antibodies to be non–cross-reactive (ref. and data not shown). (B) MDA-MB-468 cells showed quantitative ΔNp63α/TAp73 binding similar to that of HCC-1937 cells and partial dissociation following cisplatin treatment. Cells were treated as in A. Left, IP product; right, post-IP supernatant. Note the decrease in TAp73 associated with ΔNp63α following cisplatin treatment (lanes 3 and 7), despite no change in ΔNp63α or TAp73 protein levels (lanes 10 and 14). HCC-1937 cells expressed TAp73α (A), while MDA-MB-468 cells expressed both TAp73α and TAp73β (B).

Figure 6

Figure 6. Cisplatin treatment specifically induces c-Abl–dependent p73 phosphorylation.

(A) TAp73 is tyrosine phosphorylated in response to cisplatin but not doxorubicin. Immunoprecipitated p73 was probed for anti–phosphorylated tyrosine (p-Tyr) by immunoblot 6 hours following control or cisplatin (Cis) or doxorubicin (Dox) treatment (both at IC70). The same blot was then stripped and reprobed for total p73 protein. HCC-1937 cells expressed TAp73α, while MDA-MB-468 cells expressed both TAp73α and TAp73β. (B) Induction of c-Abl–dependent TAp73 phosphorylation following cisplatin treatment. Cells were pretreated with imatinib (Ima; 1 μM for 2 hours) or vehicle control as indicated and then treated with cisplatin and analyzed as in A.

Figure 7

Figure 7. TAp73 phosphorylation at Y99 is required for cisplatin-induced ΔNp63α/TAp73 dissociation and cell death in MCF-10A cells.

(A) Wild-type or Y99F TAp73α were expressed in MCF-10A cells via retrovirus. Lysates from either cisplatin-treated or untreated cells were halved and subjected to IP for either p63 or p73, followed by immunoblots as shown. Wild-type TAp73α was tyrosine phosphorylated and dissociated from ΔNp63α following cisplatin treatment (1 μM, 6 hours), while Y99F TAp73α remained unphosphorylated and bound to ΔNp63α. Note there was no change in the total level of retroviral TAp73α or endogenous ΔNp63α following cisplatin treatment. (B) TAp73 Y99 phosphorylation was required to convey cisplatin sensitivity. MCF-10A cells described in A were treated with cisplatin (1 μM) for 5 days, and cell viability was assessed by MTT. Error bars show SD for 3 independent experiments.

Figure 8

Figure 8. Imatinib treatment blocks ΔNp63α/TAp73 dissociation, TAp73-dependent transcription, and cell death induced by cisplatin.

(A) Imatinib attenuated dissociation of ΔNp63α and TAp73. In left blots (control), cells were treated with imatinib (1 μM for 8 hours) or vehicle control; in right blots, cells were pretreated with imatinib (1 μM for 2 hours) or vehicle control, then treated with cisplatin (IC70 for 6 hours) prior to IP for p63 or p73. Dissociation of TAp73 and ΔNp63α following cisplatin treatment (compare lanes 3 and 11) was attenuated by imatinib treatment (lane 15). (B and C) TAp73-dependent proapoptotic transcription required c-Abl–mediated phosphorylation. HCC-1937 cells (B) or MDA-MB-468 cells (C) were pretreated with imatinib (1 μM for 2 hours) and/or treated with cisplatin (IC80 for 6 hours) as indicated, and mRNA was analyzed by QRT-PCR. (D) c-Abl–dependent phosphorylation is important for cisplatin sensitivity. Cells were pretreated with imatinib as in C, then treated with cisplatin (IC70) and analyzed for viability by MTT at 3 days. Error bars show SD for 3 independent experiments.

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