PML promotes metastasis of triple-negative breast cancer through transcriptional regulation of HIF1A target genes
Manfredi Ponente, Letizia Campanini, Roberto Cuttano, Andrea Piunti, Giacomo A. Delledonne, Nadia Coltella, Roberta Valsecchi, Alessandra Villa, Ugo Cavallaro, Linda Pattini, Claudio Doglioni, Rosa Bernardi
Manfredi Ponente, Letizia Campanini, Roberto Cuttano, Andrea Piunti, Giacomo A. Delledonne, Nadia Coltella, Roberta Valsecchi, Alessandra Villa, Ugo Cavallaro, Linda Pattini, Claudio Doglioni, Rosa Bernardi
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Research Article Oncology

PML promotes metastasis of triple-negative breast cancer through transcriptional regulation of HIF1A target genes

Abstract

Elucidating the molecular basis of tumor metastasis is pivotal for eradicating cancer-related mortality. Triple-negative breast cancer (TNBC) encompasses a class of aggressive tumors characterized by high rates of recurrence and metastasis, as well as poor overall survival. Here, we find that the promyelocytic leukemia protein PML exerts a prometastatic function in TNBC that can be targeted by arsenic trioxide. We found that, in TNBC patients, constitutive HIF1A activity induces high expression of PML, along with a number of HIF1A target genes that promote metastasis at multiple levels. Intriguingly, PML controls the expression of these genes by binding to their regulatory regions along with HIF1A. This mechanism is specific to TNBC cells and does not occur in other subtypes of breast cancer where PML and prometastatic HIF1A target genes are underexpressed. As a consequence, PML promotes cell migration, invasion, and metastasis in TNBC cell and mouse models. Notably, pharmacological inhibition of PML with arsenic trioxide, a PML-degrading agent used to treat promyelocytic leukemia patients, delays tumor growth, impairs TNBC metastasis, and cooperates with chemotherapy by preventing metastatic dissemination. In conclusion, we report identification of a prometastatic pathway in TNBC and suggest clinical development toward the use of arsenic trioxide for TNBC patients.

Authors

Manfredi Ponente, Letizia Campanini, Roberto Cuttano, Andrea Piunti, Giacomo A. Delledonne, Nadia Coltella, Roberta Valsecchi, Alessandra Villa, Ugo Cavallaro, Linda Pattini, Claudio Doglioni, Rosa Bernardi

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Figure 1

PML is regulated by HIF1A in TNBC.

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PML is regulated by HIF1A in TNBC. (A) PML mRNA expression (centered by ...
(A) PML mRNA expression (centered by mean and log10 transformed) in 295 early-stage breast cancer samples subcategorized for high and low expression of a hypoxia-responsive gene signature of 123 common hypoxia-dependent genes. (B) Mean PML mRNA expression in breast cancer samples characterized by TCGA Network and subcategorized as estrogen receptor positive (ER+, n = 401), HER2 positive (HER2+, n = 76), TNBC (n = 88), and normal tissue (n = 22). P values are: TNBC vs. normal, P = 0.0013; TNBC vs. HER2+, P = 3.08 × 10–6; TNBC vs. ER+ P = 1.61 × 10–14. (C) RT-PCR analysis of PML expression across different breast cell lines. Non-TNBC, and TNBC cells are indicated. Data represent mean values ± SEM of 2 independent experiments. Statistical analysis was performed by comparing mean PML expression in the 4 TNBC cell lines versus mean PML expression in the 3 non-TNBC cells, respectively. (D) Immunoblot of PML and HIF1A across breast cancer cell lines: estrogen and progesterone receptor positive (ER+/PR+) cells MCF7 and ZR-75, HER2+ overexpressing MDA-MB-361 cells, and TNBC cells SUM-149, BT-549, SUM-149, and MDA-MB-231. Cropped blots are surrounded by a black line and retain important bands. β-Actin is used as loading control. (E) RT-PCR analysis of the indicated genes in MDA-MB-231 and MCF7 cells stably transduced with a control shRNA (white dots) or an HIF1A shRNA (gray dots). Data represent mean values ± SEM of 3 independent experiments. (F) Fold enrichment over normalized IgG levels of DNA immunoprecipitated by control IgG (white dots) or anti-HIF1A antibody (gray dots) and amplified with primers spanning either the promoter region or the gene body of PML in MDA-MB-231 and MCF7 cells. Data represent mean values ± SEM of 3 independent experiments. **P < 0.01, ***P < 0.001. Student’s 2-tailed t test was used to determine statistical significance.