Proapoptotic PUMA targets stem-like breast cancer cells to suppress metastasis
Qi Sun, … , David A. Cheresh, Jay S. Desgrosellier
Qi Sun, … , David A. Cheresh, Jay S. Desgrosellier
Published December 11, 2017
Citation Information: J Clin Invest. 2018;128(1):531-544. https://doi.org/10.1172/JCI93707.
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Research Article Oncology Stem cells

Proapoptotic PUMA targets stem-like breast cancer cells to suppress metastasis

Abstract

Breast cancer cells with stem cell properties are key contributors to metastatic disease, and there remains a need to better understand and target these cells in human cancers. Here, we identified rare stem-like cells in patients’ tumors characterized by low levels of the proapoptotic molecule p53-upregulated modulator of apoptosis (PUMA) and showed that these cells play a critical role in tumor progression that is independent of clinical subtype. A signaling axis consisting of the integrin αvβ3, Src kinase, and the transcription factor Slug suppresses PUMA in these cells, promoting tumor stemness. We showed that genetic or pharmacological disruption of αvβ3/Src signaling drives PUMA expression, specifically depleting these stem-like tumor cells; increases their sensitivity to apoptosis; and reduces pulmonary metastasis, with no effect on primary tumor growth. Taken together, these findings point to PUMA as a key vulnerability of stem-like cells and suggest that pharmacological upregulation of PUMA via Src inhibition may represent a strategy to selectively target these cells in a wide spectrum of aggressive breast cancers.

Authors

Qi Sun, Jacqueline Lesperance, Hiromi Wettersten, Elaine Luterstein, Yoko S. DeRose, Alana Welm, David A. Cheresh, Jay S. Desgrosellier

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Figure 3

PUMA is a critical Slug-dependent gene suppressed by αvβ3 to promote anchorage independence.

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PUMA is a critical Slug-dependent gene suppressed by αvβ3 to promote anc...
(A) qPCR analysis of non-EMT, Slug-dependent genes in β3-knockdown LM2-4 cells. Fold change (2–ΔΔCT) in β3-knockdown cells is relative to cells expressing a control nonsilencing shRNA. Cyclophilin A was used as a loading control. (B and C) Immunoblots showing PUMA protein levels in breast cancer cell lines expressing either control nonsilencing shRNA or β3 shRNA (B and C, top) or ectopic β3 cDNA (β3) or a vector-only control (C, bottom). β-Actin was used as a loading control. (D) Soft agar tumorsphere assay comparing PUMA shRNA (shPUMA) knockdown in control and β3-knockdown LM2-4 cells. Scale bar: 2 mm. (B–D) Data shown are representative of 3 independent experiments. (E and F) Quantitation of tumorsphere formation per field (E) and total number of adherent cells (F) after siRNA knockdown of PUMA (siPUMA) or NOXA (siNOXA) compared with control siRNA (siCtrl) in LM2-4 cells, with or without β3. (E) P = 0.0072 (siCtrl; shCtrl vs. shβ3); P = 0.0331 (shβ3; siCtrl vs. siPUMA no. 1); P = 0.0275 (shβ3; siCtrl vs. siPUMA no. 2); (E and F) *P < 0.05 and **P < 0.01. Statistical analysis was performed by 2-way ANOVA with Tukey’s multiple comparisons test. Data represent the mean ± SEM. n = 3 independent experiments (A, E, and F). Each sample was run in triplicate. See also Supplemental Figure 4.