Research ArticleCancer

Pentraxin-3 is a PI3K signaling target that promotes stem cell–like traits in basal-like breast cancers

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Science Signaling  21 Feb 2017:
Vol. 10, Issue 467, eaah4674
DOI: 10.1126/scisignal.aah4674
  • Fig. 1 PI3K activation triggers PTX3 expression in BLBC cells.

    (A) Fold induction of the top 10 genes up-regulated by PIK3CAH1047R (HR) expression in MCF10A cells relative to wild-type (WT) PIK3CA expression. (B) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) measurements of PTX3 mRNA abundance in MCF10A cells stably expressing WT (13) or HR. Data are means ± SEM of n = 3 independent experiments, each performed in triplicate. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) Representative Western blot analysis of MCF10A cells stably expressing WT or HR treated with BKM120 (1 μM for 24 hours) (+) or vehicle control (−) and deprived of epidermal growth factor (EGF) and insulin (n > 3). (D) Top: Representative Western blot of SUM159 cells stably expressing control (CTRL) or PIK3CA shRNAs. Bottom: qRT-PCR measurements of PTX3 mRNA normalized to GAPDH in the indicated SUM159 variants. Data are means ± SEM of n = 3 independent experiments, each performed in triplicate. (E) Representative Western blot analysis of SUM159 cells treated with vehicle (−), BEZ235 (5 μM), BKM120 (1 μM), or BYL719 (1 μM) for 24 hours (n > 3). (F) Representative Western blot analysis of MDA-MB-231 cells treated with vehicle (−) or with BEZ235 (5 μM) for 24 hours (n > 3). (G) PTX3 transcript abundance derived from microarrays of mouse tumors (GSE41118) driven by HER2 (n = 6), HR (n = 6), or HER2 + HR (n = 7). (H) Spearman’s and Pearson’s correlation analyses of PTX3 expression with that of activated PIK3CA gene signature derived from Hutti et al. (36), PTEN BIOCARTA pathway, and INPP4B gene in the UNC337, The Cancer Genome Atlas (TCGA), and in GSE5460 (66) databases, respectively. (I) Representative Western blot analysis of SUM159 cells treated with vehicle (−) or MK2206 (1 μM) for 24 hours (n > 3). (J) Representative Western blot analysis of control or HR MCF10A cells treated with compound A (Cmpd A; 5 μM) for 24 hours (n > 3). *P < 0.05 and **P < 0.01, by unpaired t test. Exp., exposure.

  • Fig. 2 PTX3 expression is specifically associated with BLBCs.

    (A) PTX3 expression (probe 206157_at) in GSE1561 analyzed using significance analysis of microarrays (SAMs) in ROCK Breast Cancer Functional Genomics database: BLBC (n = 15), HER2 (n = 8), and LumB (n = 23); FDR, 0%. (B) PTX3 expression (probe 206157_at) in GSE1456 analyzed by SAMs: BLBC (n = 25), HER2 (n = 15), LumA (n = 39), LumB (n = 23); FDR, 0%. (C) Analysis of variance (ANOVA) box plot analyses for PTX3 (probe 206157_at) in TCGA; P = 5.63 × 10−50. (D) ANOVA box plot analyses for PTX3 (probe 206157_at) in UNC337; P = 2.97 × 10−32. (E) ANOVA box plot analysis of PTX3 (probe 206157_at) in breast cancer samples collected at the Curie Institute: BLBC (n = 78), HER2 (n = 31), LumA (n = 28), and LumB (n = 32); P = 4.89 × 10−25. (F) ANOVA box plot analysis of PTX3 (probe 206157_at) in GSE7904; BLBC (n = 18) and non-BLBC (n = 21); P = 1.723 × 10−9. (G) qRT-PCR analysis of PTX3 expression in RNA from dissected BCCs lifted off slides of the indicated tumor samples: Control (n = 5), BLBC (n = 16), HER2 (n = 13), LumA (n = 25), LumB (n = 22). *P < 0.05 and **P < 0.01, by unpaired t test. (H) qRT-PCR measurements (means ± SEM of n > 3) of PTX3 mRNA abundance in the indicated BCC lines. Values were normalized to that of 18S and are displayed as arbitrary units (A.U.). (I) PTX3 and 19 most closely associated genes across all the cancer cells represented in the Broad Institute CCLE (called here “PTX3 gene signature”) were profiled in the indicated BCC lines, grouped according to breast tumor subtype. Red and blue indicate increased and diminished expression of PTX3, respectively.

  • Fig. 3 PTX3 is particularly enriched in M/MSL/CL BCCs.

    (A) PTX3 gene expression signature in the indicated BLBC subclasses from Lehmann et al. (27). (B) Representative Western blot on PTX3 in the indicated BCC lines (n > 3). (C) ANOVA box plot of PTX3 expression (NM_00282) in PDX models of indicated breast cancer subtypes. BL1/2 (n = 13), LAR (n = 2), M/MSL (n = 11), IM (n = 1); P = 0.008. (D) PTX3 gene signature across the indicated BCC lines, grouped according to molecular classification (25). (E) qRT-PCR analyses of PTX3 mRNA in HMLE cells stably expressing the transcription factors GSC, SNAIL, and TWIST, relative to that in controls. Data are means ± SEM of triplicates from ≥3 independent experiments. *P < 0.05 and **P < 0.01, by unpaired t test. (F) ANOVA box plot analyses for PTX3 expression (probe 206157_at) in BLBC and CL subtypes from the UNC337 database; P = 1.07 × 10−4.

  • Fig. 4 PTX3 is a critical and functional effector of PI3K signaling.

    (A) Controls or PTX3-expressing MCF10A cells were plated in triplicates in low-adherence plates and scored for their ability to form tumorspheres after the indicated serial passages. Data are means ± SEM of triplicates from ≥3 independent experiments. (B and C) Controls or stable PTX3-expressing MCF10A cells were analyzed for their expression of CD44 [fluorescein isothiocyanate (FITC)] and CD24 [phycoerythrin (PE)] (B) or ALDH positivity (C) (red gate) using FACS (fluorescence-activated cell sorting). Data are representative of more than three experiments. (D) qRT-PCR measurements of PTX3 mRNA abundance in SUM159 cells stably expressing control or PTX3-specific shRNA molecules. Variants expressing the indicated shRNA were analyzed for their ALDH positivity and CD44/CD24 expression using FACS. Data are means ± SEM in triplicates of more than three independent experiments. (E) ALDH positivity of controls or MCF10A cells harboring control shRNA (shctrl) or shPTX3 constructs in the WT or HR backgrounds. Data are means ± SEM from ≥3 experiments. (F) Representative images of sulforhodamine-stained, 4-day cultured MCF10A cells stably expressing HR or controls, along with shctrl or shPTX3. Viable cell count was determined colorimetrically. Data are means ± SEM of three independent biological replicates, each performed in triplicate. (G) Control or shPTX3-expressing MCF10A cells harboring control or HR expression vectors were plated in triplicates in low-adherence plates and scored for their ability to form tumorspheres at first passage. Data are means ± SEM of triplicates from ≥3 independent experiments. (H and I) Kaplan-Meier analysis of overall survival (H) and relapse-free survival (I) in UNC337 estimated on patient cohorts whose breast tumors harbored low, medium, and high PTX3 expression. (H) P = 0.00907 and (I) P = 0.00748, calculated by log-rank test. (A, C, D, E, and G) *P < 0.05, **P < 0.01, and ***P < 0.001, by unpaired t test. SSC, side scatter.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/467/eaah4674/DC1

    Fig. S1. Gene set enrichment analysis of activated PIK3CA–regulated genes in MCF10A basal-like breast cells.

    Fig. S2. PTX3 is regulated by NF-κB–dependent pathways.

    Fig. S3. PTX3 triggers CSC-like traits.

    Data file S1. PTX3 expression across multiple cell lines in the Broad Institute CCLE.

  • Supplementary Materials for:

    Pentraxin-3 is a PI3K signaling target that promotes stem cell–like traits in basal-like breast cancers

    Clémence Thomas, Whitney Henry, Benjamin G. Cuiffo, Anthony Y. Collmann, Elisabetta Marangoni, Vanessa Benhamo, Manoj K. Bhasin, Cheng Fan, Laetitia Fuhrmann, Albert S. Baldwin, Charles Perou, Anne Vincent-Salomon, Alex Toker, Antoine E. Karnoub*

    *Corresponding author. Email: akarnoub{at}bidmc.harvard.edu

    This PDF file includes:

    • Fig. S1. Gene set enrichment analysis of activated PIK3CA–regulated genes in MCF10A basal-like breast cells.
    • Fig. S2. PTX3 is regulated by NF-κB–dependent pathways.
    • Fig. S3. PTX3 triggers CSC-like traits.
    • Legend for data file S1

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

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    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft PowerPoint format). PTX3 expression across multiple cell lines in the Broad Institute CCLE.

    Citation: C. Thomas, W. Henry, B. G. Cuiffo, A. Y. Collmann, E. Marangoni, V. Benhamo, M. K. Bhasin, C. Fan, L. Fuhrmann, A. S. Baldwin, C. Perou, A. Vincent-Salomon, A. Toker, A. E. Karnoub, Pentraxin-3 is a PI3K signaling target that promotes stem cell–like traits in basal-like breast cancers. Sci. Signal. 10, eaah4674 (2017).

    © 2017 American Association for the Advancement of Science

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