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. 2013 Dec 23;8(12):e82821.
doi: 10.1371/journal.pone.0082821. eCollection 2013.

Evaluation of STAT3 signaling in ALDH+ and ALDH+/CD44+/CD24- subpopulations of breast cancer cells

Li Lin  1 , Brian Hutzen  2 , Hsiu-Fang Lee  3 , Zhengang Peng  4 , Wenlong Wang  5 , Chongqiang Zhao  5 , Huey-Jen Lin  6 , Duxin Sun  3 , Pui-Kai Li  7 , Chenglong Li  7 , Hasan Korkaya  8 , Max S Wicha  8 , Jiayuh Lin  2
Affiliations

Evaluation of STAT3 signaling in ALDH+ and ALDH+/CD44+/CD24- subpopulations of breast cancer cells

Li Lin et al. PLoS One. .

Abstract

Background: STAT3 activation is frequently detected in breast cancer and this pathway has emerged as an attractive molecular target for cancer treatment. Recent experimental evidence suggests ALDH-positive (ALDH(+)), or cell surface molecule CD44-positive (CD44(+)) but CD24-negative (CD24(-)) breast cancer cells have cancer stem cell properties. However, the role of STAT3 signaling in ALDH(+) and ALDH(+)/CD44(+)/CD24(-) subpopulations of breast cancer cells is unknown.

Methods and results: We examined STAT3 activation in ALDH(+) and ALDH(+)/CD44(+)/CD24(-) subpopulations of breast cancer cells by sorting with flow cytometer. We observed ALDH-positive (ALDH(+)) cells expressed higher levels of phosphorylated STAT3 compared to ALDH-negative (ALDH(-)) cells. There was a significant correlation between the nuclear staining of phosphorylated STAT3 and the expression of ALDH1 in breast cancer tissues. These results suggest that STAT3 is activated in ALDH(+) subpopulations of breast cancer cells. STAT3 inhibitors Stattic and LLL12 inhibited STAT3 phosphorylation, reduced the ALDH(+) subpopulation, inhibited breast cancer stem-like cell viability, and retarded tumorisphere-forming capacity in vitro. Similar inhibition of STAT3 phosphorylation, and breast cancer stem cell viability were observed using STAT3 ShRNA. In addition, LLL12 inhibited STAT3 downstream target gene expression and induced apoptosis in ALDH(+) subpopulations of breast cancer cells. Furthermore, LLL12 inhibited STAT3 phosphorylation and tumor cell proliferation, induced apoptosis, and suppressed tumor growth in xenograft and mammary fat pad mouse models from ALDH(+) breast cancer cells. Similar in vitro and tumor growth in vivo results were obtained when ALDH(+) cells were further selected for the stem cell markers CD44(+) and CD24(-).

Conclusion: These studies demonstrate an important role for STAT3 signaling in ALDH(+) and ALDH(+)/CD44(+)/CD24(-) subpopulations of breast cancer cells which may have cancer stem cell properties and suggest that pharmacologic inhibition of STAT3 represents an effective strategy to selectively target the cancer stem cell-like subpopulation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. STAT3 phosphorylation of ALDH+ subpopulation of breast cancer cells was higher than un-separated and ALDH− subpopulations.
(A) Representative flow cytometry analysis of ALDH enzymatic activity in SUM159 breast cancer cells was shown. (B) ALDH+ and ALDH− subpopulations were separated from MDA-MB-231, SUM159, and SK-BR-3 breast cancer cells by flow cytometry. Phosphorylation of STAT3 (Y705), and ERK 1/2 (T202/Y204), was detected by Western blot. (C) Breast cancer tissue microarray slides were stained using immunohistochemistry (IHC). Representative examples of ALDH1 positive/P-STAT3 (Y705) positive (>95%, sample a) and ALDH1 negative/P-STAT3 (Y705 negative (<5%, sample b) tumors were shown. The spots for ALDH1 and P-STAT3 (Y705) were from the matched tissues section from the same patient. (D) Breast cancer tissue microarray slides were double-stained with P-STAT3 and ALDH1. Representative examples of the expression of STAT3 phosphorylation and ALDH1 was shown by immufluorence (IF) staining. ALDH1 high expression tumor cells (cytoplasm, green) also expressed P-STAT3 in nuclear (red).
Figure 2
Figure 2. LLL12 and Stattic inhibited STAT3 expression.
(A) LLL12 inhibited STAT3 phosphorylation and induced apoptosis in ALDH+ breast cancer stem-like cells. (B) Stattic inhibited STAT3 but not ERK1/2 phosphorylation in ALDH+ breast cancer stem-like cells. (C) LLL12 inhibited the expression of STAT3 downstream target genes and ALDH1 in ALDH+ subpopulation of breast cancer cells. (D) STAT3 ShRNA decreased the STAT3 expression and STAT3 phosphorylation, and inhibited tumor growth in ALDH+ MDA-MB-231 breast cancer stem-like cells.
Figure 3
Figure 3. LLL12, Stattic and STAT3 ShRNA inhibited ALDH+ cell viability.
LLL12 (A) and Stattic (B) reduced the ALDH+ subpopulation of MDA-MB-231, SUM159, and SK-BR-3 breast cancer cells. Statistically significant reduction of LLL12-treated relative to the DMSO is designated by an asterisk (P<0.05). LLL12 (C), Stattic (D), and STAT3 ShRNA (E) inhibited cell viability of ALDH+ subpopulation of breast cancer cells. CTL: control lentivirus that expresses GFP. (F) LLL12 and Stattic inhibited tumorsphere formation of the ALDH+ subpopulation of breast cancer cells.
Figure 4
Figure 4. LLL12 suppressed tumor growth.
LLL12 suppressed tumor growth in (A) mouse xenografts with MDA-MB-231 breast cancer stem-like cells and (B) mammary fat pad with SUM159 breast cancer stem-like cells (ALDH+ cells). Reduction of tumor volume (Aa and Ba) and tumor weight (Ab and Bb) in all LLL12-treated mice compared to vehicle group (*P<0.05). One representative sample from tumor tissues generated from ALDH+ MDA-MB-231 cancer stem-like showing STAT3 phosphorylation were also inhibited by LLL12 treatment (Ac and Bc). (C) Immunohistochemistry staining of tumor xenografts was performed using Ki-67 and cleaved caspase-3 antibodies. The staining was visualized and photographed on a BX51 fluorescence microscope (Olympus, Tokyo, Japan) at x200 magnification (Ca). Positively stained cells in each photo were counted. LLL12 decreased the number of Ki-67 positive tumor cells (Cb) and increased the numbers of cleaved caspase-3 positive tumor cells (Cc).
Figure 5
Figure 5. LLL12 inhibited ALDH+/CD44+/CD24− subpopulations in vitro and in vivo.
ALDH+/CD44+/CD24− and ALDH−/CD44+/CD24+ subpopulations were separated from MDA-MB-231 and SUM159 breast cancer cells by flow cytometry. (A) STAT3 phosphorylation of the ALDH+/CD44+/CD24− subpopulation of breast cancer cells was higher than un-separated and the ALDH−/CD44+/CD24+ subpopulations. ALDH+/CD44+/CD24− breast cancer stem-like cells were treated with then 0.5–5 µM of LLL12 or DMSO as indicated. LLL12 inhibited STAT3 phosphorylation, induced apoptosis (B) and reduced STAT3 downstream target genes expression in ALDH+/CD44+/CD24− breast cancer stem-like cells (C). LLL12 also inhibited cell viability (D) and tumorsphere formation (E) of ALDH+/CD44+/CD24− subpopulation of breast cancer cells. (F) LLL12 suppressed tumor growth in mouse xenografts with ALDH+/CD44+/CD24− SUM-159 breast cancer stem-like cells (*P<0.05).
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