TNFalpha up-regulates SLUG via the NF-kappaB/HIF1alpha axis, which imparts breast cancer cells with a stem cell-like phenotype - PubMed (original) (raw)

. 2010 Nov;225(3):682-91.

doi: 10.1002/jcp.22264.

Pasquale Sansone, Sara Mari, Gabriele D'Uva, Simona Tavolari, Tiziana Guarnieri, Mario Taffurelli, Claudio Ceccarelli, Donatella Santini, Pasquale Chieco, Kenneth B Marcu, Massimiliano Bonafè

Affiliations

TNFalpha up-regulates SLUG via the NF-kappaB/HIF1alpha axis, which imparts breast cancer cells with a stem cell-like phenotype

Gianluca Storci et al. J Cell Physiol. 2010 Nov.

Abstract

Extracellular and intracellular mediators of inflammation, such as tumor necrosis factor alpha (TNFα) and NF-kappaB (NF-κB), play major roles in breast cancer pathogenesis, progression and relapse. SLUG, a mediator of the epithelial-mesenchymal transition process, is over-expressed in CD44(+)/CD24(-) tumor initiating breast cancer cells and in basal-like carcinoma, a subtype of aggressive breast cancer endowed with a stem cell-like gene expression profile. Cancer stem cells also over-express members of the pro-inflammatory NF-κB network, but their functional relationship with SLUG expression in breast cancer cells remains unclear. Here, we show that TNFα treatment of human breast cancer cells up-regulates SLUG with a dependency on canonical NF-κB/HIF1α signaling, which is strongly enhanced by p53 inactivation. Moreover, SLUG up-regulation engenders breast cancer cells with stem cell-like properties including enhanced expression of CD44 and Jagged-1 in conjunction with estrogen receptor alpha down-regulation, growth as mammospheres, and extracellular matrix invasiveness. Our results reveal a molecular mechanism whereby TNFα, a major pro-inflammatory cytokine, imparts breast cancer cells with stem cell-like features, which are connected to increased tumor aggressiveness.

© 2010 Wiley-Liss, Inc.

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Conflict of interest statement

Conflit of interest: All authors declare no conflict of interest.

Figures

FIGURE 1

FIGURE 1. TNFα up-regulates CD44 and Jagged-1 and down-regulates ERα expression via SLUG

(A): RT-PCR analysis of CD44, Jagged-1 and ERα mRNA level, and ERE-Luc in MCF7 cells transiently transfected with empty (pcDNA3.1) and human SLUG encoding (pSLUG) vector (1µg each, 24h); (B): NF-κB-Luc, SLUG promoter driven luciferase activity assay (SLUG-Luc) and RT-PCR analysis of SLUG mRNA in MCF7 cells exposed to TNFα (10ng/ml, 24h); (C): CD44, Jagged-1 and ERα mRNA level and ERE-Luc assay in MCF7 cells transiently transfected with SCR or SLUG specific siRNA (1µg, 48h) exposed or less to TNFα (10ng/ml, 24h). Data are presented as mean +/− S.D. of three replicates, p values of unpaired t tests: *<0.05, #<0.01 and § <0.005. β-Actin was used as reference control (RT-PCR analysis in panel A and B are normalized on the same β-Actin).

FIGURE 2

FIGURE 2. TNFα/NF-κB signalling promotes MS formation and invasive capacity of breast cancer cells via SLUG up-regulation

(A): MS forming capacity of empty/pSLUG transiently transfected (1µg, 24h) MCF7 cells and pCtoGMB/ shSLUG stably transduced MCF7 cells exposed or less to TNFα (10 ng/ml, 24h); (B): MS forming capacity of MCF7 cells exposed to the IκBα degradation inhibitors Parthenolide or Bay 11-7082 (5µM, 24h each), the specific IKKβ inhibitor sc-514 (5µM, 24h each), or stably transduced with an IKKβ specific shRNA/empty expressing retroviral vector; (C): MS forming capacity of pCtoGMB/shSLUG MCF7 cells transduced with empty or p65/IKKβ-CA encoding vector, representative MS pictures are also reported. The scale bar inset corresponds to 100µm; (D): Invasion assay in pSLUG transfected (1µg), TNFα exposed (10ng/ml, 24h), p65/IKKβ-CA transduced pCtoGMB/shSLUG MCF7 cells. Data are presented as mean +/− S.D. of three replicates, p values of unpaired t tests: *<0.05, #<0.01 and § <0.005. The scale bar represents 100 µm.

FIGURE 3

FIGURE 3. HIF1α is necessary for the SLUG-dependent stem cell-like gene signature, MS formation and invasive capacity of breast cancer cells

(A): SLUG-Luc activity assay and RT-PCR analysis of SLUG, CD44 and Jagged-1 mRNA level in MCF7 cells transiently transfected with pCDNA3.1 and HIF1α encoding vector (HIF1αODD, 1µg, 24h); (B): MS forming and invasive capacity assay of MCF7 cells transiently transfected with HIF1αODD (1µg); (C): MS forming and invasive capacity assay of pCtoGMB/shSLUG MCF7 cells transiently transfected with HIF1αODD (1µg). Representative MS pictures are also reported. Data are presented as mean +/− S.D. of three replicates, p values of unpaired t tests *<0.05; #<0.01; §<0.005. The scale bar represents 100µm. β-Actin was used as reference control.

FIGURE 4

FIGURE 4. p53 compromised cells show an over-activation of the NF-κB/HIF1α axis and SLUG expression in response to TNFα

(A): SLUG-Luc, NF-κB-Luc and HRE-Luc activity assays in pBabe/p53 dominant negative (p53D) transduced MCF7 cells exposed or less to TNFα 10 ng/ml, 24h; (B): RT-PCR analysis of CD44, Jagged-1, ERα in pBabe/p53D and pBabe/p53D exposed or less to TNFα 10ng/ml, 24h; (C): MS forming capacity of pBabe/p53D and pCtoGMB/shSLUG transduced MCF7 cells exposed or less to TNFα (10 ng/ml, 24h); representative MS pictures are also reported (D): Invasion assay of pBabe/p53D and pCtoGMB/shSLUG transduced MCF7 cells exposed or less to TNFα 0 (10 ng/ml, 24h). Data are presented as mean +/− S.D. of three replicates, p values of unpaired t tests: *<0.05; #<0.01 and §<0.005. Reference scale bar is 10µm. β-Actin was used as reference control.

FIGURE 5

FIGURE 5

Inflammatory environment activation of NF-kappaB/HIF1α/SLUG/β-Catenin axis drives the up-regulation of the basal/stem cell-like gene expression profile in breast cancer cell. p53 loss of function up-regulates the outlined interplay.

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