Korean J Physiol Pharmacol.  2014 Oct;18(5):391-396. 10.4196/kjpp.2014.18.5.391.

Inhibition of the Interleukin-11-STAT3 Axis Attenuates Hypoxia-Induced Migration and Invasion in MDA-MB-231 Breast Cancer Cells

• Lim JH

• KMID: 2285537
• DOI: http://doi.org/10.4196/kjpp.2014.18.5.391

Abstract

Although interleukin-11 (IL-11) has been reported to be elevated in hypoxic tumors and has been associated with a poor prognosis in various cancers, little is known about its precise role in promoting metastasis in hypoxic tumors. In the present study, the molecular mechanism underlying the effects of IL-11 on MDA-MB-231 breast cancer cells migration and invasion in relation to metastasis under hypoxic conditions has been defined. Inhibition of IL-11 expression or function using small interfering RNA (siRNA) or a neutralizing antibody attenuated hypoxic MDA-MB-231 breast cancer cell migration and invasion through down-regulation of matrix metalloproteinases (MMPs) and activation of epithelial-to-mesenchymal transition (EMT) related gene expression. In addition, hypoxia-induced IL-11 increased STAT3 phosphorylation and STAT3 knockdown suppressed hypoxic MDA-MB-231 breast cancer cell invasion due to reduced MMP levels and reprogrammed EMT-related gene expression. These results suggest that one of the hypoxic metastasis pathways and the regulation of this pathway could be a potential target for novel cancer therapeutics.

Keyword

Hypoxia; Interleukin-11; Invasion; Migration; STAT3

MeSH Terms

Anoxia
Antibodies, Neutralizing
Axis, Cervical Vertebra*
Breast Neoplasms*
Cell Movement
Down-Regulation
Gene Expression
Interleukin-11
Matrix Metalloproteinases
Neoplasm Metastasis
Phosphorylation
Prognosis
RNA, Small Interfering
Antibodies, Neutralizing
Interleukin-11
Matrix Metalloproteinases
RNA, Small Interfering

Figure

• 

  Fig. 1 IL-11 increases in response to hypoxia. (A) MDA-MB-231 cells were incubated under hypoxic (1% O2) or normoxic (20% O2) conditions for 24 h and indicated mRNA levels were analyzed by qRT-PCR. Values represent mean±SD of three independent experiments performed in triplicate; *p<0.05, **p<0.01 and ***p<0.001. (B) MDA-MB-231 cells were incubated under hypoxic or normoxic conditions for the indicated time. IL-11 mRNA levels were analyzed by qRT-PCR. Values represent mean±SD of three independent experiments performed in triplicate; *p<0.05 and **p<0.01. (C) Indicated cells were incubated under hypoxic or normoxic conditions for 24 h. Values represent mean±SD of three independent experiments performed in triplicate; *p<0.05 and **p<0.01. (D) Cells were incubated under hypoxic or normoxic conditions for 72 h. The cultured medium was collected to analyze the concentrations of secreted IL-11 protein as described in materials and methods. Values represent mean±SD of three independent experiments performed in triplicate; *p<0.05 and **p<0.01.

• 

  Fig. 2 IL-11 is essential for the hypoxia-induced cancer cell migration and invasion. (A) MDA-MB-231 cells were seeded in the upper side of Transwell chambers and the lower part of the chamber was filled with conditioned medium collected from MDA-MB-231 cells exposed to normoxia or hypoxia for 24 h. Photographs of migrated or invaded cells were captured at 20× magnification under a microscope (left). A quantitative analysis of the cell numbers is shown (right). Values represent mean±SD of three independent experiments performed in triplicate; *p<0.05. (B) Conditioned medium from MDA-MB-231 cells was pre-incubated with normal serum or IL-11 neutralizing antibody (0.5 µg/ml) for 1 h prior to the experiment. MDA-MB-231 cells were seeded in Matrigel-coated Transwell chamber and incubated with conditioned medium in the presence or absence of the IL-11 neutralizing antibody for 16 h. Values represent mean±SD of three independent experiments performed in triplicate; *p<0.05. (C) Transfected MDA-MB-231 cells were incubated under normoxic or hypoxic conditions for 24 h. IL-11 mRNA levels were analyzed by qRT-PCR. Values represent mean±SD of three independent experiments performed in triplicate; **p<0.01. (D) Conditioned medium was collected from MDA-MB-231 cells transfected with a siRNA against control (40 nM) or IL-11 (40 nM) under normoxic or hypoxic conditions. MDA-MB-231 cells were seeded in Matrigel-coated Transwell chamber and incubated in the presence of conditioned medium for 16 h. Values represent mean±SD of three independent experiments performed in triplicate; *p<0.05.

• 

  Fig. 3 Hypoxia-induced IL-11 leads to STAT3 phosphorylation. (A) MDA-MB-231 cells were incubated with human recombinant IL-11 for the indicated concentration, and (B) time. Indicated protein levels were analyzed by western blot analysis. (C) Conditioned medium was collected from MDA-MB-231 cells, which had been transfected with control or IL-11 siRNA and incubated under normoxic or hypoxic conditions. Non-transfected MDA-MB-231 cells were incubated with conditioned medium for 30 min as indicated. (D) MDA-MB-231 cells were incubated with conditioned medium, which had been pre-incubated with normal serum or the IL-11 neutralizing antibody (0.5 µg/ml) for 30 min. (E) MDA-MB-231 cells were incubated with recombinant IL-11 for 24 h and mRNA expression levels were then measured by qRT-PCR. Values represent mean±SD of three independent experiments performed in triplicate; *p<0.05 and **p<0.01. (F) MDA-MB-231 cells were transfected with the indicated siRNAs and incubated under normoxic or hypoxic conditions for 24 h. Values represent mean±SD of three independent experiments performed in triplicate; *p<0.05 and **p<0.01. (G) Indicated mRNA levels were measured by qRT-PCR. Values represent mean±SD of three independent experiments performed in triplicate; *p<0.05 and **p<0.01. (H) MDA-MB-231 cells were transfected with siRNA against IL-11 and incubated under normoxic or hypoxic conditions for 24 h. Values represent mean±SD of three independent experiments performed in triplicate; *p<0.05 and **p<0.01.

• 

  Fig. 4 STAT3 silencing attenuates hypoxia and IL-11-induced cancer cell invasion. (A) MDA-MB-231 cells were transfected with a siRNA against STAT3 and incubated under normoxic or hypoxic conditions for 24 h. Protein levels were analyzed by western blot. (B) Gene expression levels were analyzed by qRT-PCR. Values represent mean±SD of three independent experiments performed in triplicate; *p<0.05 and **p<0.01. (C) Cells were incubated with BSA or recombinant IL-11 (0.1 µg/ml) for 24 h. (D, E) Gene expression levels were analyzed by qRT-PCR. Values represent mean±SD of three independent experiments performed in triplicate; *p<0.05. (F) Transfected MDA-MB-231 cells with a control or a STAT3 siRNA were seeded into Matrigel-coated Transwell chamber and incubated under normoxic or hypoxic conditions for 24 h. Values represent mean±SD of three independent experiments performed in triplicate; *p<0.05. (G) Transfected MDA-MB-231 cells with a control or a STAT3 siRNA were seeded in Matrigel-coated Transwell chamber and incubated with BSA or IL-11 for 24 h. Values represent mean±SD of three independent experiments performed in triplicate; *p<0.05.

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