J Breast Cancer.  2018 Sep;21(3):259-266. 10.4048/jbc.2018.21.e42.

Hypoxia-Inducible Factor 1α Regulates the Transforming Growth Factor β1/SMAD Family Member 3 Pathway to Promote Breast Cancer Progression

• Peng J
• Wang X
• Ran L
• Song J
• Luo R
• Wang Y

• KMID: 2421366
• DOI: http://doi.org/10.4048/jbc.2018.21.e42

Abstract

PURPOSE
The transforming growth factor β1 (TGF-β1)/SMAD family member 3 (SMAD3) pathway, and hypoxia-inducible factor 1α (HIF-1α) are two key players in various types of malignancies including breast cancer. The TGF-β1/SMAD3 pathway can interact with HIF-1α in some diseases; however, their interaction in breast cancer is still unknown. Therefore, our study aimed to investigate the interactions between the TGF-β1/SMAD3 pathway and HIF-1α in breast cancer.
METHODS
Expression of HIF-1α in serum of breast cancer patients and healthy controls was detected by quantitative reverse transcription polymerase chain reaction, and the diagnostic value of HIF-1α for breast cancer was evaluated by receiver operating characteristic curve analysis. Breast cancer cell lines overexpressing SMAD3 and HIF-1α were established. Cell apoptosis and proliferation following different treatments were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and cell counting kit-8, respectively. Expression of related proteins was detected by western blot.
RESULTS
Serum levels of HIF-1α were higher in breast cancer patients than in normal controls. Both SMAD3 and HIF-1α overexpression inhibited cell apoptosis and promoted cell proliferation. Treatment with inhibitors of HIF-1α and SMAD3 promoted apoptosis in breast cancer cells and inhibited their proliferation. Overexpression of HIF-1α promoted the expression of TGF-β1 and SMAD3, while SMAD3 overexpression did not significantly affect expression of HIF-1α or TGF-β1.
CONCLUSION
HIF-1α serves as an upstream regulator of the TGF-β1/SMAD3 pathway and promotes the growth of breast cancer.

Keyword

Breast neoplasms; Hypoxia-inducible factor 1; Smad3 protein; Transforming growth factor beta1

MeSH Terms

Apoptosis
Blotting, Western
Breast Neoplasms*
Breast*
Cell Count
Cell Line
Cell Proliferation
Humans
Hypoxia-Inducible Factor 1
Polymerase Chain Reaction
Reverse Transcription
ROC Curve
Smad3 Protein
Transforming Growth Factor beta1
Transforming Growth Factors*
Hypoxia-Inducible Factor 1
Smad3 Protein
Transforming Growth Factor beta1
Transforming Growth Factors

Figure

• 

  Figure 1 Expression of hypoxia-inducible factor 1α (HIF-1α) mRNA in serum of healthy controls and patients with different stages of breast cancer. *p<0.05.

• 

  Figure 2 Potentials of hypoxia-inducible factor 1α (HIF-1α) mRNA in the diagnosis and prognosis of breast cancer. (A) Diagnostic values of serum HIF-1α mRNA for breast cancer was analyzed by receiver operating characteristic curve analysis. (B) Comparison of survival curves of patients with high and low serum level of HIF-1α mRNA was shown.

• 

  Figure 3 Effects of hypoxia-inducible factor 1α (HIF-1α) and SMAD family member 3 (SMAD3) on cell proliferation and apoptosis of breast cancer cells. (A, B) Cell proliferation of different cell lines was influenced with different treatment. (C, D) Cell apoptosis of different cell lines was influenced with different treatment. SIS3=SMAD3 inhibitor; C=control; NC=negative control; over=overexpression. *p<0.05.

• 

  Figure 4 Hypoxia-inducible factor 1α (HIF-1α) is a positive upstream regulator of transforming growth factor β1 (TGF-β1)/SMAD family member 3 (SMAD3) pathway in breast cancer. (A, B) HIF-1α overexpression significantly increased the expression levels of TGF-β 1 and SMAD3 in BT-483 and MCF-7 cell line. (C, D) SMAD3 overexpression showed no significant effects on the expression of HIF-1α or TGF-β 1 in BT-483 and MCF-7 cell line. C=control; NC=negative control; over=overexpression; GAPDH=glyceraldehyde-3-phosphate dehydrogenase. *p<0.05.

• 

  Figure 5 Effects of hypoxia-inducible factor inhibitor dimethyl-bisphenol A (DBA) on transforming growth factor β1 (TGF-β1) (A) and SMAD family member 3 (SMAD3) (B) expression. C=control; GAPDH=glyceraldehyde-3-phosphate dehydrogenase. *p<0.05.

• 

  Figure 6 Overexpression of hypoxia-inducible factor 1α (HIF-1α) and the growth of transplanted tumor in mice. (A) Representative tumors collected at 6 week after injection was shown. (B) Tumors were derived from MCF-7 cells (control), MCF-7 cells transfected with empty vector (negative control), and HIF-1α-overexpressing MCF-7 cell lines (overexpression), and the tumor growth rate was recorded and compared. (C) Comparison of tumor weight at week 6 between tumors derived from MCF-7 cells (control), MCF-7 cells transfected with empty vector (negative control), and HIF-1α-overexpressing MCF-7 cell line (overexpression). C=control; NC=negative control; over=overexpression. *p<0.05.

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