J Breast Cancer.  2015 Jun;18(2):126-133. 10.4048/jbc.2015.18.2.126.

Recombinant Human Granulocyte Colony-Stimulating Factor Promotes Preinvasive and Invasive Estrogen Receptor-Positive Tumor Development in MMTV-erbB2 Mice

• Zhao CL
• Zhang GP
• Xiao ZZ
• Ma ZK
• Lei CP
• Song SY
• Feng YY
• Zhao YC
• Feng XS

• KMID: 2374527
• DOI: http://doi.org/10.4048/jbc.2015.18.2.126

Abstract

PURPOSE
We investigated whether recombinant human granulocyte colony-stimulating factor (rhG-CSF) could promote the development of preinvasive and invasive breast cancer in mouse mammary tumor virus (MMTV-erbB2) mice with estrogen receptor-positive tumors.
METHODS
MMTV-erbB2 mice were randomly divided into three experimental groups with 20 mice in each group. MMTV-erbB2 mice were treated with daily subcutaneous injections of vehicle or rhG-CSF (low-rhG-CSF group, rhG-CSF 0.125 microg; vehicle-rhG-CSF group, normal saline 0.25 microg; and high-rhG-CSF group, rhG-CSF 0.25 microg) at 3 months of age. Cellular and molecular mechanisms of G-CSF action in mammary glands were investigated via immunohistochemistry and reverse transcription polymerase chain reaction.
RESULTS
Low, but not high, rhG-CSF doses significantly accelerated mammary tumorigenesis in MMTV-erbB2 mice. Short-term treatment with rhG-CSF could significantly promote the development of preinvasive mammary lesions. The cancer prevention effect was associated with reduced expression of proliferating cell nuclear antigen, cluster of differentiation 34, and signal transducers and activators of transcription 3 in mammary glands by >80%.
CONCLUSION
We found that G-CSF was regulated by rhG-CSF both in vitro and in vivo. Identification of G-CSF genes helped us further understand the mechanism by which G-CSF promotes cancer. Low doses of rhG-CSF could significantly increase tumor latency and increase tumor multiplicity and burden. Moreover, rhG-CSF effectively promotes development of both malignant and premalignant mammary lesions in MMTV-erbB2 mice.

Keyword

Carcinogenesis; Cell proliferation; Granulocyte colony-stimulating factor; Mammary gland

MeSH Terms

Animals
Breast Neoplasms
Carcinogenesis
Cell Proliferation
Estrogens*
Granulocyte Colony-Stimulating Factor*
Humans
Immunohistochemistry
Injections, Subcutaneous
Mammary Glands, Human
Mammary Tumor Virus, Mouse
Mice*
Polymerase Chain Reaction
Proliferating Cell Nuclear Antigen
Reverse Transcription
Transducers
Estrogens
Granulocyte Colony-Stimulating Factor
Proliferating Cell Nuclear Antigen

Figure

• 

  Figure 1 rhG-CSF promotes malignant mammary epithelial cell proliferation. (A) Effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the growth of normal and malignant breast cells (MCF-7, SKBR-3 breast cancer cells). We retreated with 0, 0.1, 1, 10, 100, 1,000 µg/L of rhG-CSF. Cell proliferation was measured by Cell Titer 96 Aqueous Cell Proliferation Assay. (B) The expression of granulocyte colony-stimulating factor (G-CSF) was measured by Western blot.

• 

  Figure 2 Chronic exposure to low granulocyte colony-stimulating factor doses had effect on pubertal mammary gland growth. (A) Representative whole mounts of mouse mammary tumor virus-erbB2 mice at 12 weeks (top panels of control; middle of low dose; and bottom of high dose; n=20 per of each group). When compared with the control group, low dose rather than high doses can significantly promote the development of pubertal mammary gland. (B) Catheter branch density in control group, low dose group and high dose group.

• 

  Figure 3 Proliferating cell nuclear antigen (PCNA) expression in mice mammary gland invasive ductal carcinoma (immunohistochemical stain for PCNA, ×200) (A) control group; (B) low-dose group; (C) the high-dose group; (D) positive percentage of proliferation cells.

• 

  Figure 4 Recombinant human granulocyte colony-stimulating factor (rhG-CSF) promote the proliferation of mammary epithelial cells in vivo. Immunohistochemical (IHC) staining of proliferating cell nuclear antigen (PCNA), CD34 and STAT3 in consecutive slides of mammary glands from vehicle- and rhG-CSF-treated mice (IHC stain for PCNA, ×200).

• 

  Figure 5 RNA expression of Cyclin D1, C-myc, vascular endothelial growth factor (VEGF), and granulocyte colony-stimulating factor (G-CSF) in the mammary glands of mammary tumor virus-erbB2 mice exposed to 0 (control), 0.125 (low recombinant human granulocyte colony-stimulating factor [rhG-CSF]), or 0.25 (high rhG-CSF) µg rhG-CSF (n=20 mice/treatment). Graphs represent mean density±SE as a percentage of the control. *p<0.05.

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