J Breast Cancer.  2018 Sep;21(3):267-276. 10.4048/jbc.2018.21.e43.

Deficiency of Follistatin-Like Protein 1 Accelerates the Growth of Breast Cancer Cells at Lung Metastatic Sites

• Zhang Y
• Xu X
• Yang Y
• Ma J
• Wang L
• Meng X
• Chen B
• Qin L
• Lu T
• Gao Y

• KMID: 2421367
• DOI: http://doi.org/10.4048/jbc.2018.21.e43

Abstract

PURPOSE
Follistatin-like protein 1 (FSTL1) is a secreted glycoprotein that has been shown to play a role in various types of cancer. However, the clinical significance and function of FSTL1 in breast cancer have not been reported. We investigated the role of FSTL1 in breast cancer in this study.
METHODS
Enzyme-linked immunosorbent assays, western blot analysis, and reverse transcription polymerase chain reaction were used to monitor the expression of FSTL1 in breast cancer tissue and in serum samples from breast cancer patients. We employed a 4T1 breast cancer model and Fstl1(+/−) mice for in vivo studies. Hematoxylin and eosin staining, western blot analysis, and RNA sequencing were used to analyze the effect of FSTL1 on primary tumor growth and lung metastasis.
RESULTS
We demonstrated that the expression of FSTL1 is reduced in both the breast cancer tissue and the serum of breast cancer patients. We showed that reduced levels of FSTL1 in serum correlate with elevated expression of Ki-67 and epidermal growth factor receptor (EGFR) in cancer tissues. Moreover, lowered expression of FSTL1 was associated with decreased survival in breast cancer patients. Experiments on the Fstl1(+/−) mouse model established that FSTL1 deficiency had no effect on primary tumor growth, but increased the lung metastases of breast cancer cells, resulting in reduced survival of tumor-bearing mice. RNA sequencing found significantly reduced expression of Egln3 and increased expression of EGFR in Fstl1(+/−) mice. Thus, our results suggest that FSTL1 may affect the expression of EGFR through Egln3, inhibiting the proliferation of breast cancer cells at lung metastatic sites.
CONCLUSION
In conclusion, we suggest a suppressor role of FSTL1 in breast cancer lung metastasis. Furthermore, FSTL1 may represent a potential prognostic biomarker and a candidate therapeutic target in breast cancer patients.

Keyword

Breast neoplasms; Follistatin-related proteins; Neoplasm metastasis; Tumor suppressor genes

MeSH Terms

Animals
Blotting, Western
Breast Neoplasms*
Breast*
Enzyme-Linked Immunosorbent Assay
Eosine Yellowish-(YS)
Follistatin-Related Proteins*
Genes, Tumor Suppressor
Glycoproteins
Hematoxylin
Humans
Lung*
Mice
Neoplasm Metastasis
Polymerase Chain Reaction
Receptor, Epidermal Growth Factor
Reverse Transcription
Sequence Analysis, RNA
Eosine Yellowish-(YS)
Follistatin-Related Proteins
Glycoproteins
Hematoxylin
Receptor, Epidermal Growth Factor

Figure

• 

  Figure 1 The expression of follistatin-like protein 1 (FSTL1) in breast cancer patients. (A) Serum levels of FSTL1 were determined by enzyme-linked immunosorbent assay in breast cancer patients (n=94) and healthy controls (n=21). (B) Western blot analysis of FSTL1 expression in cancer tissue and marched para-carcinoma tissue (n=4). (C) Real-time reverse transcription polymerase chain reaction analysis of eight paired breast carcinoma samples for the expression of FSTL1. (D) Relative expression of FSTL1 messenger RNA in cancer tissue (n=1,101) and marched para-carcinoma tissue (n=112) in breast cancer patients from the Cancer Genome Atlas (TCGA) cohort. (E) Kaplan-Meier survival analysis of public database (TCGA 2017) showed low expression of FSTL1 was associated with a worse overall survival of breast cancer patients. Each bar represents mean±SD for triplicate experiments.HR=hazard ratio. *p<0.05; †p<0.01; ‡p<0.001, unpaired t-test.

• 

  Figure 2 Generation of Fstl1+/− mice. (A) Genotyping of mice. (B) Histological analysis of follistatin-like protein 1 (FSTL1) in lung tissue of wild type and Fstl1+/− mice (n=3) by immunohistochemical (IHC) staining (IHC for FSTL1, ×400). (C) Western blot analysis of FSTL1 in lung tissue of WT and Fstl1+/− mice. (D) Western blot analysis of FSTL1 in mammary tissue of WT and Fstl1+/− mice.bp =base pair.

• 

  Figure 3 Growth of primary 4T1 tumors in wild type (WT) and Fstl1+/−mice. (A) Body weight was monitored in WT and Fstl1+/−mice for 4 weeks after the injection of 4T1 cancer cells (n=7). (B) Tumor volume was measured for 3 weeks (n=7). (C) The dissected tumors at day 7, 14, 21, and 28 (n=3). Tumors were dissected, and their weights and tumor volumes were measured. (D) Survival analysis of WT (n=37) and Fstl1+/−mice (n=44). Each bar represents the mean±SD for triplicate experiments.W=WT; Fstl1+/−. *p<0.05, unpaired t-test.

• 

  Figure 4 Increased lung metastasis in Fstl1+/−mice. (A) Thirty-one days after injection of 4T1 cancer cells, the mice were euthanized and analyzed for lung metastasis. The arrows were pointed to the lung metastasis in fresh lung tissue. (B) Thirty-one days after injection of 4T1 cancer cells, the mice were euthanized and analyzed for lung metastasis of formalin fixation. (C) Representative H&E stained images of the lung. (D) Number of lung metastatic foci were assessed in wild type (WT) (n=6) and Fstl1+/−mice (n=7). (E) Number and size of lung metastatic foci were assessed in WT (n=6) and Fstl1+/−mice (n=7). Each bar represents mean±SD for triplicate experiments.*p<0.05, unpaired t-test.

• 

  Figure 5 The epidermal growth factor receptor (EGFR) signaling in lung metastatic tumor tissue of Fstl1+/−mice. (A) Differential gene expression in lung metastatic tumor tissue of wild type (WT) (n=3) and Fstl1+/−mice (n=3). (B) Relative expression of Egln3 was decreased in lung metastatic tumor tissue of Fstl1+/−mice by real-time reverse transcription polymerase chain reaction (n=7). (C) Relative expression of EGFR was increased in lung metastatic tumor tissue of Fstl1+/−mice by transcription analysis (n=7). (D) Western blot analysis of phosphorylation of extracellular signal-regulated kinase (p-ERK) expression in lung metastatic tumor tissue of WT and Fstl1+/−mice. (E) Hypothetical model of follistatin-like protein 1 (FSTL1)-regulated cancer cell proliferation. Each bar represents the mean±SD for triplicate experiments.*p<0.05; †p<0.001, unpaired t-test.

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