J Breast Cancer.  2019 Dec;22(4):562-578. 10.4048/jbc.2019.22.e56.

Notch1 in Tumor Microvascular Endothelial Cells and Tumoral miR-34a as Prognostic Markers in Locally Advanced Triple-Negative Breast Cancer

• Kim D
• Lee J
• Kang J
• Kim SH
• Yoo TK
• Oh S
• Lee A

• KMID: 2470893
• DOI: http://doi.org/10.4048/jbc.2019.22.e56

Abstract

PURPOSE
Triple-negative breast cancer (TNBC) is associated with poor prognosis with limited treatment options. Angiogenesis is known to be involved in the progression of TNBC, and targeting this pathway results in modest clinical benefits. In this study, we analyzed the role of tumor microvascular endothelial Notch1 (EC Notch1) and tumoral miR-34a as prognostic markers in patients with TNBC.
METHODS
The expression of miR-34a was analyzed using archival tumor tissues from 114 patients with TNBC. Simultaneously, archival tumor tissues were also checked for the expression of CD34 and Notch1 by immunostaining. The ratio of Notch1-microvascular density (MVD) to CD34-MVD was defined as EC Notch1. The association between the expression of miR-34a or EC Notch1 and clinicopathological characteristics was analyzed.
RESULTS
In the overall patient population, patients with low expression of EC Notch1 was associated with better overall survival (OS, p = 0.041) than those with high expression of EC Notch1. In lymph node-positive TNBC patients, high levels of miR-34a and low levels of EC Notch1 correlated significantly with higher survival benefits in terms of OS (p = 0.026), disease-free survival (p = 0.009), and metastasis-free survival (p = 0.038) relative to that in other patients. Decreased expression of EC Notch1 and increased expression of miR-34a also showed a survival benefit in locally advanced TNBC.
CONCLUSION
The fact that miR-34a and EC Notch1 are associated with the angiogenesis suggests that angiogenesis may play a role in the development and progression of TNBC.

Keyword

MIRN34 microRNA; Receptor, Notch1; Triple negative breast neoplasms

MeSH Terms

Disease-Free Survival
Endothelial Cells*
Humans
Prognosis
Receptor, Notch1
Triple Negative Breast Neoplasms*
Receptor, Notch1

Figure

• 

  Figure 1 Quantitative real-time polymerase chain reaction analysis of miR-34a expression levels in 114 TNBC tissues. The relative miR-34a expression level (value of 2−∆Ct) in each sample was calculated based on a comparative Ct method with normalization to RNU6B RNA. Each data point represents the mean 2−∆Ct values from 3 independent assays. miR-34a = miRNA-34a; TNBC = triple-negative breast cancer; RNU6B = U6 small nuclear 6B.

• 

  Figure 2 Immunohistochemical staining of CD34 and Notch1 in microvessels of TNBC tissues. Representative staining of Notch1 immunostaining (DAB substrate, brown) in (A) EC Notch1 low group and (B) EC Notch1 high group (original magnification ×200). Representative staining of CD34 immunostaining (DAB substrate, brown) in (C) EC Notch1 low group and (D) EC Notch1 high group (original magnification ×200). Arrowheads mark microvessels (Notch1-positive). TNBC = triple-negative breast cancer; DAB = diaminobenzidine; EC Notch1 = the ratio of Notch1 positive MVD to CD34 MVD; EC = endothelial cell; MVD = microvessel density.

• 

  Figure 3 (A) The distribution of EC Notch1 and (B) expression level of EC Notch1 in 114 TNBC patients. EC Notch1 = the ratio of Notch1 positive MVD to CD34 MVD; EC = endothelial cell; MVD = microvessel density; TNBC = triple-negative breast cancer. *p < 0.001.

• 

  Figure 4 Relation between miR-34a expression and EC Notch1 in matched clinical samples. miR-34a = miRNA-34a; EC Notch1 = the ratio of Notch1 positive MVD to CD34 MVD; EC = endothelial cell; MVD = microvessel density; RNU6B = U6 small nuclear 6B.

• 

  Figure 5 Association between EC Notch1 and survival outcomes in TNBC. (A) OS, (B) DFS, (C) MFS in patients with TNBC; based on EC Notch1 expression level. (D) OS, (E) DFS, (F) MFS in patients with locally advanced TNBC; based on EC Notch1 expression level. EC Notch1 = the ratio of Notch1 positive MVD to CD34 MVD; EC = endothelial cell; MVD = microvessel density; TNBC = triple-negative breast cancer; OS = overall survival; DFS = disease-free survival; MFS = metastasis-free survival; LN = lymph node.

• 

  Figure 6 Association between miR-34a expression and survival outcomes in TNBC. (A) OS, (B) DFS, (C) MFS in patients with TNBC; based on miR-34a expression level. (D) OS, (E) DFS, (F) MFS in patients with locally advanced TNBC; based on miR-34a expression level. miR-34a = miRNA-34a; TNBC = triple-negative breast cancer; OS = overall survival; DFS = disease-free survival; MFS = metastasis-free survival; LN = lymph node.

• 

  Figure 7 DFS of patients with TNBC based on miR-34a expression level from TCGA data. DFS = disease-free survival; TNBC = triple-negative breast cancer; miR-34a = miRNA-34a; TCGA = The Cancer Genomic Atlas.

• 

  Figure 8 Kaplan–Meier analysis of survival of patients with TNBC stratified according to the expression of miR-34a and EC Notch1. (A) OS, (B) DFS, (C) MFS in patients with TNBC; based on the expression of miR-34a and EC Notch. (D) OS, (E) DFS, (F) MFS in patients with locally advanced TNBC; based on the expression of miR-34a and EC Notch1. TNBC = triple-negative breast cancer; miR-34a = miRNA-34a; EC Notch1 = the ratio of Notch1 positive MVD to CD34 MVD; EC = endothelial cell; MVD = microvessel density; OS = overall survival; DFS = disease-free survival; MFS = metastasis-free survival; LN = lymph node.

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