Serum exosomal-annexin A2 is associated with African-American triple-negative breast cancer and promotes angiogenesis

Abstract

Background: Limited information is available on biomarker(s) for triple-negative breast cancer (TNBC) that can address the higher incidence and aggressiveness of TNBC in African-American (AA) women. Our previous studies have demonstrated annexin A2 (AnxA2) association with exosomes which promotes angiogenesis and metastasis. Therefore, our goal was to examine the expression and function of exosomal-annexin A2 (exo-AnxA2) derived from the serum samples of breast cancer patients.

Methods: The expression of serum exo-AnxA2 and its association with clinicopathological features of the breast cancer patients were determined. The role of serum exo-AnxA2 to promote angiogenesis was determined by an in vivo Matrigel plug assay.

Results: Our results show that the expression of serum exo-AnxA2 in breast cancer patients (n = 169; 83.33 ± 2.040 ng/mL, P < 0.0001) is high compared to non-cancer females (n = 68; 34.21 ± 2.238 ng/mL). High expression of exo-AnxA2 levels in breast cancer was significantly associated with tumor grade (P < 0.0001), poor overall survival (hazard ratio (HR) 2.802; 95% confidence intervals (CI) = 1.030-7.620; P = 0.0353), and poor disease-free survival (HR 7.934; 95% CI = 1.778-35.398; P = 0.0301). The expression of serum exo-AnxA2 levels was significantly elevated in TNBC (n = 68; 109.1 ± 2.905 ng/mL; P < 0.0001) in comparison to ER⁺ (n = 50; 57.35 ± 1.545 ng/mL), HER2⁺ (n = 59; 78.25 ± 1.146 ng/mL), and non-cancer females (n = 68; 34.21 ± 2.238 ng/mL). Exo-AnxA2 showed diagnostic values with a maximum AUC as 1.000 for TNBC, 0.8304 for ER⁺, and 0.9958 for HER2⁺ compared to non-cancer females. The expression of serum exo-AnxA2 was significantly elevated in AA women with TNBC (n = 29; 118.9 ± 4.086 ng/mL, P < 0.0001) in comparison to Caucasian-American TNBC (n = 27; 97.60 ± 3.298 ng/mL) patients. Our in vivo results suggest a role of serum exo-AnxA2 in angiogenesis and its association with aggressiveness of TNBC in AA women.

Conclusions: Our results demonstrated that the expression of serum exo-AnxA2 is high in AA women with TNBC and promotes angiogenesis. These findings suggest that exo-AnxA2 holds promise as a potential prognosticator of TNBC and may lead to an effective therapeutic option.

Keywords: And racial disparity; Angiogenesis; Annexin A2; Breast cancer; Exosomes.

Fig. 1

Expression of AnxA2 in exosomes derived from serum samples of breast cancer patients. a Size analysis of exosomes. Representative image of the average size of exosomes isolated from serum samples of cancer patients were analyzed by Malvern Zetasizer. Studied exosomes range of size is 52.06–122.3 nm in diameter with an average size of 87.85 ± 21.30 nm. b Western blot analysis for the expression of AnxA2 and exosomal markers CD9, TSG101, and flotillin-1 in lysates of exosomes purified from breast cancer patients and non-cancer patients. MDA-MB-231 cell lysate was used as a positive control for the expression of AnxA2, CD9, TSG101, flotillin-1, and calnexin. Calnexin (endoplasmic reticulum marker) was analyzed as a marker absent in exosomes. Coomassie blue staining was performed as an equal loading control for exosomes derived from serum samples of patients. c Exosomes purified from serum samples of breast cancer patients were immunoprecipitated with an antibody to AnxA2, EpCAM, or isotype mouse/rabbit immunoglobulin (M-IgG/ Rb-IgG). The immunoprecipitated whole exosomes were lysed with RIPA lysis buffer and analyzed for the expression of AnxA2, EpCAM (endothelial marker), exosomal markers (CD9, TSG101, and flotillin-1), calnexin (endoplasmic reticulum marker), and GM130 (cis-Golgi marker) by Western blot analysis. Calnexin and GM130 were used as a negative control for exosomes

Fig. 2

Exo-AnxA2 analysis in serum samples of breast cancer patients and non-cancer females. a Scatter plot analysis of exo-AnxA2 protein concentration obtained through ELISA analysis from serum of non-cancer (n = 68) females and breast cancer (n = 169) patients. Each point represents the mean of triplicates. The data are expressed as the mean ± SEM (****, P < 0.0001; two-tailed Student’s t test). b Matrigel plug assay with serum exosomes derived from non-cancer females and breast cancer patients along with incubation with LCKLSL AnxA2 inhibitory or LGKLSL control peptides was performed in athymic nude mice (n = 3). Representative images of the Matrigel plugs are shown. Peptide concentration: 5 μmol/L. c Quantification of hemoglobin estimation of homogenized Matrigel plugs from non-cancer and breast cancer serum exosomes by Drabkin’s method (n = 3; *, P < 0.05; ****, P < 0.0001; one-way ANOVA followed by Tukey’s multiple comparison test)

Fig. 3

The expression of exo-AnxA2 levels and its association with clinicopathological features. a Scatter plot analysis of expression of serum exo-AnxA2 levels in non-cancer females (n = 68) and different grades of breast tumor patients (grade I, n = 16; grade II, n = 49; and grade III, n = 94). The data are expressed as the mean ± SEM (^ns, non-significant; ****, P < 0.0001; one-way ANOVA followed by Tukey’s multiple comparison test). Kaplan-Meier survival analysis for exo-AnxA2 levels in serum samples of breast cancer patients. b Overall survival of patients with breast cancer based on serum exo-AnxA2 expression using Kaplan-Meier curves (n = 169). c Diseases-free survival of patients with breast cancer on serum exo-AnxA2 expression (n = 107). The P value was calculated using the log-rank test

Fig. 4

Exo-AnxA2 expression among breast cancer subtypes. Scatter plot analysis of serum exo-AnxA2 concentration in non-cancer (n = 68), ER⁺ (n = 50), HER2⁺ (n = 59), and TNBC (n = 58) breast cancer patients. The data are expressed as the mean ± SEM (****, P < 0.0001; one-way ANOVA followed by Tukey’s multiple comparison test)

Fig. 5

Serum exo-AnxA2 expression in breast cancer subtypes and its association with race and tumor grade: a The concentration of serum exo-AnxA2 expression levels among race in subtypes of breast cancer patients and non-cancer females. The data are expressed as the mean ± SEM (*, P < 0.05; ****, P < 0.0001; two-tailed Student’s t test). b Scatter plot analysis of serum exo-AnxA2 levels in ER⁺, HER2⁺, and TNBC breast cancer patients of different tumor grades. The data are expressed as the mean ± SEM (*, P < 0.05; one-way ANOVA followed by Bonferroni’s multiple comparison test)

Fig. 6

Serum exo-AnxA2 promotes angiogenesis. a Representative images of Matrigel plugs containing serum exosomes derived from non-cancer, ER⁺, HER2⁺, TNBC breast subtypes, and their impact on angiogenesis. b Quantification of angiogenesis formation through hemoglobin estimation by Drabkin’s method (n = 3; ****, P < 0.0001; one-way ANOVA followed by Tukey’s multiple comparison test). c Representative images of Matrigel plugs containing serum exosomes derived from AA and CA TNBC patients that show comparison of angiogenesis between AA and CA TNBC patients. LCKLSL (AnxA2 inhibitory peptide) and LGKLSL (control peptide) were used to demonstrate the functional role of AnxA2 in contributing to angiogenesis. Peptide concentration: 5 μmol/L. d Quantification of angiogenesis formation through hemoglobin estimation by Drabkin’s method (n = 3; **, P < 0.01; ****, P < 0.0001; one-way ANOVA followed by Tukey’s multiple comparison test)

Fig. 7

Diagnostic outcome for the prediction of aggressive breast cancer. a Receiver operating characteristic (ROC) curve analysis using serum exo-AnxA2 for discriminating breast cancer patients (n = 169) from non-cancer females (n = 68). b ROC curve analysis for discriminating ER⁺ (n = 50), HER2⁺ (n = 59), and TNBC (n = 58) patients from non-cancer females (n = 68) using serum exo-AnxA2. The AUC values are shown on the graphs