Fig 1: High expression of anterior gradient 3 (AGR3) was associated with high risk of recurrence or metastasis in luminal B subtype of invasive ductal carcinoma (IDC) patients of grade I-II. Compared the proportion of AGR3 highly expressed patients in the group developing metastasis or recurrence (MR group) and the group disease-free (DF group). Besides, compared the proportion of AGR3 highly expressed patients in the group developing metastasis or recurrence within 5 years (MR 5 years group) and the group disease-free over 5 years (DF 5 years group). The proportion of every group was as follows. (A) MR, 55.1%; DF, 33.7%; MR 5 years, 50.0%; DF 5 years, 33.9%. (B) Luminal of MR, 70.8%; luminal of DF, 40.9%; luminal of MR 5 years, 69.4%; luminal of DF 5 years, 42.2%. (C) Luminal A of MR, 56.9%; luminal A of DF, 37.7%; luminal A of MR 5 years, 72.7%; luminal A of DF 5 years, 40.0%. (D) Luminal B of MR, 68.6%; luminal B of DF, 42.2%; luminal B of MR 5 years, 68.0%; luminal B of DF 5 years, 43.0%. (E) Non-luminal of MR, 19.0%; non-luminal of DF, 10.4%; non-luminal of MR 5 years, 15.0%; non-luminal of DF 5 years, 2.6%. (F) Human epidermal growth factor receptor 2 (HER2)-overexpressing of MR, 13.0%; HER2-overexpressing of DF, 0%; HER2-overexpressing of MR 5 years, 0%; HER2-overexpressing of DF 5 years, 7.7%. (G) Triple-negative breast cancer (TNBC) of MR, 23.5%; TNBC of DF, 9.1%; TNBC of MR 5 years, 18.7%; TNBC of DF 5 years, 0%. (H) Grade ?-? of MR, 64.0%; grade ?-? of DF, 36.5%; grade ?-? of MR 5 years, 58.5%; grade ?-? of DF 5 years, 37.0%. (I) Luminal of grade ?-? in MR, 75.68%; luminal of grade ?-? in DF, 43.13%; luminal of grade ?-? in MR 5 years, 72.41%; luminal of grade ?-? in DF 5 years, 44.44%. (J) Luminal A of grade ?-? in MR, 72.72%; luminal A of grade ?-? in DF, 40.91%; luminal A of grade ?-? in MR 5 years, 70.0%; luminal A of grade ?-? in DF 5 years, 44.8%. (K) Luminal B of grade ?-? in MR, 76.9%; luminal B of grade ?-? in DF, 43.9%; luminal B of grade ?-? in MR 5 years, 73.7%; luminal B of grade ?-? in DF 5 years, 44.3%. (L) Non-luminal of grade ?-? in MR, 30.8%; non-luminal of grade ?-? in DF, 8.9%; non-luminal of grade ?-? in MR 5 years, 17.0%; non-luminal of grade ?-? in DF 5 years, 0%. (M) TNBC of grade ?-? in MR, 36.4%; TNBC of grade ?-? in DF, 10.3%; TNBC of grade ?-? in MR 5 years, 30.0%; TNBC of grade ?-? in DF 5 years, 0%. (N) Grade ? of MR, 30.8%; grade ? of DF, 20.0%; grade ? of MR 5 years, 20.7%; grade ? of DF 5 years, 22.2%.
Fig 2: High expression of anterior gradient 3 (AGR3) was associated with poor prognosis in luminal B subtype of invasive ductal carcinoma (IDC) patients of grade I-II. (A) Overall survival (OS) and progression-free survival (PFS) curves of 330 cases of IDC patients were shown, respectively. (B) OS and PFS of 236 luminal patients were shown, respectively. (C) OS and PFS of 66 luminal A patients were shown, respectively. (D) OS and PFS of 170 luminal B patients were shown, respectively. (E) OS and PFS of 88 non-luminal patients were shown, respectively. (F) OS and PFS of 61 triple-negative breast cancer (TNBC) patients were shown, respectively. (G) OS and PFS curves of 261 IDC patients of grade ?-? were shown, respectively. (H) OS and PFS of 197 luminal patients of grade ?-? were shown, respectively. (I) OS and PFS of 55 luminal A patients of grade ?-? were shown, respectively. (J) OS and PFS of 142 luminal B patients of grade ?-? were shown, respectively. (K) OS and PFS of 58 non-luminal patients of grade ?-? were shown, respectively. (L) OS and PFS of 40 TNBC patients of grade ?-? were shown, respectively.
Fig 3: Breast cancer cells with anterior gradient 3 (AGR3) high expression were resistant to taxane but sensitive to 5-fluoropyrimidines (5-FU). (A) Compared the proportion of AGR3 highly expressed patients in groups with different drug sensitivities. Taxane sensitive group was 25% and resistant group was 64.1% (p=0.000, upper panel in left). Upper panel in right was representative immunohistochemistry (IHC) images of AGR3 expression in taxane sensitive and resistant group. 5-FU sensitive group was 73.3% and resistant group was 42.1% (p=0.004, lower panel in left). Lower panel in right were representative IHC images of AGR3 expression in 5-FU sensitive and resistant group. (B) With the treatment of taxane, cell viability of 3×Flag-AGR3-HA/MDA-MB-231 cells was much higher than 3×Flag-vector/MDA-MB-231 cells (*p < 0.05, ** p < 0.01, upper panel). Taxane IC50 values of 3×Flag-AGR3-HA/MDA-MB-231 cells in MTT and ATP assay were much higher than 3×Flag-vector/MDA-MB-231 cells (MTT, p=0.015; ATP, p=0.038, upper panel). With the treatment of 5-FU, cell viability of 3×Flag-AGR3-HA/MDA-MB-231 cells was much lower than 3×Flag-vector/MDA-MB-231 cells (*p < 0.05, **p < 0.01, lower panel). 5-FU IC50 value of 3×Flag-AGR3-HA/MDA-MB-231 cells in MTT and ATP assay were much lower than 3×Flag-vector/MDA-MB-231 cells (MTT, p=0.025; ATP, p=0.009, lower panel). (C) With the treatment of taxane, cell viability of siAGR3/T47D cells was much lower than scr/T47D cells (*p < 0.05, ** p < 0.01, upper panel). Taxane IC50 values of siAGR3/T47D cells in MTT and ATP assay were much lower than scr/T47D cells (MTT, p=0.033; ATP, p=0.006, upper panel). With the treatment of 5-FU, cell viability of siAGR3/T47D cells was much higher than scr/T47D cells (*p < 0.05, **p < 0.01, ***p < 0.001, lower panel). 5-FU IC50 value of siAGR3/T47D cells in MTT and ATP assay were much higher than scr/T47D cells (MTT, p=0.004; ATP, p=0.017, lower panel).
Fig 4: Anterior gradient 3 (AGR3) promoted proliferation and invasion ability of breast cancer cells. (A) Western blot analysis of AGR3 expression in MDA-MB-231 and T47D cells (patient tissues with AGR3 high expression as positive control) (left panel). AGR3 expression was detected by primary flag antibodies in 3×Flag-vector/MDA-MB-231 and 3×Flag-AGR3-HA/MDA-MB-231 cells (middle panel). AGR3 expression was detected by primary AGR3 antibodies in T47D, scr/T47D and siAGR3/T47D cells (right panel). (B) Proliferation ability was detected by ATP and sulforhodamine B (SRB) assays in 3×Flag-vector/MDA-MB-231 and 3×Flag-AGR3-HA/MDA-MB-231 cells, respectively (upper panel in left and middle). Proliferation assay was repeated by using Nikon ECLIPSE Ti microscope to count cell number in 3×Flag-vector/MDA-MB-231 and 3×Flag-AGR3-HA/MDA-MB-231 cells (upper panel in right). Proliferation ability was also detected by ATP and SRB assays in scr/T47D and siAGR3/T47D cells, respectively (lower panel in left and middle). Proliferation assay was also repeated by using Nikon ECLIPSE Ti microscope to count cell numbers in scr/T47D and siAGR3/T47D cells (lower panel in right). Bars are mean±SD. All experiments were performed 3 times independently (*p < 0.05, **p < 0.01). (C) Migration ability was detected by Matrigel Boyden chamber assays in 3×Flag-vector/MDA-MB-231 and 3×Flag-AGR3-HA/MDA-MB-231 cells (upper panel). Migration ability was detected by wound healing assays in scr/T47D and siAGR3/T47D cells (lower panel). Bars are mean±SD. All experiments were performed 3 times independently (*p < 0.05, ***p < 0.001). (D) Invasion ability was detected by Matrigel Boyden chamber assays in 3×Flag-vector/MDA-MB-231 and 3×Flag-AGR3-HA/MDA-MB-231 cells (upper panel). Invasion ability was detected by Matrigel Boyden chamber assays in scr/T47D and siAGR3/T47D cells (lower panel). Bars are mean±SD. All experiments were performed 3 times independently (*p < 0.05, ***p < 0.001).
Fig 5: Anterior gradient 3 (AGR3) expression increased with the development of breast tumor malignancy and was negatively correlated with histological grade but positively correlated with estrogen receptor (ER)/progesterone receptor (PR) status. (A) Representative immunohistochemistry (IHC) images of AGR3 low expression group (upper) and AGR3 high expression group (lower). (B) AGR3 expression increased with progression of malignancy degree of lesions. AGR3 expression of benign lesions was low, ductal carcinoma in situ (DCIS) was higher and IDC was the highest. (C) Representative IHC images of AGR3 expression in different histological grades of IDC (left panel). Western blot analysis of AGR3 expression in the frozen breast tumor specimens consisting of normal tissues, grade I, grade II, and grade III. Every type of tissues had 6 cases. ß-actin was used as a loading control (right panel). (D) The expression of AGR3, ER, and PR were detected by IHC in serial paraffin sections. The upper panel was representative IHC images of AGR3 and ER. The lower was AGR3 and PR. (E) Representative IHC images of AGR3 expression in disease-free group and recurrence or metastasis group.
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