Endosomal protein expression of γ1-adaptin is associated with tumor growth activity and relapse-free survival in breast cancer

Abstract

Background: γ1-Adaptin is a subunit of adaptor protein complex-1 (AP-1), which regulates intracellular transport between the trans-Golgi network (TGN) and endosomes. Since expression levels of AP-1 subunits have been reported to be associated with cell proliferation and cancer malignancy, we investigated the relationships between the immunohistochemical expression of γ1-adaptin and both clinicopathological factors and relapse-free survival (RFS) in breast cancer tissue.

Materials and methods: SK-BR-3 cell line depleted of γ1-adaptin was used for cell proliferation, migration, and invasion assay. Intracellular localization of γ1-adaptin was examined with immunohistochemistry (IHC) using an antibody against γ1-adaptin, and with double immunohistofluorescence (IHF) microscopy using markers for the TGN and endosome. γ1-Adaptin intensities in IHC samples from 199 primary breast cancer patients were quantified and assessed in relation to clinicopathological factors and RFS.

Results: Cell growth, migration, and invasion of SK-BR-3 cells were significantly suppressed by the depletion of γ1-adaptin. Although the staining patterns in the cancer tissues varied among cases by IHC, double IHF demonstrated that γ1-adaptin was mainly localized in EEA1-positive endosomes, but not in the TGN. γ1-Adaptin intensity was significantly higher in the tumor regions than in non-tumor regions. It was also higher in patients with Ki-67 (high), ER (-), PgR (-), and HER2 (+). Among subtypes of breast cancer, γ1-adaptin intensity was higher in HER2 than in luminal A or luminal B. The results of the survival analysis indicated that high γ1-adaptin intensity was significantly associated with worse RFS, and this association was also observed in group with ER (+), PgR (+), HER2 (-), Ki-67 (high), or luminal B. In addition, the Cox proportional hazards model showed that high γ1-adaptin intensity was an independent prognostic factor.

Conclusion: These results suggest that the endosomal expression of γ1-adaptin is positively correlated with breast cancer malignancy and could be a novel prognostic marker.

Keywords: AP-1; Adaptor protein complex-1; Clathrin adaptor; Endosome; γ1-Adaptin.

Fig. 1

Effect of γ1-adaptin depletion on cell growth, migration, and invasion. a Western blot analysis of control (Ctrl) and γ1-adaptin-depleted SK-BR-3 cells (sh-γ1) using antibodies against EGFR, γ1-adaptin (γ1), and GAPDH. Molecular weight is indicated on the right. b Ctrl and sh-γ1 cells were cultured for 1, 4, 7, and 10 days, and their cell growth activity was assessed. The ratio to the value at day1 was plotted as the means ± SD of three experiments. The statistical differences between Ctrl and sh-γ1 at each time point were analyzed using Student’s t test. ***p < 0.001. c, d Cell migration (c) and invasion (d) of Ctrl and sh-γ1 cells were analyzed. The ratio of sh-γ1 to Ctrl was plotted as mean ± SD of three experiments. The statistical difference was analyzed using Student’s t test (*p < 0.05)

Fig. 2

Protein expression of γ1-adaptin in breast cancer tissues. a Lysates of 14 cases of breast cancer were examined by Western blot analysis using anti-γ1-adaptin (γ1) and GAPDH antibodies. Subtypes are indicated on the top. Lum: Luminal. Molecular weight (Kd) is indicated on the right. b The band intensity of γ1 was normalized to that of GAPDH, and the ratio to the value of No. 7 is plotted as mean ± SD (from three experiments). The case numbers correspond to those in (b). c To validate anti-γ1 antibody in IHC, cell pellets of MCF-7 cells transfected with control siRNA (si-ctrl) or siRNA for γ-adaptin (si-γ1) were immunostained using anti-γ1 (anti-γ1) (red). The nuclei were stained with Hoechst 33342 (blue). Bar: 20 µm. d Localization of γ1-adaptin in the non-cancer and cancer regions of breast cancer tissues are shown. For the cancer regions, four staining patterns were observed: Neg (negative), PN (perinuclear), SC (scattered), and Dif (diffuse). The nuclei were stained with hematoxylin. Boxed regions are magnified and shown in insets. Bars: 20 µm

Fig. 3

Subcellular localization of AP-1 in breast cancer tissues. Double IHF was performed in cases of Neg (a), PN (b), SC (c), and Dif (d), and non-cancer regions (e) using a combination of anti-γ1-adaptin (anti-γ1, red) and anti-EEA1 (endosomal marker, green), or anti-TGN46 (TGN marker, green). Merged images are shown on the right. Boxed regions are magnified and shown in insets. Bars: 5 µm

Fig. 4

Quantification of γ1-adaptin intensity and comparison with clinicopathological factors. a In an original image of ROI (i), the area mainly containing tumor cells were manually enclosed (dotted line), converted to gray scale and color-inverted (ii). Extracted thresholded pixels are red (iii). b Correlation analysis between the γ1 adaptin intensities by IHC and values obtained from the Western blot analysis (WB) in Fig. 1b. c γ1-Adaptin intensity was compared between the cancer and non-cancer regions, and between groups divided according to Ki-67, ER, PgR, and HER2. *p < 0.05, **p < 0.01, ***p < 0.001 (Mann–Whitney U test). (d) γ1-adaptin intensity was compared among the subtypes. *p < 0.05, **p < 0.01 (Steel–Dwass test)

Fig. 5

Survival time analysis. a ROC analysis for obtaining an optimum cut-off value for γ1-adaptin intensity. b, c Cases were divided into two groups by the cut-off value, and recurrent free survival (RFS; b) and overall survival (c) were evaluated using the Kaplan–Meier method. d Cases were divided into two groups according to ER, PgR, HER2, and Ki-67, and then, RFS was evaluated using the Kaplan–Meier method. e RFS was also analyzed for cases of luminal A and luminal B types. *p < 0.05, **p < 0.01 (log-rank test)