Pharmacological induction of cell surface GRP78 contributes to apoptosis in triple negative breast cancer cells

Abstract

Breast cancer tumor with triple-negative receptors (estrogen, progesterone and Her 2, receptors) is the most aggressive and deadly subtype, with high rates of disease recurrence and poor survival. Here, we show that induction in cell surface GRP78 by doxorubicin and tunicamycin was associated with CHOP/GADD153 upregulation and increase in apoptosis in triple negative breast cancer tumor cells. GRP78 is a major regulator of the stress induced unfolded protein response pathway and CHOP/GADD153 is a pro-apoptotic transcription factor associated exclusively with stress induced apoptosis. The blocking of cell surface GRP78 by anti-GRP78 antibody prevented apoptosis, suggesting that induction of cell surface GRP78 by doxorubicin and tunicamycin is required for apoptosis. A better understanding of stress induction of apoptotic signaling in triple negative breast cancer cells may help to define new therapeutic strategies.

Figure 1. GRP78 expression: Comparison between MDAMB468 and BT474 breast cancer cell lines

(A) Representative histograms by FACS analysis showing MDAMB468 cell line negative for cell surface Her2 (red line), ER (green line) and GRP78 (blue line) and BT474 positive for cell surface Her2, ER and GRP78. Isotype control is represented by a black line. (B) Effect of doxorubicin and taxotere on the percent of cell surface GRP78. Doxorubicin and taxotere significantly increased the percent of cell surface GRP78 in MDAMB468 triple negative cells (*p < 0.001) but not in BT474 cells. (C) The effect of the different drugs on cell survival. BT474 and MDAMB468 were demonstrated to be sensitive to doxorubicin. The effect of taxotere was greater in BT474 than in MDAMB468 cells. The addition of taxotere significantly decreased cell survival in BT474 cells (*p < 0.001) and but was less effective, though significant in MDAMB468 cells (**p < 0.05).

Figure 2. Tumorigenic effect of doxorubicin and tunicamycin on cell surface GRP78 negative cell lines

(A) The 4T1 breast cancer mouse cell line expressed a low percent of cell surface GRP78 similar to MDAMB468. Doxorubicin and tunicamycin induced a significant increase in cell surface GRP78 (*p < 0.001). (B) Colony formation by MDAMB468 and 4T1 TNBC cells treated with doxorubicin and tunicamycin was inhibited significantly (*p < 0.001). (C) 10-week-old Balb/C nude mice were inoculated subcutaneously in the right flank with 1 × 10⁶ 4T1 cells in 100 μL PBS or with 4T1 pre-incubated with 0.1 μg/ml doxorubicin or with 10 μg/ml tunicamicin (10 mice per group). Mice from the same group uniformly developed relatively small tumors after doxorubicin or tunicamycin treatment compared to non treated mice cells (p < 0.02). (D) 4T1 cells extracted from mice xenografts, 31 days after tumor inoculation, showed significant increased cell surface GRP78 pre-incubated with doxorubicin (0.1 μg/ml) or tunicamycin (10 μg/ml) (*p < 0.004).

Figure 3. The effect of doxorubicin and tunicamycin on apoptosis

(A) Determination of the effect of doxorubicin and tunicamycin on apoptosis by FACS analysis. Doxorubicin (0.1 μg/ml) significantly induced apoptosis in 4T1 cells (**p < 0.001). Tunicamycin induced apoptosis significantly at 10 μg/ml (*p < 0.05). On the upper right, representative FACS analysis of apoptosis by AnnexinV/PI of cells incubated with doxorubicin and tunicamycin compared to non-treated cells. (B) Caspase 3 activity was significantly increased by these drugs (**p < 0.001). (C) Cytochrome c release was increased significantly by these drugs (***p < 0.006). (D) CHOP/GADD153 expression was significantly increased by doxorubicin and tunicamycin (**p < 0.001). Representative FACS analysis of caspase 3 activity, cytochrome c detection and CHOP/GADD153 expression in 4T1 cells treated with doxorubicin and tunicamycin compared to non-treated cells. Black line: Isotype. Blue line: Non treated control cells. Red line: Doxorubicin treated cells. Green line: Tunicamycin treated cells.

Figure 4. Blocking of cell surface GRP78 by anti-GRP78 antibody diminished doxorubicin and tunicamycin induction of apoptosis

(A) Cell surface GRP78 in doxorubicin (d) and tunicamycin (t) treated cells was inhibited significantly by blocking of cell surface GRP78 by anti-GRP78 antibody (+ab) (*p < 0.01). No changes were observed in control cells (c). (B) Apoptosis was assessed by FACS analysis using Annexin/PI. Blocking of cell surface GRP78 (+ab) diminished apoptosis in doxorubicin (d) and tunicamycin (t) treated cells (**p < 0.008). In the upper right flank, representative FACS analysis of cell surface GRP78. Black line: Isotype, filled colored line: drug treated cells, dashed line: cell surface GRP78 blocked by anti GRP78 antibody.

Figure 5. Anti GRP78 antibodies in the serum of mice

Analysis of anti-GRP78 IgG antibodies titer in serum of mice by ELISA on day 31 after 4T1 inoculation shows a significant decrease in antibody titer of mice injected with 4T1 cells treated either with doxorubicin or tunicamycin in comparison to tumor control mice (*p < 0.001).