Anti-progranulin/GP88 antibody AG01 inhibits triple negative breast cancer cell proliferation and migration

Abstract

Background: Triple negative breast cancer (TNBC) is characterized by invasiveness and short survival. Identifying novel TNBC-targeted therapies, to potentiate standard of care (SOC) therapy, is an unmet need. Progranulin (PGRN/GP88) is a biological driver of tumorigenesis, survival, and drug resistance in several cancers including breast cancer (BC). PGRN/GP88 tissue expression is an independent prognostic factor of recurrence while elevated serum PGRN/GP88 level is associated with poor outcomes. Since PGRN/GP88 expression is elevated in 30% TNBC, we investigated the involvement of progranulin on TNBC.

Methods: The effect of inhibiting PGRN/GP88 expression in TNBC cells by siRNA was investigated. The effects of a neutralizing anti-human PGRN/GP88 monoclonal antibody AG01 on the proliferation and migration of two TNBC cell lines expressing PGRN/GP88 were then examined in vitro and in vivo.

Results: Inhibition of GP88 expression by siRNA and AG01 treatment to block PGRN/GP88 action reduced proliferation and migration in a dose-dependent fashion in MDA-MB-231 and HS578-T cells. Western blot analysis showed decreased expression of phosphorylated protein kinases p-Src, p-AKT, and p-ERK upon AG01 treatment, as well as inhibition of tumor growth and Ki67 expression in vivo.

Conclusion: PGRN/GP88 represents a therapeutic target with companion diagnostics. Blocking PGRN/GP88 with antibody treatment may provide novel-targeted solutions in TNBC treatment which could eventually address the issue of toxicity and unresponsiveness associated with SOC.

Keywords: Anti-progranulin antibody; Ki67; Migration; Progranulin SiRNA; Progranulin/GP88; Proliferation; Triple negative breast cancer.

Conflict of interest statement

Conflict of interest Binbin Yue, Jianping Dong, and Ginette Serrero are employees of A&G Pharmaceutical. Rupa Guha and Aditi Banerjee have no conflicts to declare.

Figures

Fig. 1

Effect of progranulin silencing on cell proliferation, migration, and protein phosphorylation in MDA-MB-231 cells. Progranulin silencing in MDA-MB-231 cells was performed separately with two commercially available GRN SiRNA pools as described in the method section. a, b Western blot analysis showed a dose-dependent reduction in progranulin (PGRN) expression in aGRN siRNA smartpool and bGRN siRNA ON-targetplus-transfected MDA-MB-231 cell lysates as compared to lysates from control siRNA-transfected cells. The quantification of proteins bands intensity was done using LabWorks 4 software. The protein bands were first normalized against GAPDH expression as a loading internal control for each lane. The results for GRN SiRNA treatment were then expressed as % of values in control SiRNA-transfected cells. c Bright-field microscopic representative view (×100) of MDA-MB-231 cells 48 h after siRNA transfection (smartpool GRN SiRNA). d Progranulin silencing using GRN SiRNA smartpool in MDA-MB-231 showing a dose-dependent reduction in cell proliferation. Data points represent fold increase in live cell numbers over the inoculation density in control SiRNA and GRN SiRNA-transfected cells ± SD. e Progranulin silencing using ON- targetplus GRN SiRNA pool in MDA-MB-231 cells showing a dose-dependent reduction in cell proliferation. Data points represent fold increase in live cell numbers over the inoculation density in control SiRNA and GRN SiRNA-transfected cells ± SD. fGRN SiRNA (smart pool) transfection in MDA-MB-231 cells results in a dose-dependent decrease in % migration (24 h) as compared to control siRNA-transfected cells by transwell migration assay. gGRN SiRNA (ON-targetplus smart pool) transfection in MDA-MB-231 cells resulted in a dose-dependent decrease in % migration compared to control SiRNA-transfected cells by transwell migration assay. Data points represent mean of % migration ± SD. h Photomicrographs (×100) of migration transwells as obtained from GRN siRNA (ON-targetplus) and control siRNA-transfected MDA-MB-231 cells. i Western blot analysis of the expression of several signaling and attachment proteins in MDA-MB-231 cell lysate upon progranulin silencing (Smartpool). Western blots are representative of two replicates of the experiments. GraphPad Prism P value: 0.01 to 0.05 (*), 0.001 to 0.01 (**), 0.0001 to 0.001 (***)

Fig. 2

Effect of AG01 on the proliferation, migration, and signaling in two TNBC cell lines MDA-MB-231 and HS578-T. Data from a–d are obtained from two independent experiments run in duplicates and are represented as mean ± SD. a Effect of AG01 (100 μg/ml) on MDA-MB-231 cell proliferation compared to human IgG (100 μg/ml) and vehicle (PBS) as controls. Cells were counted after 72 h treatment by trypsinization and trypan blue exclusion method. b Proliferation of MDA-MB-231 cells treated for 72 h with different concentrations of AG01 (25–300 μg/ml) and determined by cell counting. c Effect of AG01 (100 μg/ml) on HS578-T cell proliferation compared to human IgG (100 μg/ml) and vehicle (PBS) as controls. d Proliferation of HS578-T cells treated with different concentrations of AG01 (25–300 μg/ml) for 48 h. e, f Ki67 expression by immunohistochemistry in MDA-MB-231 and HS578-T cells. Cells were harvested onto glass microscope slides with a cytospin centrifuge at a density of 105 cells/slide. Cells were fixed, permeabilized, and incubated with anti-Ki67 antibody (Appendix 1—Supplementary Material) in 1% BSA-PBS. Following incubation with secondary antibody, the slides were stained with freshly prepared DAB solution and counterstained by Mayer’s Hematoxylin solution. Ki67-positive cells were counted and the numbers were normalized to the total number of cells counted in the same field (approximately 200). e Photomicrograph images (×100) of MDA-MB-231 cells stained by immunohistochemistry with Ki67 antibody. MDA-MB-231 cells were treated with control or AG01 at 100 μg/ml for 72 h. f Ki67-positive nuclear count was evaluated by counting at least 200 MDA-MB-231 cells from three different fields. Ki67-positive nuclei counts were normalized to the total cell count and represent the average of two experiments run in duplicate. Data are provided as mean positive nuclei ± SD. g Photomicrograph images (×100) of HS578-T cell stained with Ki67 antibody. HS578-T cells were treated with control or AG01 at 100 μg/ml for 48 h. Ki67-positive nuclear count was evaluated by counting at least 200 HS578-T cells from three different fields, h Ki67-positive nuclei count was normalized with the total cell count and represent the average of two experiments run in duplicate (P < 0.0001). GraphPad Prism P value: 0.01 to 0.05 (*), 0.001 to 0.01 (**), 0.0001 to 0.001 (***), < 0.0001 (****), ns (not significant)

Fig. 3

Effect of AG01 antibody on TNBC cell migration and invasion. Data points represent mean ± SD from triplicates of three independent experiments. a Effect of AG01 (100 μg/ml) on MDA-MB-231 cell migration compared to human IgG (100 μg/ml) and vehicle (PBS) as controls. b AG01 treatment of MDA-MB-231 cells at 25, 50, and 100 μg/ml showed a dose-dependent reduction in % cell migration as compared to control-treated cells. c Effect of AG01 (100 μg/ml) on HS578-T cell migration compared to human IgG (100 μg/ml) and vehicle (PBS) as controls. d AG01 treatment in HS578-T cells at 50, 75, and 100 μg/ml showed a dose-dependent reduction in % cell migration as compared to control cells. e AG01 treatment of MDA-MB-231 cells shows a dose-dependent reduction in % cell invasion as compared to control cells. f Photomicrographs (×100) of cells invasion through transwell after crystal violet staining. GraphPad Prism P value: 0.001 to 0.01 (**), 0.0001 to 0.001 (***), < 0.0001 (****), ns (not significant)

Fig. 4

Effect of AG01 treatment of TNBC cells on the phosphorylation of regulatory proteins in MDA-MB-231 cells. MDA-MB-231 cells were treated with AG01 (100 μg/ml) or control for 24 and 72 h before preparing cell lysates for western blot analysis. The quantification of proteins bands intensity was done using LabWorks 4 software. The protein bands were first normalized against GAPDH expression as a loading internal control for each lane. In order to calculate the ratio of phosphoprotein/total corresponding protein, the normalized density of phosphoprotein was divided by the normalized corresponding total protein intensity. The results for AG01 treatment were then expressed as % of values in untreated control. a Western blot analysis of Akt and p-Akt expression Graphs represent normalized densitometric analysis of p-Akt and p-Akt/Akt ratio. b Western blot analysis of ERK1/2 and p-ERK1/2 upon AG01 treatment for 24 and 72 h compared to controls. Graphs represent normalized densitometric analysis of p-ERK and p-ERK/ERK ratio. c Western blot analysis of Src and p-Src. Graphs represent normalized densitometric analysis of p-Src and p-Src/Src ratio. d Western blot analysis of FAK and p-FAK. Graphs represent densitometric analysis of p-FAK and p-FAK/FAK ratio. Western blots are representative of at least two replicate experiments. Data were analyzed and expressed as % change in densitometric readings as compared to control. The left graph represents the % relative expression in phosphoprotein and the right graph represents the ratio of normalized phosphoprotein/total corresponding protein. Data point represents mean ± SEM. P < 0.05*, P < 0.009**, P < 0.0009***

Fig. 5

Effect of AG01 treatment on the phosphorylation of signaling proteins in HS578-T cells. a Western blot analysis of Akt and p-Akt expression in HS578-T cells treated with 100 μg/ml AG01 for 24 and 48 h compared to controls at the same time points. Graph representing densitometric analysis of p-Akt and p-Akt/Akt ratio. b Western blot analysis of ERK1/2 and p-ERK1/2 expression in HS578-T cell lysate after AG01 treatment for 24 and 48 h compared to controls. Graph represents the densitometric analysis of p-ERK and p-ERK/ERK ratio. c Western blot analysis of Src and p-Src expression in HS578-T cells upon 24 and 48 h AG01 treatment. Graphs represent the densitometric analysis of p-Src and p-Src/Src ratio in AG01-treated cells compared to control. d Western blot analysis of FAK and p-FAK after 24 and 48 h of HS578-T cells and densitometric analysis of p-FAK and p-FAK/FAK ratio. Western blots are representative of at least two replicate experiments. Data analyzed as % change in densitometric readings as compared to control. The right panel represents the % relative expression in phosphoprotein and phosphoprotein/total corresponding protein compared to control. Data point represents mean ± SEM. P < 0.05 *, P < 0.009**, P < 0.0009***

Fig. 6

AG01 treatment of MDA-MB-231 cells leads to inhibition in the expression of proteins in oncology array. a Expression of proteins in Human XL oncology array was performed with MDA-MB-231 cell lysate upon 72 h AG01 treatment compared to control-treated cells. Circles identify dots corresponding to 9 representative proteins with decreased expression in AG01-treated cells. Inset provides the identities of these proteins. b Graph representing densitometric analysis of the expression of SPARC, progranulin, proteins associated with metastasis and angiogenesis, obtained from the analysis of the duplicate protein array results for control cells (black bar) and AG01-treated cells (light gray bar). c Western blot analysis of the expression of SNAIL, c-Met, ICAM-1, and AXL in MDA-MB-231 and HS578-T cell lysate upon AG01 treatment (light gray bar) as compared to controls (black bar). Graphs represent the densitometric analysis of SNAIL, c-Met, ICAM-1, and AXL. d Western blot analysis of progranulin from MDA-MB-231 and HS578-T cell lysate upon AG01 treatment as compared to parallel PBS controls. Graph representing densitometry analysis. e Western blot analysis of fibronectin from MDA-MB-231 upon AG01 treatment as compared to parallel PBS controls. Graph representing densitometry analysis. Results are representative of at least two replicates of the experiment. Data were analyzed as % change in densitometry readings as compared to parallel control. Data point represents mean ± SEM. P < 0.05*, P < 0.009**, P < 0.0009***, ns non-significant

Fig. 6

AG01 treatment of MDA-MB-231 cells leads to inhibition in the expression of proteins in oncology array. a Expression of proteins in Human XL oncology array was performed with MDA-MB-231 cell lysate upon 72 h AG01 treatment compared to control-treated cells. Circles identify dots corresponding to 9 representative proteins with decreased expression in AG01-treated cells. Inset provides the identities of these proteins. b Graph representing densitometric analysis of the expression of SPARC, progranulin, proteins associated with metastasis and angiogenesis, obtained from the analysis of the duplicate protein array results for control cells (black bar) and AG01-treated cells (light gray bar). c Western blot analysis of the expression of SNAIL, c-Met, ICAM-1, and AXL in MDA-MB-231 and HS578-T cell lysate upon AG01 treatment (light gray bar) as compared to controls (black bar). Graphs represent the densitometric analysis of SNAIL, c-Met, ICAM-1, and AXL. d Western blot analysis of progranulin from MDA-MB-231 and HS578-T cell lysate upon AG01 treatment as compared to parallel PBS controls. Graph representing densitometry analysis. e Western blot analysis of fibronectin from MDA-MB-231 upon AG01 treatment as compared to parallel PBS controls. Graph representing densitometry analysis. Results are representative of at least two replicates of the experiment. Data were analyzed as % change in densitometry readings as compared to parallel control. Data point represents mean ± SEM. P < 0.05*, P < 0.009**, P < 0.0009***, ns non-significant

Fig. 7

Effect of AG01 on the tumorigenicity of MDA-MB-231 xenograft in athymic nude mice. MDA-MB-231 cells were injected subcutaneously in the flank of female athymic nude mice as described in the method section. When the tumor became visible, the mice were randomized to experimental groups (8 mice per group and with 4 mice per cage) and treated with either human IgG or AG01 at 10 mg/kg twice weekly by i.p injection. Tumor size was determined with a caliper and tumor volume calculated as described in the method section. a Graph representing changes in tumor volume during treatment. Each data point represents mean ± SEM. b Mouse body weight in the two treatment groups at the end of the experiment showing that there were no changes in body weight with AG01 treatment. c Tumor weight in Human IgG and AG01 groups as the end of the experiments. d Photomicrograph images (×200) of Ki67 immunohistochemistry obtained from tumor sections of Human IgG control (HuIgG) and AG01-treated mice. e Ki67 immunohistochemistry images were evaluated using Image J software in five different fields. Each bar represents mean ± SEM and expressed as % relative change compared to the HuIgG control. f Photomicrograph images (×200) of dividing cells (at any stage of cell division) obtained after haematoxylin eosin staining of tumor sections of control (HuIgG) and AG01-treated groups. g Mitotic index was determined by counting diving cells out of total cells from five different fields from three independent sections from three independent tumors. Values were expressed as mean ± SEM and then normalized to the HuIgG control. h Photomicrograph images (×200) of microvessels obtained after H&E staining of tumor sections of control (PBS and Human IgG) and AG01-treated groups. Images were evaluated by counting number of microvessels from 3 different fields. i Values were expressed as mean ± SEM and normalized to the HuIgG control