Cisplatin-induced increase in heregulin 1 and its attenuation by the monoclonal ErbB3 antibody seribantumab in bladder cancer

Cisplatin-based combination chemotherapy is the foundation for treatment of advanced bladder cancer (BlCa), but many patients develop chemoresistance mediated by increased Akt and ERK phosphorylation. However, the mechanism by which cisplatin induces this increase has not been elucidated. Among six patient-derived xenograft (PDX) models of BlCa, we observed that the cisplatin-resistant BL0269 express high epidermal growth factor receptor, ErbB2/HER2 and ErbB3/HER3. Cisplatin treatment transiently increased phospho-ErbB3 (Y1328), phospho-ERK (T202/Y204) and phospho-Akt (S473), and analysis of radical cystectomy tissues from patients with BlCa showed correlation between ErbB3 and ERK phosphorylation, likely due to the activation of ERK via the ErbB3 pathway. In vitro analysis revealed a role for the ErbB3 ligand heregulin1-β1 (HRG1/NRG1), which is higher in chemoresistant lines compared to cisplatin-sensitive cells. Additionally, cisplatin treatment, both in PDX and cell models, increased HRG1 levels. The monoclonal antibody seribantumab, that obstructs ErbB3 ligand-binding, suppressed HRG1-induced ErbB3, Akt and ERK phosphorylation. Seribantumab also prevented tumor growth in both the chemosensitive BL0440 and chemoresistant BL0269 models. Our data demonstrate that cisplatin-associated increases in Akt and ERK phosphorylation is mediated by an elevation in HRG1, suggesting that inhibition of ErbB3 phosphorylation may be a useful therapeutic strategy in BlCa with high phospho-ErbB3 and HRG1 levels.


Supplementary Figure 2. BL0269 tumors collected after 3-and 17-days following cisplatin treatment showed little alteration in EGFR phosphorylation.
Mice bearing BL0269 tumors were treated with vehicle or cisplatin and sacrificed after 3 or 17 days. Tumors were extracted, formalin fixed, and paraffin embedded. Then the FFPE samples sectioned and stained for various phospho-proteins as indicated (bar: 50 µm).

Supplementary Figure 3: Effects of cisplatin treatment of cisplatin on bladder cancer cell lines.
A). T24, RT4, TCCSUP and J82 cells were treated with platinum binding buffer (3 mM NaCl and 1 mM sodium phosphate) (CBB) or 200 nM cisplatin in CBB for 72 hours. Immunoblots were subjected to antiphospho-ErbB3 (Y1289) antibody or total ErbB3. Note that ErbB3 levels were much higher in RT4 cells compared to the rest, and therefore, we are showing here both an exposure that shows the expression of ErbB3 in T24 and J82 cells (TCCSUP did not appear to express detectable levels of ErbB3) and another exposure that showed the relative levels of ErbB3 in RT4 cells. B). Correlation of ERK phosphorylation with patient death, which was determined at the time of analysis. ERK phosphorylation was scored separately in the nucleus and the cytoplasm. Patient death correlated with nuclear ERK phosphorylation but not phosphorylation of cytoplasmic ERK1/2, indicating that it is the genomic effects of ERK phosphorylation that regulate patient survival.

Supplementary Figure 4. Increased expression of HRG1 in cisplatin resistant T24 and RT4 cells compared to cisplatin sensitive J82 and TCCSUP. (A)
The four cell lines were pelleted and the cell pellets formalin fixed and paraffin embedded. The FFPE cell pellets were then sectioned and stained for HRG1. Results indicate that T24 and RT4 cells express higher HRG1 (brown staining, see inset) compared to J82 and TCCSUP (bar: 50 uM). (B) Extrinsic HRG1 localizes to the cell membrane. Cells were cultured in medium containing charcoal stripped serum to remove all steroids and growth factors including HRG1. Then the cells were treated for 72 hours with 200 nM cisplatin, which increased intrinsic HRG1 (localized to the cytosol). However, at the end of that period, 50 ng/ml extrinsic HRG1 was added to the medium for 15 minutes, then the cells were washed and fixed. Fixed cells were stained for HRG1. The extrinsic HRG1 localized to the cell membrane (bar: 15 µM).

Supplementary Figure 5. Effect of the combination of cisplatin and seribantumab/MM-121 on ErbB3 protein levels in BL0269 tumors. A)
Log-rank (Mantel Cox) test for the comparison of survival groups. B) Immunocompromised mice bearing BL0269 tumors treated with vehicle (control), 2 mg/Kg cisplatin, 10 mg/Kg seribantumab (MM-121) and the combination of the two were formalin fixed and paraffin embedded. Sections were stained for ErbB3 levels by immunohistochemistry-(bar: 25 µm). C). Representative tumors from the four groups that were obtained following euthanasia of the mice at the end of the experiment. The tumors were formalin fixed and paraffin embedded, after which the blocks were sectioned and stained for ErbB3 phosphorylation (Y1289) (bar: 50 µm). Inset: Enlarged figures showing ErbB3 phosphorylation in the four groups.
Supplementary Figure 6. Seribantumab, but not trastuzumab, reduces tumor growth in the cisplatin sensitive BL0440 PDX model and in cisplatin resistant T24 cells. A). J82 cells were treated with vehicle, 200 nM cisplatin, 2 µM seribantumab or the combination for 72 hours, after which cells were trypsinized, and unfixed cells were stained with propidium iodide (PI) and Annexin V (AV). Stained cells were then assayed by flow cytometry. Data obtained was analyzed to obtain fractions that represent live cells (unstained), early apoptosis (Annexin V stained), late apoptosis (both AV and PI treated) and in necrosis (PI only). B, C) Mice dually implanted with BL0440 PDX tumors were treated with 10 mg/kg seribantumab, 10 mg/kg Herceptin (trastuzumab), or placebo twice weekly for three weeks and then monitored for up to 10 days. B). Corresponding box and whisker plots of tumor size. C). Immunohistochemical staining of ErbB3 of BL0440 PDX tumors treated with vehicle or trastuzumab. Bar: 50 μm.