Abstract
Bladder cancer is among the leading causes of cancer in the Western and developing world. Left undetected and untreated, this disease progresses from its initial stages into muscle-invasive bladder cancer (MIBC) which is significantly more aggressive and difficult to treat. Current treatment for MIBC is platinum-based chemotherapy, which is highly toxic to the patient. This chemotherapeutic regimen utilizes cisplatin in combination with two to four other chemotherapeutics. In addition to being highly toxic, this regimen is sometimes ineffective due to intrinsic or acquired cisplatin resistance. Due to this, new therapeutic targets have been sought. One likely candidate is the ErbB family of receptor tyrosine kinases, which have been shown to be upregulated in MIBC. Due to their role in cell survival, and their utility as therapeutic targets in other epithelial cancers, many clinical trials have been conducted to assess the utility of ErbB inhibitors in the treatment of MIBC. Unfortunately, none of these studies has shown any added benefit over standard platinum-based chemotherapy. This study was undertaken to elucidate the mechanisms by which the ErbB family and cisplatin interact within the bladder cancer cell. Four established bladder cancer cell lines, T24, TCCSUP, J82, and RT4, were used for this study to provide a broader representation of MIBC. The T24 and TCCSUP cell lines were derived from women, while the J82 and RT4 cell lines were derived from men. Cells were treated with various ErbB inhibitors as well as cisplatin. Cells were also transfected with siRNA against EGFR, ErbB2, and ErbB3 to knockdown expression of these ErbB family members in these cells. Cells were analyzed for growth via MTT assay, as well as apoptosis and cell cycle distribution by flow cytometry. Cell lysates were further probed by Western Blot for protein expression levels and protein activation. Western Blot results revealed that the cell lines chosen had ErbB family expression consistent with the literature, except the RT4 cell line which expressed very low levels of all four ErbB family members. MTT assay demonstrated that the T24, TCCSUP, and RT4 cell lines were all resistant to cisplatin treatment, while the J82 cell line was sensitive to cisplatin. MTT assay further showed that knockdown of EGFR via siRNA was more effective at inhibiting cellular growth than ErbB2 knockdown. While flow cytometry revealed that neither siRNA had a major effect on apoptosis in any cell line, it also revealed that cisplatin induced a growth arrest in the J82 cell line by causing a G2 arrest of the cell cycle. While ErbB3 siRNA was ineffective at knocking down ErbB3 expression in all cell lines except J82, this was determined to be caused by an increase in ErbB3 protein stability in these cell lines. In the J82 cell line, ErbB3 knockdown significantly inhibited cellular growth, and trended towards an increase in apoptosis. Probing of cellular lysates revealed that no common pattern of ErbB family phosphorylation sites was activated between the four cell lines. However, cisplatin-resistant cell lines did trend toward an increase in activation of the downstream effector ERK upon treatment with cisplatin. However, ERK phosphorylation was not able to be reliably inhibited by ErbB inhibitors during ligand-induced activation. While growth assays demonstrated the ability of various combinations of ErbB inhibitors to retard cellular growth, no one drug combination was determined to be the best. In addition, no drug combination tested was observed to have a significant effect on apoptosis or cell cycle distribution of bladder cancer cell lines. The current study points to the importance of the ErbB family in the broad cancer treatment landscape, and demonstrates the desperate need for new therapies which target this family by unconventional methods. Taking advantage of this information, in combination with current treatment regimens and cancer knowledge, will help the medical community to better serve the population of MIBC patients.