BIIB021, A Novel Hsp90 Inhibitor, Sensitizes Esophageal Squamous Cell Carcinoma To Radiation

BackgroundEsophageal carcinoma is the eighth most common cancer and the sixth leading cause of cancer deaths in the world. In China and other East Asian countries, more than 90% of cases are esophageal squamous cell carcinoma (ESCC).Radiotherapy is generally used as preoperative and postoperative treatment that has a crucial role in improving local control and survival of ESCC. However, intrinsic tumor radioresistance accounts for the high recurrence and poor 5-year survival of ESCC patients. Therefore, identification of reliable radiosensitizers to ESCC cells would be urgently desirable. Heat shock protein 90 (HSP90) is a molecular chaperone that assists the conformational maturation, folding and refolding of client proteins during stress and protects them from degradation. More than 200 "client proteins" have been identified so far, including tyrosine kinases, transcription factors, structural proteins and hormone receptors.It has been confirmed that EGFR, Akt, Raf-1, HER2 are associated with the radioresponse, thereby protecting radiation-induced cell death. HSP90 is abundantly expressed in esophageal cancer as well as in esophageal cancer cell lines.Therefore, HSP90 represents a potential therapeutic target in the treatment of patients with ESCC. Inhibition of HSP90 leads to degradation of these radioresistant proteins thereby enhancing tumor cell death in a variety of cell lines and tumor models, including ESCC. Inhibition of HSP90 may not only provide a unique therapeutic pathway, but also promote the efficacy of radiotherapy. BIIB021 is a novel, orally available inhibitor of HSP90 that is currently in phase Ⅰ/Ⅱ clinical trials. Preclinical studies have shown that 17-AAG significantly sensitized the ESCC cell lines for irradiation. However,17-AAG is poor in pharmaceutical properties and is difficult to be formulated, which significantly affects its therapeutic efficacy in clinical practiceln this report, we investigated a fully synthetic and bioavailable HSP90 inhibitor, BIIB021 in ESCC cell lines, either as single agent or in combination with radiation. We found that BIIB021 showed significantly antitumor activity.Objective:The aim of this study is to investigate the effect of BIIB021 on the proliferation and radiosensitivity of ESCC cell lines.Methods:1. CCK-8 assay is used to evaluate the growth inhibition of Eca109 and Eca9706 cell lines treated with increasing concentrations of BIIB021Logarithmically growing cells Eca109 and Eca9706 were counted and seeded in 96-well plates and treated with increasing concentrations of BIIB021 (OnM,65nM, 125nM,250nM,500nM, 1μM,2μM). Following drug addition, the plates were incubated for 5 days. Cell growth inhibition was examined by CCK-8 assay. Percentage of viable cells=(OD450 Of treated sample-OD450 of blank sample)/ (OD450 of control sample-OD450 of blank sample) ×100.2. Clonogenic assay is used to evaluate the effect of BIIB021 in combination with radiation on Eca109 and Eca9706 cell linesEca109 and Eca9706 were seeded in 6-well plates. After allowing cells to attach, BIIB021 was added about the IC25 of each cell line (Eca109:97nM; Eca9706:33nM). For BIIB021 in combination with radiation,16 hr after adding BIIB021, cells were irradiated with a single dose of 2,4,6,8Gy. Four hours after radiation, all plates were aspirated and fresh medium were added.14 days after seeding, colonies were stained with crystal violet, and the number of colonies containing at least 50 cells was counted.3. Apoptosis and cell cycle are detected by flow cytometry analysis (control, BIIB021 alone, radiation alone, BIIB021 in combination with radiation)Eca109 and Eca9706 cells were plated and exposed to either 1μM BIIB021 or 6Gy radiation for 24 hr. For combination, cells were treated with 1μM BIIB021 for 16 hr, and then treated with a single dose of 6Gy of radiation. Cells were collected 24 hr after radiation. Apoptosis analysis was performed and cell cycle distribution was measured.4. The expression of proteins which are associated with radioresponse and apoptosis (pEGFR, pAkt, Raf-1, cleaved PARP) are examined by western blotting. The time course for the effect of BIIB021 on the expression of pEGFR and pAkt was also done by western blotting.Results:1. BIIB021 inhibited the growth of Eca109 and Eca9706 in a dose-dependent manner. Eca9706 was more sensitive to BIIB021 than Eca109.2. BIIB021 radiosensitized ESCC cell lines.3. BIIB021 in combination with radiation significantly increased the apoptosis rate and enhanced G2 phase arrest of ESCC cell lines compared with other treatment alone.4. BIIB021 induced degradation of Hsp90 key client proteins associated with radioresistance and induced degradation of pAkt and pEGFR in a time-dependent manner in Eca109 and Eca9706 cell lines.Conclusions:1.All the results indicated that BIIB021 exhibited strong antitumor activity in ESCC cell lines, either as a single agent or in combination with fractionated radiation.2. Radiotherapy is generally used to treat advanced unresectable tumors or used as preoperative and postoperative treatment, that has a crucial role in improving local control and survival of ESCC. However, intrinsic tumor radioresistance accounts for the high recurrence and poor 5-year survival of ESCC patients. Our results suggests that this synthetic and bioavailable HSP90 inhibitor BIIB021 simultaneously affects multiple pathways involved in tumor development and progression in the ESCC setting and may represent a better strategy for the treatment of ESCC patients, either as a monotherapy or a radiosensitizer.