Role Of Zoledronic Acid Plays In Hypoxia Inducible Factor-1 Alpha Induced Endocrine Therapy Resistance In Breast Cancer

ObjectiveEstrogen receptor positive breast cancer accounts for 70-80% of all kinds of breast cancer for which endocrine therapy (including anti-estrogen agent tamoxifen and aromatase inhibitor AI) is the standard therapy. However, a majority of patients would eventually develop primary or acquired endocrine resistance. Hypoxia is common to solid tumors including breast cancer. Central to hypoxia reaction is hypoxia inducible factor HIF-1 alpha which has been found that being related to tumor infiltration and invasion, resistant to radiotherapy and chemotherapy. The relationship between HIF-1α and endocrine therapy has not yet identified, especially lack of direct in vivo evidence. Zoledronic acid is a nitrogen-containing bisphosphonate and currently the standard therapy of breast cancer with bone metastasis. Studies demonstrate that zoledronic acid has direct anti-tumor effect. We have treated an elderly patient with endocrine resistance, which occurred after 32 months’neo-adjuvant endocrine therapy (Letrozole 2.5mg daily). A second core needle biopsy was performed and immunohistochemistry showed that HIF-1α expression was significantly higher in second biopsy tissue than those in baseline. The patient was given zoledronic acid treatment once owing to severe osteoporosis and surgery was performed one month later. The surgical specimen was found that HIF-1α and ki67 expression was significantly reduced. Therefore, we hypothesized that zoledronic acid may play an important role in endocrine resistance. Until now, relationship between zoledronic acid and endocrine resistance is rarely investigated. The study is to identify the relationship between HIF-1 alpha and endocrine therapy and the role of zoledronic acid plays in HIF-1 alpha induced endocrine therapy and relevant molecular mechanism.MethodsPart Ⅰ1. Immunohistochemistry was performed in second core needle biopsy tissue and surgical tissue after zoledronic acid treatment.2. Postmenopausal patients with ER-alpha positive breast cancer were treated with Letrozole (2.5mg daily) for at least 3 months.3. The expression of HIF-1-alpha was assessed by immunohistochemistry in paired samples from core needle biopsy at baseline and treated with letrozole for three months and surgical samples dissected after zoledronic acid therapy.Part II1. The full-length HIF-la gene was introduced into human ER-alpha-positive breast cancer MCF-7 cells, cells stably express HIF-1α gene were obtained after screening.2. The proliferation of MCF-7 cells stably express HIF-1 alpha gene and their sensitivity to fulvestrant were investigated by CCK-8 assay.3. The human ER-alpha-positive MCF-7 breast cancer cells were cultured under long-term chronic intermittent hypoxia at least three months to get a stable growth of hypoxic cells.4. Effects of chronic intermittent hypoxia on the proliferation were investigated by CCK-8 assay.5. The shRNA targeting HIF-1α was infected in intermittent hypoxic cells and clonogenic assay was used to observe its sensitivity to fulvestrant.6. Western blot was used to detect expression level of ER-alpha and HIF-1α.7. ER positive human breast cancer cells stably express HIF-1 alpha gene and empty plasmid were implanted orthotopically into BALB/c female nude mice to determine the effect of HIF-1 alpha on the tumor growth. Fulvestrant (Astrazeneca) was administered to BALB/c female nude mice mentioned above and tumor growth was tested to demonstrate the effect of HIF-1 alpha on resistance to endocrine therapy in vivo.8. Effects of zoledronic acid and fulvestrant combined with zoledronic acid treatment on the proliferation of MCF-7 cells stably express HIF-1 alpha gene were investigated by CCK-8 assay.9. Western blot was used to detect the effect zoledronic acid on the expression level of HIF-la and established resistant signal pathway proteins under normoxia and hypoxia. Real-time PCR was used to determine HIF-1 alpha mRNA expression level.10. ER positive human breast cancer cells stably express HIF-1 alpha gene and empty plasmid were implanted orthotopically into BALB/c female nude mice. Fulvestrant (Astrazeneca). zoledronic acid (Novartis), fulvestrant combined with zoledronic acid were administered to BALB/c female nude mice mentioned above, respectively and tumor growth was tested to demonstrate the effect of HIF-] alpha on resistance to endocrine therapy in vivo.ResultsPart Ⅰ1. HIF-la expression level was significantly higher in the second core needle tissue, while the expression of HIF-1α was significantly reduced after zoledronic acid treatment for this patient.2. Samples and clinicopathologic data were collected from 20 patients who were treated with letrozol (2.5mg, daily) at least 3 months during 2011-2013, obtain from cancerhospital, Fudan University.3. Higher baseline HIF-1-alpha level was correlated with poor clinical response (P<0.05, Spearman correlation test).4. The expression level of HIF-1α was universally decreased in post-zoledronic acidtreatment residual tumors compared with that in second biopsy samples (P=0.001, Wilcoxon t test),Part Ⅱ1. Wild type MCF-7 cells were infected with full-length HIF-1 alpha gene and cells stably expressing HIF-1 alpha gene (MCF-7/hif) were obtained after screening.2. MCF-7/hif was resistant to fulvestrant treatment compaired with control cells (P<0.01). Chronic intermittent hypoxia significantly promoted cancer cell Proliferation in MCF-7 lines (P<0.05).3. Chronic iniermittent hypoxia up-regulated the expression of HIF-la, while down-regulated ER-alpha protein.4. The sensitivity of intermittent hypoxic MCF-7cells to fulvestrant was significantly increased after HIF-1α gene was knocked down (P<0.01).6. MCF-7/hif cell xeograft grow faster and larger than those of control group (P<0.05).7. Fulvestrant was effective in suppressing tumor growth of mice implanted orthotopically with control cells rather than MCF-7/hif ones (P<0.05).8. Zoledronic acid significantly inhibits the expression of HIF-1 alpha, RAS, pMAPK and pmTOR (P<0.01), while it has no significant effect on the expression of PI3K and pAKT (P>0.05). Real-time PCR indicated that zoledronic acid significantly inhibit the HIF-1 alpha mRNA expression (P<0.01).9. Single agent zoledronic acid did not inhibit the proliferation of MCF-7/hif (P>0.05), while zoledronic acid combined with fulvestrant significantly inhibit the proliferation of MCF-7/hif (P<0.01).10. Single agent zoledronic acid treatment did not exert an effect on the growth of MCF-7/hif cell xeograft, while zoledronic acid combined with fulvestrant significantly suppress tumor growth of MCF-7/hif cell xeograft (P<0.01).Conclusion Results of this pilot study indicated that hypoxia inducible factor-1 alpha might be one of the critical factors involving in endocrine resistance of human breast carcinoma. Cells that over-expressed HIF-1 alpha were not sensitive to fulvestrant treatment. Zoledronic acid may improve endocrine resistance via inhibiting RAS/MAPK/mTOR/HIF-1 pathway. New treatment strategy targeting HIF-1 alpha should be considered to overcome or reverse endocrine resistance in breast cancer.