Her-4 Mediates Anoikis Resistance, Chemoresistance, And Metastatic Potential In Osteosarcoma

Osteosarcoma (OS) is the most common primary bone cancer in children, and despite more than 20 years of clinical trials, the 5-year survival for OS patients remains essentially unchanged (∼70% for non-metastatic patients and 30% for metastatic patients), with the vast majority deaths arising from metastasis to lungs or bones. We wanted to identify signaling pathways that mediate tumor survival and metastasis in OS, and we focused on Her-4 (ERBB4), which promoted survival on neuroblastoma cells in our previous work. Purpose: To evaluate the role of Her-4 in OS cell survival, metastasis and “stemness”. Methods: Her-4 expression was measured by flow cytometry, western blot and Q-PCR. Validated OS cell lines were manipulated with shRNA to knock-down Her-4 (compared to scrambled control). In vitro responses were measured by varying culture conditions (normal, high-density or anchorage-independent growth), serum starvation, hypoxia or chemotherapy (methotrexate, cisplatin, doxorubicin and 4-OH-ifosfamide) and measuring proliferation, cell cycle, and apoptosis (by both flow cytometry and PARP cleavage). A sarcosphere assay assessed expression of “stemness” markers, or tumor initiating cells (TICs), and Her-4 expression. The luciferase-labeled CCH-OS-O xenograft model was used to assess the impact of Her-4 knock-down on metastatic potential, with weekly luciferase measures (IVIS 100, Xenogen) assessing tumor burden. Immunohistochemistry (IHC) assessed Her-4 expression in archival material and murine xenografts. Results: Most OS cells express Her-4 which upregulated with increased culture density and with serum starvation. RT-PCR showed that the proportion of cleavable isoforms increased with increasing cell density (p 0.01). Knock-down of Her-4 in OS cell lines reduced proliferation and increased apoptosis (by sub-diploid DNA and PARP cleavage) for cells grown in conventional culture, high density or spheres. Her-4 knock-down increased sensitivity to methotrexate (60% increase in apoptosis) but not other chemotherapies. To assess the role of Her-4 in metastasis, NOD/SCID/IL2Rg-/- mice were injected in the tibia with CCH-OD-O cells expressing luciferase and either Her-4 shRNA or scrambled control. With this model, a “primary” tumor arises in the tibia, with spontaneous metastasis to the lungs. Non-invasive imaging showed reduced growth of primary tumors in knock-down mice and complete elimination of tumor signal from lungs. Xenograft lungs showed a significant reduction in metastasis in knock-down mice. IHC for Her-4 showed that, while tibial tumors of knock-down mice still expressed no Her-4, the tumors present in these mice had re-expressed Her-4 despite the shRNA. Stress resistance, chemotherapy resistance and metastatic potential are TIC-associated behaviors, so we examined the association of Her-4 with other described TIC markers for OS. Sarcosphere culture induced expression of both Her-4 and stemness markers CD117 and Stro1 over a period of 2-7 days. Her-4 expression preceded upregulation of other markers by 1-2 days. Conclusions: Expression of Her-4 confers a survival benefit to OS cells under stressed conditions, including anoikis, serum starvation and methotrexate exposure. Her-4 is necessary for OS metastasis. Her-4 expression increases in sarcophere cultures and may be a mediator of stemness. Therapies targeting Her-4 in OS should be evaluated in further preclinical models. Citation Format: Rocio K. Rivera-Valentin, Yingqi Hua, Yi Zhang, Nupur Lala, Yanwen Yang, Dennis P.M. Hughes. Her-4 mediates anoikis resistance, chemoresistance, and metastatic potential in osteosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A71.