Date of Graduation
Biochemistry and Molecular Biology
Masters of Science (MS)
Joya Chandra, Ph.D
Gary Gallick, Ph.D
Andrew Gladden, Ph.D
Winston Huh, MD
Patrick Zweidler-McKay, MD, Ph.D
ERBB4 (Her-4) is a member of the ERBB family of Class I receptor tyrosine kinases. ERBB4 is unique within the ERBB family for alternate splicing in its juxtamembrane and cytoplasmic regions, which produces four juxtamembrane isoforms and two cytoplasmic isoforms. The cleavable juxtamembrane isoforms, Jm-a and Jm-d, can undergo proteolytic cleavage and produce an 80-kDa cytoplasmic fragment referred to as “p80,” which has been demonstrated to enhance cellular survival and malignant behavior in many solid tumors . ERBB4 was almost exclusively 80 kDa in size and localized to the nucleus in primary tumors and metastases in studies characterizing ERBB4 expression in osteosarcoma, indicating that ERBB4 may be cleaved to enhance malignant behavior in osteosarcoma. ERBB4 protein was significantly up-regulated from monolayer to sphere cultures in osteosarcoma, leading to the hypothesis that there would be an up-regulation of the cleavable isoforms Jm-a and Jm-d from monolayer to sphere cultures of osteosarcoma.
The first aim of this thesis was to measure if the proportion of cleavable juxtamembrane isoform expression in osteosarcoma tumor spheres from monolayer culture. All cell lines on the panel showed a significant increase in expression, by proportion or up-regulation, of at least one of the cleavable juxtamembrane isoforms Jm-a or Jm-d in sphere culture. We hypothesized that there would be a significant reduction of lung metastases with shRNA specific for Her-4 in osteosarcoma cells in xenograft mice. Immunohistochemical staining for ERBB4 was performed on sections of lungs from the experimental mice injected with CCH-OS-O cells with shRNA against ERBB4 and controls, with adjacent sections stained with vimentin as a counterstain for total osteosarcoma metastases. There were significant reductions in formation of ERBB4-negative macrometastases (> 200 microns) in ERBB4 control mice and of ERBB4-negative micrometastases (6 cells-200 microns) in ERBB4 control and ERBB4 knockdown mice over two experiments. There was not a significant difference in the numbers of oligometastases (1-5 cells) in ERBB4 knockdown and control mice. Taken together, the immunohistochemistry suggests that ERBB4 expression may be important to metastatic progression, or formation of detectable foci, in osteosarcoma.
Osteosarcoma, ERBB signaling, metastasis, ERBB4, Her4, cancer biology, solid tumors