| Abstract |
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.
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