Essential erbB family phosphorylation in osteosarcoma as a target for CI-1033 inhibition.
BACKGROUND
The role of erbB tyrosine kinases, especially Her-2, in osteosarcoma has engendered intense debate. Some investigators identified an association between low-level Her-2 expression, compared to none, and poor patient outcome. Others questioned the importance of apparent cytoplasmic expression of Her-2, since membranous overexpression is associated with poor outcome in carcinomas. We previously demonstrated that primary osteosarcoma cells express cell-surface EGFR and Her-2, with the p80 isoform of Her-4 localized to the nucleus. We wished to determine if erbB kinases in osteosarcoma were phosphorylated, and if this was required for growth.


METHODS
We cultured early passage osteosarcoma cell lines in the presence or absence of the pan-erbB inhibitor CI-1033 and examined the phosphorylation status of EGFR, Her-2, and Her-4 by immunohistochemistry, cell-based ELISA, flow cytometry and two dimensional Western blot. We also assessed the impact of CI-1033 upon osteosarcoma growth and survival in vitro.


RESULTS
EGFR, Her-2, and Her-4 were constitutively phosphorylated in early passage osteosarcoma cells cultured in vitro. CI-1033 abrogated erbB receptor phosphorylation and caused growth inhibition and apoptosis in a titratible fashion with concentrations of 1 muM or more.


CONCLUSIONS
EGFR, Her-2, and Her-4 are constitutively phosphorylated in early passage osteosarcoma cells in tissue culture, and erbB signaling provides essential growth and anti-apoptotic signals to osteosarcoma cells. This suggests that erbB overexpression is not required for erbB to promote malignancy, but rather that overexpression is one of several mechanisms that generate unregulated erbB signaling.
Hughes
Dennis P M
DP
Division of Pediatrics, MD Anderson Cancer Center, Houston, Texas 77030, USA. dphughes@mdanderson.org
Thomas
Dafydd G
DG
Giordano
Thomas J
TJ
McDonagh
Kevin T
KT
Baker
Laurence H
LH
D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D001859:Bone Neoplasms / Q000188:drug therapy / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007124:Immunoenzyme Techniques; D009025:Morpholines / Q000494:pharmacology*; D012516:Osteosarcoma / Q000188:drug therapy* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D066247:Receptor, ErbB-4; D014407:Tumor Cells, Cultured
D066246:ErbB Receptors; D009025:Morpholines; D018719:Erb-b2 Receptor Tyrosine Kinases; D066247:Receptor, ErbB-4; C420268:Canertinib; C587689:ERBB4 protein, human