Heregulin beta1 drives gefitinib-resistant growth and invasion in tamoxifen-resistant MCF-7 breast cancer cells.
BACKGROUND
Resistance to anti-epidermal growth factor receptor (anti-EGFR) therapies is an emerging clinical problem. The efficacy of anti-EGFR therapies can be influenced by the presence of heregulins (HRGs), which can bind erbB3/4 receptors and can activate alternative signalling pathways. In the present study we have examined whether HRG signalling can circumvent EGFR blockade in an EGFR-positive tamoxifen-resistant MCF-7 (Tam-R) breast cancer cell line.


METHODS
Tam-R cells, incubated with the selective EGFR tyrosine kinase inhibitor gefitinib ('Iressa', ZD1839), were exposed to HRGbeta1 and the effects on erbB receptor dimerization profiles and on activation of associated downstream signalling components were assessed by immunoprecipitation, western blotting and immunocytochemistry. The effects of HRGbeta1 on gefitinib-treated Tam-R cell growth and invasion were also examined, and HRGbeta1 expression levels were assessed in breast cancer tissue by immunohistochemistry to address the potential clinical relevance of such a resistance mechanism.


RESULTS
In Tam-R cells, HRGbeta1 promoted erbB3/erbB2 and erbB3/EGFR heterodimerization, promoted ERK1/2 and AKT pathway activation and increased cell proliferation and invasion. Gefitinib prevented HRGbeta1-driven erbB3/EGFR heterodimerization, ERK1/2 activation and Tam-R cell proliferation, but HRGbeta1-driven erbB3/erbB2 heterodimerization, AKT activation and Tam-R cell invasion were maintained. A combination of gefitinib and the phosphatidylinositol 3-kinase inhibitor LY294002 effectively blocked HRGbeta1-mediated intracellular signalling activity, growth and invasion in Tam-R cells. Similarly, targeting erbB2 with trastuzumab in combination with gefitinib in Tam-R cells reduced HRGbeta1-induced erbB2 and ERK1/2 activity; however, HRGbeta1-driven AKT activity and cell growth were maintained while cell invasion was significantly enhanced with this combination. In clinical tissue all samples demonstrated cytoplasmic tumour epithelial HRGbeta1 protein staining, with expression correlating with EGFR positivity and activation of both AKT and ERK1/2.


CONCLUSIONS
HRGbeta1 can overcome the inhibitory effects of gefitinib on cell growth and invasion in Tam-R cells through promotion of erbB3/erbB2 heterodimerization and activation of the phosphatidylinositol 3-kinase/AKT signalling pathway. This may have implications for the effectiveness of anti-EGFR therapies in breast cancer as HRGbeta1 is enriched in many EGFR-positive breast tumours.
Tenovus Centre for Cancer Research, Welsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff CF10 3XF, UK. hutchesonir@cf.ac.uk
Iain R
IR
Hutcheson
Janice M
JM
Knowlden
Steve E
SE
Hiscox
Denise
D
Barrow
Julia M W
JM
Gee
John F
JF
Robertson
Ian O
IO
Ellis
Robert I
RI
Nicholson
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D061067:Antibodies, Monoclonal, Humanized; D018931:Antineoplastic Agents, Hormonal / Q000494:pharmacology*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor / Q000187:drug effects / Q000378:metabolism; D049109:Cell Proliferation; D019281:Dimerization; D019008:Drug Resistance, Neoplasm; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism; D005260:Female; D000077156:Gefitinib; D006801:Humans; D047468:Immunoprecipitation; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009361:Neoplasm Invasiveness; D020890:Neuregulin-1 / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011799:Quinazolines / Q000494:pharmacology*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D020893:Receptor, ErbB-3 / Q000378:metabolism; D015398:Signal Transduction; D013629:Tamoxifen / Q000494:pharmacology*; D000068878:Trastuzumab
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D018931:Antineoplastic Agents, Hormonal; D066246:ErbB Receptors; D000077156:Gefitinib; D020928:Mitogen-Activated Protein Kinases; D020890:Neuregulin-1; D019869:Phosphatidylinositol 3-Kinases; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D011799:Quinazolines; D018719:Erb-b2 Receptor Tyrosine Kinases; D020893:Receptor, ErbB-3; D013629:Tamoxifen; D000068878:Trastuzumab; C087230:heregulin beta1