Celecoxib analogues disrupt Akt signaling, which is commonly activated in primary breast tumours.
BACKGROUND
Phosphorylated Akt (P-Akt) is an attractive molecular target because it contributes to the development of breast cancer and confers resistance to conventional therapies. Akt also serves as a signalling intermediate for receptors such as human epidermal growth factor receptor (HER)-2, which is overexpressed in 30% of breast cancers; therefore, inhibitors to this pathway are being sought. New celecoxib analogues reportedly inhibit P-Akt in prostate cancer cells. We therefore examined the potential of these compounds in the treatment of breast cancer. The analogues were characterized in MDA-MB-453 cells because they overexpress HER-2 and have very high levels of P-Akt.


METHODS
To evaluate the effect of the celecoxib analogues, immunoblotting was used to identify changes in the phosphorylation of Akt and its downstream substrates glycogen synthase kinase (GSK) and 4E binding protein (4EBP-1). In vitro kinase assays were then used to assess the effect of the drugs on Akt activity. Cell death was evaluated by poly(ADP-ribose) polymerase cleavage, nucleosomal fragmentation and MTS assays. Finally, tumour tissue microarrays were screened for P-Akt and HER-2 expression.


RESULTS
OSU-03012 and OSU-O3013 inhibited P-Akt and its downstream signalling through 4EBP-1 and GSK at concentrations well below that of celecoxib. Disruption of P-Akt was followed by induction of apoptosis and more than 90% cell death. We also noted that the cytotoxicity of the celecoxib analogues was not significantly affected by serum. In contrast, the presence of 5% serum protected cells from celecoxib induced death. Thus, the structural modification of the celecoxib analogues increased P-Akt inhibition and enhanced the bioavailability of the drugs in vitro. To assess how many patients may potentially benefit from such drugs we screened tumour tissue microarrays. P-Akt was highly activated in 58% (225/390) of cases, whereas it was only similarly expressed in 35% (9/26) of normal breast tissues. Furthermore, HER-2 positive tumours expressed high levels of P-Akt (P < 0.01), supporting in vitro signal transduction.


CONCLUSIONS
We determined that Celecoxib analogues are potent inhibitors of P-Akt signalling and kill breast cancer cells that overexpress HER-2. We also defined an association between HER-2 and P-Akt in primary breast tissues, suggesting that these inhibitors may benefit patients in need of new treatment options.
Kucab
Jill E
JE
British Columbia Research Institute for Children's and Women's Health, Department of Pediatrics, University of British Columbia, Vancouver, British Columbia, Canada. kucab@interchange.ubc.ca
Lee
Cathy
C
Chen
Ching-Shih
CS
Zhu
Jiuxiang
J
Gilks
C Blake
CB
Cheang
Maggie
M
Huntsman
David
D
Yorida
Erika
E
Emerman
Joanne
J
Pollak
Michael
M
Dunn
Sandra E
SE
D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000473:pathology / Q000503:physiopathology*; D000068579:Celecoxib; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002867:Chromones / Q000494:pharmacology*; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D009025:Morpholines / Q000494:pharmacology*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism / Q000502:physiology*; D011720:Pyrazoles / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013449:Sulfonamides / Q000494:pharmacology*
D000894:Anti-Inflammatory Agents, Non-Steroidal; D002867:Chromones; D016861:Cyclooxygenase Inhibitors; D004791:Enzyme Inhibitors; D009025:Morpholines; D011720:Pyrazoles; D013449:Sulfonamides; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D051057:Proto-Oncogene Proteins c-akt; D000068579:Celecoxib