p14ARF expression in invasive breast cancers and ductal carcinoma in situ--relationships to p53 and Hdm2.
BACKGROUND
p14ARF stabilises nuclear p53, with a variable expression of p14ARF mRNA in breast cancers. In vitro, nuclear p14ARF binds Hdm2 to block Hdm2-dependent nucleocytoplasmic shuttling of p53, which is required before cytoplasmic degradation of p53. p14ARF is negatively regulated by p53 and through p53-independent pathways. No studies have yet examined levels of p14ARF protein expression in breast cancer and their relationship to Hdm2/p53 immunoreactivity or subcellular localisation. Previously, immunohistochemical expression of cytoplasmic p14ARF, p53 and Hdm2 has been described. HER-2 (c-erbB2/neu) predicts prognosis and interacts with the p14ARF/Hdm2 pathway to inactivate p14ARF and to influence Hdm2 activity and localisation. This study examined p14ARF and p53/Hdm2 expression and subcellular localisation by using immunohistochemistry in a series of invasive ductal breast cancers (IDCs) with concomitant ductal carcinoma in situ (DCIS), to evaluate whether findings in vitro were related to clinicopathological parameters such as HER-2 and their effect on patient outcome.


METHODS
The 4C6 anti-p14ARF monoclonal antibody and Dako Envision Plus system were used to evaluate p14ARF expression in 103 patients; p53/Hdm2 staining was performed.


RESULTS
p14ARF was evaluable in 96 patients, with nuclear p14ARF expression (modified Quick-score > or = 3) in 79% (n = 76) of IDCs and in associated DCIS in 74 patients. Cytoplasmic p14ARF was detectable in 23 breast cancers. Nuclear and cytoplasmic p14ARF showed no correlation with p53 subcellular immunoreactivity. Increasing levels of cytoplasmic p14ARF were associated with nuclear and cytoplasmic Hdm2 expression (P < 0.001). Subcellular ARF expression was not associated with clinicopathological parameters, and although not an independent prognosticator, these preliminary findings suggest that cytoplasmic p14ARF might be associated with a better overall survival (P = 0.09; log rank). The association between HER-2 positivity and nuclear p14ARF (P = 0.038), as well as nuclear Hdm2 (P = 0.019), reflects the in vitro findings of HER-2 interaction with the ARF/Hdm2 pathway. Cytoplasmic p53 and Hdm2 expression might have biological implications, through an association of cytoplasmic p53 with increased tumour proliferation (P = 0.005), and an improved overall survival (P = 0.002, log rank) in cytoplasmic Hdm2-expressing tumours, that independently predict favourable overall survival (P = 0.02) and disease-free survival (P = 0.03).


CONCLUSIONS
Nuclear p14ARF expression is similar in IDCs and DCIS and is associated with Hdm2 immunoreactivity. Nuclear p14ARF and Hdm2 might be regulated by HER-2. Clearly, our findings in vivo suggest a complexity of p14ARF/Hdm2 and p53 pathways in which consideration of cytoplasmic p14ARF and Hdm2 might have tumorigenic implications.
Vestey
S B
SB
University of Bristol Division of Surgery, Bristol Royal Infirmary, Bristol, UK. vesteyhaynes@hotmail.com
Sen
C
C
Calder
C J
CJ
Perks
C M
CM
Pignatelli
M
M
Winters
Z E
ZE
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001943:Breast Neoplasms / Q000378:metabolism* / Q000401:mortality / Q000473:pathology; D018270:Carcinoma, Ductal, Breast / Q000378:metabolism* / Q000401:mortality / Q000473:pathology; D002285:Carcinoma, Intraductal, Noninfiltrating / Q000378:metabolism* / Q000401:mortality / Q000473:pathology; D006801:Humans; D007150:Immunohistochemistry; D008875:Middle Aged; D009687:Nuclear Proteins / Q000378:metabolism; D011379:Prognosis; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D016019:Survival Analysis; D025803:Tumor Suppressor Protein p14ARF / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D051736:Proto-Oncogene Proteins c-mdm2; D018719:Erb-b2 Receptor Tyrosine Kinases; D025803:Tumor Suppressor Protein p14ARF; D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human