RUNX3 inactivation by frequent promoter hypermethylation and protein mislocalization constitute an early event in breast cancer progression.
BACKGROUND
We had previously established that inactivation of RUNX3 occurs by frequent promoter hypermethylation and protein mislocalization in invasive ductal carcinomas (IDC) of breast. Here, we hypothesize that inactivation of RUNX3 occurring in ductal carcinoma in situ (DCIS) represent early event in breast carcinogenesis.


METHODS
The study cohort of 40 patients included 17 pure DCIS cases and 23 cases of DCIS with associated IDC (DCIS-IDC). The DCIS and IDC components of mixed cases were manually microdissected to permit separate evaluation. All the 63 samples including 17 pure DCIS, 23 samples each of DCIS and IDC of DCIS-IDC cases were analyzed for RUNX3 protein expression using R3-6E9 monoclonal antibody as well as promoter methylation status by methylation specific PCR.


RESULTS
Compared to matched normal breast samples (4 of 40, 10%), DCIS (35 of 40, 88%) and IDC (21 of 23, 91%) exhibited significant RUNX3 inactivation (P<0.001) in the form of negative or weak nuclear staining. In contrast to normal breast tissues (1/10, 10%), promoter hypermethylation of RUNX3 was significantly higher in the neoplastic breast samples (46 of total 61, 75%) including 30 of 40 (75%) DCIS and 16 of 21 (76%) IDC samples (P=0.009). Overall, promoter hypermethylation correlated with RUNX3 inactivation in 42 of 46 (91%) methylated samples (P=0.03). Mislocalized cytoplasmic expression also accounted for RUNX3 inactivation in majority of DCIS (33/40, 83%) and IDC (20/23, 87%) samples independent of promoter hypermethylation.


CONCLUSIONS
Our data suggest that RUNX3 inactivation by promoter hypermethylation and protein mislocalization constitute an early event in breast cancer progression.
Subramaniam
Manish Mani
MM
Oncology Research Institute, National University of Singapore, Singapore, Republic of Singapore.
Chan
Jason Yongsheng
JY
Soong
Richie
R
Ito
Kosei
K
Ito
Yoshiaki
Y
Yeoh
Khay Guan
KG
Salto-Tellez
Manuel
M
Putti
Thomas Choudary
TC
D001943:Breast Neoplasms / Q000235:genetics* / Q000401:mortality / Q000473:pathology* / Q000601:surgery; D002278:Carcinoma in Situ / Q000235:genetics / Q000473:pathology; D018270:Carcinoma, Ductal, Breast / Q000235:genetics / Q000473:pathology; D050677:Core Binding Factor Alpha 3 Subunit / Q000037:antagonists & inhibitors* / Q000235:genetics*; D019175:DNA Methylation; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000235:genetics; D018450:Disease Progression; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D008408:Mastectomy; D042282:Microdissection; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic
D050677:Core Binding Factor Alpha 3 Subunit; D017931:DNA Primers; D004273:DNA, Neoplasm; C493729:Runx3 protein, human