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RUNX3 inactivation by frequent promoter hypermethylation and protein mislocalization constitute an early event in breast cancer progression

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Abstract

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. Conclusion Our data suggest that RUNX3 inactivation by promoter hypermethylation and protein mislocalization constitute an early event in breast cancer progression.

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Abbreviations

DCIS:

Ductal carcinoma in situ

IDC:

Invasive ductal carcinoma

DCIS-IDC:

DCIS associated with IDC

IHC:

Immunohistochemistry

MSP:

Methylation specific PCR

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Acknowledgements

We thank Ms. Cheong Sok Lian, the research nurse and Mr. Ramakrishna, the operation associate for collection of clinico-pathological data and retrieval of the cases from the pathology files of the department of pathology, National University of Singapore.

Funding Information

T.C. Putti is supported by a grant from the National Healthcare Group SIG06/027.

M Salto-Tellez receives funding support from SCS Grants MN-05 & MN-77, awarded by the Singapore Cancer Syndicate, Agency for Science, Technology and Research, Singapore.

K.G. Yeoh is funded by the grant for identification of novel biomarkers for screening of gastric cancer (R-364-000-049-305).

Competing Interests

None.

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Authors and Affiliations

  1. Oncology Research Institute (ORI), National University of Singapore (NUS), Singapore, Republic of Singapore

    Manish Mani Subramaniam, Jason Yongsheng Chan, Richie Soong, Kosei Ito, Yoshiaki Ito & Manuel Salto-Tellez

  2. Department of Pathology, National University of Singapore (NUS), 5 Lower Kent Ridge Road, Singapore, 119074, Republic of Singapore

    Richie Soong, Manuel Salto-Tellez & Thomas Choudary Putti

  3. Institute of Molecular and Cell Biology, Proteos, Singapore

    Kosei Ito & Yoshiaki Ito

  4. Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Republic of Singapore

    Khay Guan Yeoh

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  1. Manish Mani Subramaniam
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Correspondence to Thomas Choudary Putti.

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Subramaniam, M.M., Chan, J.Y., Soong, R. et al. RUNX3 inactivation by frequent promoter hypermethylation and protein mislocalization constitute an early event in breast cancer progression. Breast Cancer Res Treat 113, 113–121 (2009). https://doi.org/10.1007/s10549-008-9917-4

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