We measured the mitochondrial oxidative phosphorylation (mtOXPHOS) activities of all five complexes and determined the activity and gene expression in detail of the Complex III subunits in human breast cancer cell lines and primary tumors. Our analysis revealed dramatic differences in activity of complex III between normal and aggressive metastatic breast cancer cell lines. Determination of Complex III subunit gene expression identified over expression and co-regulation of UQCRFS1 (encoding RISP protein) and UQCRH (encoding Hinge protein) in 6 out of 9 human breast tumors. Analyses of UQCRFS1/RISP expression in additional matched normal and breast tumors demonstrated an over expression in 14 out of 40 (35%) breast tumors. UQCRFS1/RISP knockdown in breast tumor cell line led to decreased mitochondrial membrane potential as well as a decrease in matrigel invasion. Furthermore, reduced matrigel invasion was mediated by reduced ROS levels coinciding with decreased expression of NADPH oxidase 2, 3, 4 and 5 involved in ROS production. These studies provide direct evidence for contribution of impaired mtOXPHOS Complex III to breast tumorigenesis.
Background: We recently described a number of DNA copy number changes that differentiated low grade ductal carcinoma in situ (DCIS) lesions with and without associated invasive carcinoma. One of the invasion-associated candidate genes was Nuclear Corepressor 2/Silencing Mediator of Retinoid and Thyroid Receptors (NCOR2/SMRT). This gene is a component of a repressing nuclear receptor complex and a mediator of ER activity. It facilitates recovery from DNA double strand breaks. Previous studies showed that downregulation of the gene was associated with transformation of non-Hodgkin lymphomas.Materials and Methods: Genomic DNA was extracted from microdissected paraffin sections of formalin fixed low grade DCIS lesions with an invasive component (n=42), as well as morphologically similar DCIS lesions without associated invasion within at least 5 years (n=38). Comparative PCR was performed with GAPDH as the reference gene. In addition, we assayed tissue microarrays (TMAs) by immunohistochemistry using two anti-Smrt antibodies. Three TMAs included 162 interpretable cases of invasive breast carcinoma from Roswell Park Cancer Institute (RPCI). One additional TMA included 73 interpretable breast cancers from a previously described cohort from Vienna, Austria. Nuclear staining in neoplastic cells was scored as positive, reduced (significantly weaker compared to admixed benign cells), and negative. Ncor2/Smrt expression was correlated with various patho-biologic variables.Results: Allelic loss of NCOR2/SMRT was observed in 79% (33/42) of DCIS lesions with associated invasion, but in only 26% (10/38) of pure DCIS cases (p<0.01). In the RPCI breast cancer cohort, nuclear Ncor2/Smrt protein was absent or downregulated in 73% (119/162) of tumors. In the Vienna cohort, there was no or markedly reduced nuclear staining in 48% (35/73) of cases. In both cohorts, loss of nuclear reactivity was more common in ER-negative breast cancers. In the Vienna series, Ncor2/Smrt loss was more frequent in PR-negative tumors as well. In the RPCI cohort, Ncor2/Smrt downregulation was more common in ductal (81%, 109/135) than in lobular (37%, 10/27) carcinomas. Neither series showed a significant association between Ncor2/Smrt expression and tumor grade or HER2/neu status.Discussion: Allelic loss of NCOR2/SMRT was significantly more common in DCIS lesions associated with stromal invasion. In invasive tumors, the protein downregulation rate was between 48% and 73%. The gene was more commonly downregulated in ER-negative carcinomas, consistent with its role as a modulator of ER function. Our data provide new evidence that NCOR2/SMRT may play an important part in early breast cancer progression. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3150.
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