Deregulation of cofactor of BRCA1 expression in breast cancer cells

Abstract

Cofactor of BRCA1 (COBRA1) is an integral component of the human negative elongation factor (NELF), a four-subunit protein complex that inhibits transcription elongation. Previous in vivo work indicates that COBRA1 and the rest of the NELF complex repress estrogen-dependent transcription and the growth of breast cancer cells. In light of the COBRA1 function in breast cancer-related gene expression, we sought to examine regulation of COBRA1 expression in both established breast cancer cell lines and breast carcinoma tissues. We found that COBRA1 expression was inversely correlated with breast cancer progression, as tumor samples of patients who had distant metastasis and local recurrence expressed very low levels of COBRA1 mRNA when compared to those who were disease free for over 10 years (P = 0.0065 and 0.0081, respectively). Using both breast and prostate cancer cell lines, we also explored the possible mechanisms by which COBRA1 expression is regulated. Our results indicate that the protein abundance of COBRA1 and the other NELF subunits are mutually influenced in a tightly coordinated fashion. Small interfering RNA (siRNA) that targeted at one NELF subunit dampened the protein levels of all four subunits. Conversely, ectopic expression of COBRA1 in the knockdown cells partially rescues the co-depletion of the NELF subunits. In addition, our study suggests that a post-transcriptional, proteasome-independent mechanism is involved in the interdependent regulation of the NELF abundance. Furthermore, a lack of COBRA1 expression in breast carcinoma may serve as a useful indicator for poor prognosis.

Fig. 1.

NELF expression in established cell lines and clinical tissues. A: Western blot analysis of NELF expression in multiple cell lines. The blots were probed with anti-NELF-A, anti-COBRA1 (NELF-B), anti-NELF-C, and anti-NELF-E antibodies, respectively. Ran was used as a loading control. B: Normal tissue array was immunostained with a COBRA1-specific polyclonal antibody. Several representative images of selective tissues are shown on the right. The images were taken at ×100 magnification. Abbreviations are as following: GFEN, endocervix; GFES, endometrium, secretory; GIGA, gastric mucosa, antral; GISI, small intestine, mucosa; GFFT, fallopian tube; GIC, colon, mucosa; BE, breast epithelium; and HPG, gallbladder. C: Representative staining pattern of COBRA in normal mammary (panels A,B) and in tumor tissue (panels C,D). Mammary epithelial cells showed a strong nuclear pattern of staining of COBRA, whereas breast cancer cells showed a markedly reduced staining compared with normal epithelial cells. The panels are shown with ×100 magnification, and the insets with ×400 magnification. The arrows indicate the areas where the magnified insets come from.

Fig. 2.

Quantitative RT-PCR analysis of COBRA1 mRNA expression in human ductal carcinoma tissues. A: Relationship between COBRA1 expression and clinical outcome over a 10-year follow-up period. There is a strong correlation between low COBRA1 levels and patients with metastasis or local recurrence (*P = 0.0065 and 0.0081, respectively). B: Comparison between those patients who remained disease free and those who developed further disease progression (metastasis, recurrence, and motality combined). C: Kaplan–Meier survival curve for a disease free survival.

Fig. 3.

Protein levels of NELF subunits in control and NELF knockdown cells. T47D (A) and LNCaP (B) cells were transfected with siRNA oligos for the control or the individual NELF subunits. Whole cell extracts were prepared 72 h after transfection and an equal amount of the extract was resolved by 10% SDS–PAGE. Protein levels of each NELF subunits were determined by Western blot analysis with the corresponding antibodies. α-tubulin was used as the loading control.

Fig. 4.

Further characterization of the interdependency of the NELF subunits. A: Time course study of the NELF subunits following COBRA1 siRNA knockdown. T47D cells were transfected with the control or COBRA1 siRNA duplex and harvested for whole cell extract preparation at 24, 48, 72, and 96 h after transfection. Protein levels of the individual NELF subunits were determined as described in Figure 3. B: Partial rescue of the NELF co-depletion with ectopic expression of COBRA1. T47D cells were first transfected with siRNA duplex for 48 h. Transfected cells were then infected with the recombinant adenovirus for the control protein LacZ or COBRA1. Whole cell extract was analyzed for the expression of the NELF subunits.

Fig. 5.

Examination of the mRNA levels of the NELF subunits in control and NELF knockdown cells. T47D cells were transfected with control or siRNA specific for the individual NELF subunits. Total RNA was extracted and analyzed for mRNA expression of each subunit by quantitative RT-PCR. Relative expression level was determined by normalization against the levels of GAPDH mRNA.

Fig. 6.

Mechanistic study of the interdependency of the NELF subunits. A: Evaluation of the protein half-lives of NELF-A, -C, and -E in the control and COBRA1 knockdown T47D cells. T47D cells were first transfected with control or COBRA1 siRNA. Seventy-two hours post-transfection, cells were treated with cycloheximide (100 μg/ml) for 0, 2, 4, 6, 8, and 12 h. Whole cell extract was prepared and analyzed for the levels of the individual NELF subunits by Western blot analysis. Protein bands were quantified with ImageQuant V5.2 and relative intensity was used for plotting the graphs. B: Effect of proteasome and lysosome inhibitors on the stability of the NELF subunits. T47D cells were first transfected with control or COBRA1 siRNA, and then treated with proteasome inhibitor MG132 (5 μM) or lysosome inhibitor ALLN (200 μM) for 8 h. Whole cell extract was prepared and protein level of each NELF subunits was determined by Western blot analysis. c-Jun was used as a positive control for the drug effects. α-tubulin was used as a loading control.