BRCA1 Plays a Role in the Hypoxic Response by Regulating HIF-1α Stability and by Modulating Vascular Endothelial Growth Factor Expression

Abstract

A recent study of breast cancer patients with and without BRCA1 gene mutations found significantly lower levels of VEGF in serum from patients with BRCA1 mutations (Tarnowski, B., Chudecka-Glaz, A., Gorski, B., and Rzepka-Gorska, I. (2004) Breast Cancer Res. Treat. 88, 287–288). Here, we describe a possible mechanistic explanation for this correlation. Because hypoxia in tumors stimulates VEGF expression and secretion we hypothesized that altered BRCA1 protein levels in breast tumors could affect hypoxia-stimulated VEGF promoter activity. This possibility was tested in cells transfected with various combinations of expression plasmids for BRCA1, BRCA1 specific inhibitory RNAs (BRCA1-siRNAs), HIF-1α, and a VEGF promoter-reporter and then incubated in normoxia (21%, O2) or hypoxia (0.1%, O2). As predicted, increased BRCA1 levels enhanced both hypoxia-stimulated VEGF promoter activity and the amounts of VEGF mRNA, as determined by semiquantitative RT-PCR and quantitative real time PCR. Using the ChIP assay, we discovered that BRCA1 could be recruited to the endogenous human VEGF promoter along with HIF-1α following hypoxia. An interaction between BRCA1 and HIF-1α was found in human breast cancer cells. We also found that hypoxia-stimulated VEGF promoter activity and secretion was reduced in cells containing reduced amounts of endogenous BRCA1 protein (obtained by transfecting with BRCA1 siRNAs). A mechanistic explanation for these effects is provided by our finding a reduced half-life and reduced accumulation of HIF-1α in hypoxic cells transfected with BRCA1-siRNAs and that proteasome inhibitors blocked these effects of BRCA1-siRNAs. Thus, our results suggest that normal amounts of BRCA1 function in hypoxia to regulate HIF-1α stability, probably by interacting with HIF-1α. A recent study of breast cancer patients with and without BRCA1 gene mutations found significantly lower levels of VEGF in serum from patients with BRCA1 mutations (Tarnowski, B., Chudecka-Glaz, A., Gorski, B., and Rzepka-Gorska, I. (2004) Breast Cancer Res. Treat. 88, 287–288). Here, we describe a possible mechanistic explanation for this correlation. Because hypoxia in tumors stimulates VEGF expression and secretion we hypothesized that altered BRCA1 protein levels in breast tumors could affect hypoxia-stimulated VEGF promoter activity. This possibility was tested in cells transfected with various combinations of expression plasmids for BRCA1, BRCA1 specific inhibitory RNAs (BRCA1-siRNAs), HIF-1α, and a VEGF promoter-reporter and then incubated in normoxia (21%, O2) or hypoxia (0.1%, O2). As predicted, increased BRCA1 levels enhanced both hypoxia-stimulated VEGF promoter activity and the amounts of VEGF mRNA, as determined by semiquantitative RT-PCR and quantitative real time PCR. Using the ChIP assay, we discovered that BRCA1 could be recruited to the endogenous human VEGF promoter along with HIF-1α following hypoxia. An interaction between BRCA1 and HIF-1α was found in human breast cancer cells. We also found that hypoxia-stimulated VEGF promoter activity and secretion was reduced in cells containing reduced amounts of endogenous BRCA1 protein (obtained by transfecting with BRCA1 siRNAs). A mechanistic explanation for these effects is provided by our finding a reduced half-life and reduced accumulation of HIF-1α in hypoxic cells transfected with BRCA1-siRNAs and that proteasome inhibitors blocked these effects of BRCA1-siRNAs. Thus, our results suggest that normal amounts of BRCA1 function in hypoxia to regulate HIF-1α stability, probably by interacting with HIF-1α. The gene encoding the breast cancer susceptibility gene 1 (BRCA1), 2The abbreviations used are: BRCA1, breast cancer susceptibility gene-1; ARNT, aryl hydrocarbon receptor nuclear translocator; ChIP, chromatin immunoprecipitation; qRT, quantitative reverse transcriptase; HBC, hereditary breast cancer; HIF-1α, hypoxia-inducible transcription factor 1α; IP, immunoprecipitation; VEGF, vascular endothelial growth factor; VHL, von Hippel-Lindau gene; WB, Western blotting assays; CHX, cycloheximide; GFP, green fluorescent protein; HRE, hypoxia responsive element; Luc, luciferase; wt, wild type. 2The abbreviations used are: BRCA1, breast cancer susceptibility gene-1; ARNT, aryl hydrocarbon receptor nuclear translocator; ChIP, chromatin immunoprecipitation; qRT, quantitative reverse transcriptase; HBC, hereditary breast cancer; HIF-1α, hypoxia-inducible transcription factor 1α; IP, immunoprecipitation; VEGF, vascular endothelial growth factor; VHL, von Hippel-Lindau gene; WB, Western blotting assays; CHX, cycloheximide; GFP, green fluorescent protein; HRE, hypoxia responsive element; Luc, luciferase; wt, wild type. which is frequently mutated in the germline of women with a genetic predisposition to breast and ovarian cancers, was cloned in 1994 (1Miki Y. 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Full Text Full Text PDF PubMed Scopus Google VEGF and VEGF reverse As we used of for regions of the VEGF promoter that not have known as for and reverse and for and reverse VEGF transfected with or as I. Fan S. Meng Q. J. Goldberg I.D. Rosen E.M. Cancer Res. PubMed Scopus Google Scholar). The the was to cells to hypoxic (0.1%, O2) for an when the for a VEGF secretion assay, which was a human VEGF The to the protein in the cell from cells a The in a hypoxia with a or with containing and K. H. K. T. K. Res. PubMed Scopus Google Scholar). BRCA1 of BRCA1 are shown in The which is found in a with a significantly increased for BRCA1 breast A. J. Genet. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, D. L. I. N. G. M. J. N. T. J. Genet. 1997; Google Scholar, S. M. M. Brody L.C. M.A. N. J. 1997; PubMed Scopus Google a protein of the domain The a BRCA1 protein containing a in the that activity (5Monteiro A.N. August A. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13595-13599Crossref PubMed Scopus (427) Google Scholar). The a BRCA1 protein that is the This activity. The a protein the domain function S. Wang J.A. Ma Y.X. Yuan R.Q. Meng Q. Erdos M.R. Pestell R.G. Goldberg I.D. Rosen E.M. Oncogene. 2001; 20: 77-87Crossref PubMed Scopus (224) Google Scholar). of or or of determined from of the The used in the and and of BRCA1 HIF-1α this a of was used to hypoxia. This hypoxic is for some breast cancer have to O2) K. C. M. Cancer Res. Google Scholar, Rosen Int. J. 2003; 19: PubMed Scopus Google Scholar, G.M. Google Scholar). BRCA1 VEGF we used a VEGF promoter-reporter with HIF-1α and activity was of hypoxia. activity our hypoxic was in of cells in of cells the HIF-1α expression of cells with BRCA1 and the HIF-1α expression enhanced activity from the VEGF promoter The of this the of BRCA1 DNA Thus, a activity was when HIF-1α was and this was significantly enhanced when BRCA1 was also this of BRCA1 to VEGF promoter activity the of a functional in the VEGF cells with BRCA1, HIF-1α, and a VEGF containing a or an containing a and then incubated hypoxic and for activity. The results that BRCA1 to activity from a VEGF promoter hypoxic when this promoter a its to HIF-1α This finding suggests that BRCA1 may VEGF promoter activity by directly whether the VEGF activity in increased we the of BRCA1 endogenous VEGF this cells transfected with BRCA1 or and to hypoxic for when RNA was and used for semiquantitative RT-PCR A and and quantitative real time As we found that BRCA1 increased the amounts of the endogenous of VEGF and VEGF in cells to hypoxic for of cells transfected with BRCA1 of BRCA1 the endogenous levels of and cells transfected with or the BRCA1 expression incubated for and then to hypoxia (0.1%, O2) for when RNA was used for semiquantitative The is of quantitative of of the was and of these BRCA1 significantly enhanced and expression for of cells transfected with or without BRCA1 in O2). of time in in experiments to the shown in A and B. was used as the for semiquantitative RT-PCR and to the quantitative real time The results to the in and are as the The of BRCA1 VEGF expression is for both VEGF BRCA1 to VEGF was used to and of proteins with specific Because BRCA1 is known to regulate activity by directly interacting with various transcription it is possible that BRCA1 other are recruited to the endogenous VEGF promoter hypoxia. significantly increased binding of HIF-1α, ARNT, and BRCA1 to the promoter of the endogenous VEGF promoter was found in both and cells or of hypoxia A and for protein in cells incubated in hypoxia The could not amounts of chromatin containing VEGF promoter these results suggest that the of VEGF promoter in by specific increased interaction between the proteins and VEGF promoter of and from regions of the VEGF promoter that not have known or binding not amounts of VEGF promoter DNA of the tested BRCA1 for the VEGF of BRCA1 important for regulating activity hypoxic we used the of BRCA1 shown in of the BRCA1 and significantly BRCA1 to VEGF expression or was and in VEGF These be as in BRCA1 protein levels by the expression BRCA1 and HIF-1α BRCA1 can regulate transcription activity interacting with various transcription we for between endogenous BRCA1 and endogenous HIF-1α by with the proteins by HIF-1α protein was in the in from cells incubated in the hypoxic This interaction not significantly affect BRCA1 protein levels as hypoxia HIF-1α levels in this As a to be that our could the BRCA1 expression was with a expression cells. and with an or Western that BRCA1 was in with the and that was in with the protein was in with or in transfected with that not BRCA1 and HIF-1α from to directly or BRCA1 VEGF and VEGF inhibitory RNA to whether endogenous BRCA1 protein levels affect VEGF gene expression and secretion We BRCA1-siRNAs and to regions of the BRCA1 to from Cell. 2003; PubMed Scopus Google Scholar). transfected with for then with the and incubated for and then to our hypoxic for an when activity was significantly reduced activity when compared with the In addition, endogenous BRCA1 levels by also reduced secretion of endogenous VEGF protein following hypoxia this reduced VEGF secretion may reduced VEGF in these hypoxic cells. that BRCA1 not significantly or levels of VEGF VEGF promoter in the of hypoxia. The for the BRCA1 of VEGF levels, not been BRCA1 HIF-1α is known to regulate HIF-1α protein S. J. J. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). In the of HIF-1α that normoxia is blocked hypoxic S. J. J. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). These suggest a for BRCA1 levels could affect activity BRCA1 HIF-1α hypoxic this we the of BRCA1-siRNAs the levels of endogenous HIF-1α In the of BRCA1 or basal HIF-1α hypoxic and as of A and or and significantly reduced HIF-1α in nuclear or in cell for of cells transfected with We then tested whether the of BRCA1 HIF-1α accumulation hypoxic could altered HIF-1α transfected with BRCA1-siRNAs to hypoxia for and then with various proteasome inhibitors for an hypoxic significantly increased HIF-1α accumulation to in cells containing reduced endogenous BRCA1 levels This finding suggests that BRCA1 levels hypoxic to a of HIF-1α. directly whether reduced BRCA1 levels in hypoxic cells is with decreased HIF-1α stability, we HIF-1α this cells transfected with expression plasmids encoding or BRCA1-siRNAs then to hypoxia and then with for to the endogenous BRCA1 a and the of HIF-1α hypoxic for the between hypoxic cells with and without BRCA1 time endogenous BRCA1 levels also significantly the HIF-1α half-life hypoxic reduced levels of BRCA1 HIF-1α hypoxic we also tested whether the of BRCA1, by the of endogenous HIF-1α. this we transfected cells with expression for or the BRCA1 and HIF-1α hypoxic as for the The not VEGF promoter activity hypoxic as as BRCA1 also in the increased of is with a increased half-life of the endogenous HIF-1α when compared with cells the This half-life is also increased about when compared with cells a normal with of endogenous BRCA1 levels the half-life of HIF-1α in hypoxic cells. of cells transfected with expression for then to hypoxia for and then with for the time hypoxic used for Western blotting with and of the HIF-1α from experiments was from to and to a transfected not that was amounts of the HIF-1α to the in the of CHX, and used to from of experiments not and from the BRCA1 significantly reduced the HIF-1α half-life In addition, the amounts of HIF-1α in to the time is reduced significantly time in the hypoxic cells in BRCA1 cells of BRCA1 the of endogenous HIF-1α in hypoxic cells. accumulation of HIF-1α protein in of cells transfected with BRCA1 or the for and then to hypoxia for was Western amounts of HIF-1α to and then to the amounts from experiments from and as as in A that cells as in that the are from experiments with cells. and experiments with cells transfected with BRCA1 or the and to hypoxia as in A and then incubated with for the by Western and and as in to BRCA1 significantly increased the HIF-1α half-life In addition, the amounts of HIF-1α in to the time are reduced significantly time in the hypoxic cells in cells The suggest a role for BRCA1 in the of VEGF expression in hypoxia. BRCA1 with HIF-1α hypoxic and increased BRCA1 protein levels can the of the VEGF promoter to hypoxia in a HIF-1α hypoxic functional is for the of HIF-1α and for normal amounts of VEGF expression and secretion Thus, our suggest that BRCA1 an important role in expression of VEGF and BRCA1 also be in the of other studies that of BRCA1 and to activity a in the domain the of BRCA1 to activity in human breast cancer cells These results suggest that both the and of BRCA1 are to VEGF of the for the interaction between BRCA1 and HIF-1α may our of these BRCA1 gene The results of our studies and that reduced endogenous BRCA1 protein levels the of hypoxia to VEGF suggest that our may be to sporadic breast that also reduced amounts of BRCA1 the of this is not BRCA1 levels both VEGF expression and it is to BRCA1 that both BRCA1 activity and angiogenesis, possibility for the effects of reduced BRCA1 levels is suggested by recent that the survival of cells and/or or the inhibition of is by hypoxia X. 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Google Scholar). expression of BRCA1 not affect p300 protein levels in of the cell used and not Here, the ChIP assay, we a new role for BRCA1 in HIF-1α BRCA1 is recruited to it is of containing HIF-1α and ARNT, in the endogenous VEGF promoter These suggest that BRCA1 is a important of HIF-1α hypoxic showed that significantly HIF-1α in hypoxic cells transfected with of BRCA1-siRNAs. Because BRCA1 not significantly HIF-1α levels in normoxia or hypoxia not decreased accumulation of HIF-1α protein hypoxia is not to by decreased HIF-1α gene the of HIF-1α protein was not by finding that proteasome to an increased accumulation of HIF-1α protein in transfected hypoxic cells suggests that normal amounts of BRCA1 of HIF-1α hypoxia. This suggests that BRCA1 may have a role in in regulating HIF-1α and/or other of the BRCA1 and HIF-1α hypoxic it is that the BRCA1 HIF-1α accumulation is a a Because the von Hippel-Lindau tumor protein HIF-1α the we whether BRCA1 HIF-1α M.S. 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