Our findings suggest that overexpression of the BRCA1 gene inhibits TERT expression and telomerase enzymatic activity in various cell types and causes telomere shortening. On the other hand, BRCA1 had little or no effect on the expression of a subset of other components of the telomerase holoenzyme (TP1, HSP90, hTR, and dyskerin), c-Myc, or two telomeric repeat-binding proteins (TRF1 and TRF2). The inhibition of TERT expression is due, in part, to the inhibition of c-Myc-mediated transactivation of the TERT promoter, as demonstrated using TERT promoter and E-box-luciferase reporter assays. Moreover, while BRCA1 inhibited c-Myc-induced TERT promoter activity, it did not inhibit Sp1- or NF-κB-induced promoter activity.
It is unlikely that inhibition of cell proliferation contributed significantly to the reduced TERT expression and telomerase activity or the shortened telomeres observed in wtBRCA1-transfected cell lines. Thus, the BRCA1-overexpressing DU-145 and T47D cell clones had only a slight reduction in doubling times compared with the control (neo) clones or parental cells, and induction of BRCA1 overexpression in the TCN-regulated DU-145 cell line had no effect on cell proliferation. The reduction in TERT promoter activity and c-Myc E-box activity occurred rapidly upon expression of wtBRCA1. Thus, each of these was observed in transient transfection assays in which the end points were determined 24 h after an overnight transfection of the wtBRCA1 vector. Similarly, in HCC1937 breast cancer cells, which contain only mutant BRCA1 (5382insC), wtBRCA1 inhibited telomerase enzymatic activity in a transient transfection assay. In these cells, transient transfection of wtBRCA1 has no effect on cell proliferation and protects these cells against cytotoxicity and apoptosis induced by a DNA damaging agent (adriamycin) (18
). Human primary cell cultures usually exhibit very low TERT expression and telomerase activities. Whether or not the mechanism of telomerase suppression in human primary cells is related to that in wtBRCA1-overexpressing cancer cells remains to be determined.
Stable DU-145 and T47D wtBRCA1 cell clones were able to maintain telomeres, although these were significantly shorter than in the control clones. The ability to maintain short telomeres may be explained by residual telomerase activity, since neither TERT expression nor telomerase activity was abolished by wtBRCA1. Alternatively, the telomere maintenance could be due to a telomerase-independent mechanism for the alternative lengthening of telomeres, a process that is often observed in tumor cells (6
The studies of DU-145/wtBRCA1/Tet-Off cells suggest that up-regulation of BRCA1 causes progressive shortening of telomeres over about 60 days (60 doublings) but that the initial phase of the loss of longer subpopulations of telomeres occurred very rapidly, over 3 days (three to four doublings). This finding was consistent with evidence of significant telomere erosion only 4 days after transient transfection of a wtBRCA1 gene in DU-145 cells demonstrated by the telo-FISH assay. The rapidity of telomere loss induced by exogenous BRCA1 suggest the possibility that telomerase inhibition is not the only mechanism by which BRCA1 causes telomere shortening. Similarly rapid telomere shortening (0.85 kb per day) due to telomere degradation was observed in cells expressing a TRF2 protein targeted to telomeres (1
). And very rapid telomere loss was precipitated by exogenous RB94 (an isoform of RB1 lacking amino acids 1 to 112) in telomerase-positive bladder cancer and immortalized urothelial cells (67
). Whether BRCA1 promotes telomere degradation is conjectural, but this seems likely since rapid telomere shortening is not characteristic of cells with absent or very low telomerase activity.
It is interesting that the RMN complex (Rad50-Mr
e11-p95NBS1), which is implicated in DNA double-strand break repair, was found to interact with TRF2 and to localize at telomeres (69
). It was also reported that BRCA1 interacts and colocalizes with this complex following DNA damage (61
). It remains to be discovered if BRCA1 is present at telomeres or exerts a telomerase-independent telomere regulatory function.
Unlike normal fibroblasts, DU-145 and T47D wtBRCA1 clones and DU-145/wtBRCA1/Tet-Off cells induced to overexpress BRCA1 were able to proliferate in long-term culture without entering into senescence or apoptosis, despite severe telomere shortening. Critically short telomeres may cause senescence, in part, by activating a p53/p21WAF1/Cip1
pathway and/or a p16/Rb1 pathway (55
). DU-145 cells contain inactivating mutations in both p53 (double point mutation) and Rb1 (deletion of exon 21), and T47D cells contain mutant p53 but have wild-type Rb1. Thus, defects in telomere damage signaling pathways may contribute to the ability of these cells to survive and grow despite having very short telomeres. Our studies suggest that wtBRCA1 does not alter the expression of the telomere repeat binding factors TRF1 and TRF2 (at least at the mRNA level). However, given that the telomeres are considerably shorter in wtBRCA1 than control cell clones, it is possible that the same levels of TRF1 and TRF2 could help to protect the shorter telomeres.
Interestingly, subclones derived from immortal populations of HeLa cells (which had stable telomere populations with a mean TRF length of 3.7 kb), showed various telomere lengths, the smallest of which was about 1.7 kb (7
), similar to that observed in DU-145/wtBRCA1/Tet-Off cells that were induced to express BRCA1.
The studies using c-Myc−/−
fibroblasts revealed a very low level of TERT promoter activity (
10% of that found in c-Myc+/+
cells), which was further suppressed by wtBRCA1. This finding suggests that wtBRCA1 can also cause c-Myc-independent inhibition of TERT promoter activity, although most of the endogenous, BRCA1-suppressible activity appears to be c-Myc dependent in the cell types studied. While exogenous c-Myc caused a significant increase in TERT and E-box-Luc activity in c-Myc−/−
cells, the activity levels in these cells were well below the endogenous activity levels in c-Myc+/+
cells. This finding suggests that the c-Myc−/−
cells have developed an additional mechanism(s) to down-regulate the TERT/Myc activity.
Our findings are consistent with a previous study in which BRCA1 was found to interact with c-Myc and to repress c-Myc transcriptional and transformational activity (60
). In that study, the helix-loop-helix region of c-Myc, the same region involved in c-Myc-Max heterodimerization, was required for the BRCA1-c-Myc interaction. Amino acids 175 to 303 and 443 to 511 of BRCA1 were required for the interaction with c-Myc, as determined by glutathione S
-transferase capture assays (60
). In another study, it was reported that BRCA1 inhibits c-Myc-inducible TERT promoter activity through a three-way interaction involving BRCA1, c-Myc, and Nmi (N-Myc interactor) (35
). In that study, wtBRCA1 was insufficient to inhibit TERT promoter activity, and addition of exogenous Nmi was required to inhibit TERT. In our study, wtBRCA1 alone was sufficient to inhibit TERT promoter activity, TERT expression, and telomerase activity. The finding that wtBRCA1 inhibited TERT promoter activity in c-Myc−/−
rat fibroblasts suggests that BRCA1 can also inhibit c-Myc-independent TERT promoter activity. However, a caveat is that unlike the case in most human primary cells, TERT and telomerase are constitutively expressed in rodent cells, thus indicating that their regulation in rodents and humans is not identical.
We found that the carboxyl terminus of BRCA1 is dispensable for inhibition of TERT and E-box reporter activity. BRCA1 genes encoding amino acids 1 to 771 (ΔKpn
I) and 1 to 1313 (ΔBam
HI) (but not amino acids 1 to 302 (ΔEco
RI]) partially or strongly inhibited these reporters. However, one mutant BRCA1 (T300G) failed to inhibit telomerase or TERT-Luc activity but gave significant, though incomplete, inhibition of E-box-Luc activity. T300G is a breast cancer-associated mutant that codes for a full-length protein with a point mutation (61
Cys→Gly) that disrupts the RING domain. This finding suggests that an intact RING domain is required for inhibition of TERT. The T300G mutation (which affects BRCA1 amino acid 61) should not disrupt the BRCA1-c-Myc interaction, which involves amino acids 175 to 303 and 443 to 511 of BRCA1 (60
). These findings suggest that inhibition of the c-Myc transactivation function is insufficient to inhibit TERT or that more complete inhibition of c-Myc is required to inhibit the TERT promoter. They also suggest that BRCA1 binding to c-Myc is necessary but not sufficient to suppress TERT promoter activity. Similarly, we found that the T300G mutation did not disrupt BRCA1 binding to the estrogen receptor but inhibited its ability to prevent estrogen receptor transcriptional activity (17
Most of these studies were conducted using a BRCA1 overexpression model. However, several considerations suggest that the findings may be relevant to the physiological regulation of TERT. First, knockdown of BRCA1 significantly increased TERT promoter activity in MCF-7 cells, although the extent was modest (30 to 35%). The small extent of the increase in TERT activity may be due, in part, to the partial recovery of BRCA1 protein levels by the time the cells were harvested for luciferase assays. The BRCA1 knockdown model may mimic some sporadic breast and ovarian cancers; a significant proportion of these tumors exhibit loss of BRCA1 expression (50
). Secondly, endogenous BRCA1 was detectable at transiently transfected mouse and human TERT promoters, and the quantity of BRCA1 at the promoter sites was increased in cells transfected with a wtBRCA1 gene. Interestingly, the quantity of endogenous c-Myc at the TERT promoter was also increased in cells transfected with wtBRCA1. These findings suggest that BRCA1 may recruit c-Myc to the TERT promoter or stabilize its interaction with the promoter, although the precise mechanism remains to be determined.
Finally, an overexpression model may be very relevant to understanding some of the physiological actions of BRCA1 as a breast cancer tumor suppressor. Thus, during normal mouse development, BRCA1 is especially highly expressed in proliferating mammary epithelial cells that are undergoing differentiation during puberty and pregnancy (36
). And cultured mammary epithelial cells induced to undergo differentiation in vitro also show greatly increased BRCA1 expression. It has been suggested that overexpression of BRCA1 during these particular periods may contribute to its tumor suppressor activity.