BRIT1 regulates p53 stability and functions as a tumor suppressor in breast cancer
Advance Access publication June
BRIT1 regulates p53 stability and functions as a tumor suppressor in breast cancer
Bo Zhang 2
Edward Wang 1
Hui Dai 1
Ruozhen Hu 1
Yulong Liang 0
Kaiyi Li 0
Guobin Wang 2
Guang Peng 2 3
Shiaw-Yih Lin 1
0 Department of Surgery, Baylor College of Medicine , Houston, TX 77030 , USA
1 Department of Systems Biology, The University of Texas MD Anderson Cancer Center , Houston, TX 77054 , USA
2 Department of General Surgery, Union Hospital, Tongji Medical College, The University of Huazhong Science & Technology , Wuhan, Hubei Province 430022 , People's Republic of China
3 Department of Clinical Cancer Prevention, The University of Texas MD Anderson Cancer Center , Houston, TX 77030 , USA
In humans, the gene encoding the BRCA1 C terminus-repeat inhibitor of human telomerase expression 1 (BRIT1) protein is located on chromosome 8p23.1, a region implicated in the development of several malignancies, including breast cancer. Previous studies by our group and others suggested that BRIT1 might function as a novel tumor suppressor. Thus, identifying the molecular mechanisms that underlie BRIT1's tumor suppressive function is important to understand cancer etiology and to identify effective therapeutic strategies for BRIT1-deficient tumors. We thus investigated the role of BRIT1 as a tumor suppressor in breast cancer by using genetic approaches. We discovered that BRIT1 functions as a post-transcriptional regulator of p53 expression. BRIT1 regulates p53 protein stability through blocking murine double minute 2-mediated p53 ubiquitination. To fully demonstrate the role of BRIT1 as a tumor suppressor, we depleted BRIT1 in normal breast epithelial cells. We found that knockdown of BRIT1 caused the oncogenic transformation of normal mammary epithelial cells. Furthermore, ectopic expression of BRIT1 effectively suppressed breast cancer cell proliferation and colony formation in vitro and tumor growth in vivo. Taken together, our study provides new insights into the biological functions of BRIT1 as a tumor suppressor in human breast cancer.
Introduction
Breast cancer 1 (BRCA1) C terminus-repeat inhibitor of human
telomerase expression 1 (BRIT1) is a gene initially identified by a
functional genomic screen as a novel repressor of human telomerase
and later matched to a putative disease gene called microcephalin 1
(MCPH1) (
1,2
). Our previous studies further showed that expression
of BRIT1 is indeed decreased in several types of human cancers. We
found significant decreases in BRIT1 gene copy number in 72% of 54
breast cell lines and 40% of 87 advanced epithelial ovarian cancers.
In one of the 10 cancer specimens studied, we also identified a BRIT1
Abbreviations: ATM, ataxia telangiectasia mutated; BRCA1, breast cancer
1; BRCT, BRCA1 C terminus; BRIT1, BRCA1 C terminus-repeat inhibitor of
human telomerase expression 1; CHK2, checkpoint kinase 2; CHX,
cycloheximide; DDR, DNA damage response; FBS, fetal bovine serum; MDM2, murine
double minute 2; MTT, 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetra-zo
lium bromide; shRNA, small hairpin RNA; siRNA, small interfering RNA;
TBST, Tris-buffered saline–0.1% Tween-20; UV, ultraviolet.
†These authors contributed equally to this work.
gene deletion that led to the loss of BRIT1 function in DNA damage
response (DDR) (
3
). More recently, a study of breast cancer samples
from 319 patients showed that BRIT1 protein expression was reduced
in 29% of breast cancers, particularly in higher grade tumors 4().
Another study conducted in epithelial ovarian cancers found that low
expression of BRIT1 protein correlated with low patient survival5().
Collectively, our studies and studies from other groups suggest that
BRIT1 may function as a novel tumor suppressor. Thus, identifying
the molecular mechanisms that underlie BRIT1’s tumor suppressive
function is important not only to understand cancer etiology but also
to identify effective therapeutic strategies for BRIT1-deficient tumors.
To understand its biological function, we analyzed the protein
structure of BRIT1 and found that human BRIT1 protein contains
three BRCA1 C terminus (BRCT) domains: one in its N terminus and
two in its C terminus (
6–10
). BRCT domains are conserved in many
important molecules involved in DDR and tumor suppression, such
as BRCA1, mediator of DNA damage checkpoint protein 1 (MDC1)
and Nijmegen breakage syndrome 1 (
11
). DDR is an
evolutionarily conserved pathway in cells to detect, signal and repair damaged
DNA and thereby to assure the integrity of their genome. Ataxia
telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) are
two central kinases in the DDR pathway that can phosphorylate and
activate a variety of downstream proteins for cell cycle arrest, DNA
repair or apoptosis if the damage is irreparable (
12–15
). It has been
well established that an intact and effective DDR is essential for the
maintenance of genomic stability and that the DDR acts as a
critic (...truncated)