CTDP1 regulates breast cancer survival and DNA repair through BRCT-specific interactions with FANCI.

BRCA1 C-terminal domains are found in a specialized group of 23 proteins that function in the DNA damage response to protect genomic integrity. C-terminal domain phosphatase 1 (CTDP1) is the only phosphatase with a BRCA1 C-terminal domain in the human proteome, yet direct participation in the DNA damage response has not been reported. Examination of the CTDP1 BRCA1 C-terminal domain-specific protein interaction network revealed 103 high confidence interactions enriched in DNA damage response proteins, including FANCA and FANCI that are central to the Fanconi anemia DNA repair pathway necessary for the resolution of DNA interstrand crosslink damage. CTDP1 expression promotes DNA damage-induced FANCA and FANCD2 foci formation and enhances homologous recombination repair efficiency. CTDP1 was found to regulate multiple aspects of FANCI activity, including chromatin localization, interaction with γ-H2AX, and SQ motif phosphorylations. Knockdown of CTDP1 increases MCF-10A sensitivity to DNA interstrand crosslinks and double-strand breaks, but not ultraviolet radiation. In addition, CTDP1 knockdown impairs in vitro and in vivo growth of breast cancer cell lines. These results elucidate the molecular functions of CTDP1 in Fanconi anemia interstrand crosslink repair and identify this protein as a potential target for breast cancer therapy.

Recent insights into the molecular basis of Fanconi anemia: genes, modifiers, and drivers.

Fanconi anemia (FA), the most common form of inherited bone marrow failure, predisposes to leukemia and solid tumors. FA is caused by the genetic disruption of a cellular pathway that repairs DNA interstrand crosslinks. The impaired function of this pathway, and the genetic instability that results, is considered the main pathogenic mechanism behind this disease. The identification of breast cancer susceptibility genes (for example, BRCA1/FANCS and BRCA2/FANCD1) as being major players in the FA pathway has led to a surge in molecular studies, resulting in the concept of the FA-BRCA pathway. In this review, we discuss recent advances in the molecular pathogenesis of FA from three viewpoints: (a) new FA genes, (b) modifier pathways that influence the cellular and clinical phenotypes of FA and (c) non-canonical functions of FA genes that may drive disease progression independently of deficient DNA repair. Potential therapeutic approaches for FA that are relevant to each will also be proposed.

Ubiquitination-Linked Phosphorylation of the FANCI S/TQ Cluster Contributes to Activation of the Fanconi Anemia I/D2 Complex.

Repair of interstrand crosslinks by the Fanconi anemia (FA) pathway requires both monoubiquitination and de-ubiquitination of the FANCI/FANCD2 (FANCI/D2) complex. In the standing model, the phosphorylation of six sites in the FANCI S/TQ cluster domain occurs upstream of, and promotes, FANCI/D2 monoubiquitination. We generated phospho-specific antibodies against three different S/TQ cluster sites (serines 556, 559, and 565) on human FANCI and found that, in contrast to the standing model, distinct FANCI sites were phosphorylated either predominantly upstream (ubiquitination independent; serine 556) or downstream (ubiquitination-linked; serines 559 and 565) of FANCI/D2 monoubiquitination. Ubiquitination-linked FANCI phosphorylation inhibited FANCD2 de-ubiquitination and bypassed the need to de-ubiquitinate FANCD2 to achieve effective interstrand crosslink repair. USP1 depletion suppressed ubiquitination-linked FANCI phosphorylation despite increasing FANCI/D2 monoubiquitination, providing an explanation of why FANCD2 de-ubiquitination is important for function of the FA pathway. Our work results in a refined model of how FANCI phosphorylation activates the FANCI/D2 complex.

FANCI Regulates Recruitment of the FA Core Complex at Sites of DNA Damage Independently of FANCD2.

The Fanconi anemia (FA)-BRCA pathway mediates repair of DNA interstrand crosslinks. The FA core complex, a multi-subunit ubiquitin ligase, participates in the detection of DNA lesions and monoubiquitinates two downstream FA proteins, FANCD2 and FANCI (or the ID complex). However, the regulation of the FA core complex itself is poorly understood. Here we show that the FA core complex proteins are recruited to sites of DNA damage and form nuclear foci in S and G2 phases of the cell cycle. ATR kinase activity, an intact FA core complex and FANCM-FAAP24 were crucial for this recruitment. Surprisingly, FANCI, but not its partner FANCD2, was needed for efficient FA core complex foci formation. Monoubiquitination or ATR-dependent phosphorylation of FANCI were not required for the FA core complex recruitment, but FANCI deubiquitination by USP1 was. Additionally, BRCA1 was required for efficient FA core complex foci formation. These findings indicate that FANCI functions upstream of FA core complex recruitment independently of FANCD2, and alter the current view of the FA-BRCA pathway.

Interactions between MYC and transforming growth factor alpha alter the growth and tumorigenicity of liver progenitor cells.

The MYC oncogene induces both cell proliferation and apoptosis. The apoptotic function of MYC is thought to inhibit carcinogenesis; thus, when disrupted, tumorigenic potential is increased. Both MYC and transforming growth factor alpha (TGFalpha) are commonly over-expressed in hepatocellular carcinomas, and transgenic mice expressing these genes rapidly develop tumors via the suppression of MYC-induced apoptosis by the growth factor. However, the nature of the interactions between MYC and TGFalpha are not well understood. Specifically, it is unclear whether TGFalpha acts only as an anti-apoptotic factor in its interactions with MYC or whether it has substantial effects on cell growth. We investigated whether TGFalpha can provide additional mitogenic signals if it is not required to act as an anti-apoptotic factor. We demonstrate that expression of MYC and TGFalpha in liver progenitor cells (known as oval cells) results in enhanced cell proliferation in culture and the generation of poorly differentiated tumors after inoculation into nude mice. We further demonstrate that while the apoptosis-deficient T58A and S71F alleles of MYC retain their ability to promote oval cell proliferation, they have opposite growth interactions with TGFalpha. The T58A allele has a stimulatory effect on both proliferation and tumorigenicity. In contrast, co-expression of the S71F allele reduces proliferation and slows tumor development. We conclude that the tumorigenic growth effects of MYC in TGFalpha-expressing liver progenitor cells are not solely dependent on its apoptotic activity.