Eunmi Park

Point mutations in the yeast histone H4 gene prevent silencing of the silent mating type locus HML.

The N-terminal serine and four conserved lysine residues near the N-terminus of yeast histone H4 are acetylated. We found that a mutation that changed the fourth lysine to alanine resulted in specific derepression of the silent mating type locus HML, while mutations that altered the N-terminal serine or the first three lysines had only minor phenotypic effects. Our results support an active role for histone H4 in the silencing of gene expression at this locus.

ARD1 and NAT1 proteins form a complex that has N-terminal acetyltransferase activity.

Two yeast genes, ARD1 and NAT1, are required for the expression of an N‐terminal protein acetyltransferase. This activity is required for full repression of the silent mating type locus HML, for sporulation, and for entry into G0. While the NAT1 gene product is thought to be the catalytic subunit of the enzyme, the role of the ARD1 protein has remained unclear. We have used epitope tagged derivatives of ARD1 and NAT1 to provide biochemical evidence for the formation of an ARD1‐NAT1 complex, and to show that both proteins are required for the N‐terminal acetyltransferase activity. We also present evidence for the formation of ARD1‐ARD1 homodimers. Deletion analysis suggests that the C‐terminal region of ARD1 may be involved in the formation of both ARD1‐ARD1 and ARD1‐NAT1 complexes.

Inhibition of the Nedd8 system sensitizes cells to DNA interstrand cross-linking agents.

The Fanconi anemia pathway is required for repair of DNA interstrand cross-links (ICL). Fanconi anemia pathway–deficient cells are hypersensitive to DNA ICL–inducing drugs such as cisplatin. Conversely, hyperactivation of the Fanconi anemia pathway is a mechanism that may underlie cellular resistance to DNA ICL agents. Modulating FANCD2 monoubiquitination, a key step in the Fanconi anemia pathway, may be an effective therapeutic approach to conferring cellular sensitivity to ICL agents. Here, we show that inhibition of the Nedd8 conjugation system increases cellular sensitivity to DNA ICL–inducing agents. Mechanistically, the Nedd8 inhibition, either by siRNA-mediated knockdown of Nedd8-conjugating enzymes or treatment with a Nedd8-activating enzyme inhibitor MLN4924, suppressed DNA damage–induced FANCD2 monoubiquitination and CHK1 phosphorylation. Our data indicate that inhibition of the Fanconi anemia pathway is largely responsible for the heightened cellular sensitivity to DNA ICLs upon Nedd8 inhibition. These results suggest that a combination of Nedd8 inhibition with ICL-inducing agents may be an effective strategy for sensitizing a subset of drug-resistant cancer cells. Mol Cancer Res; 10(3); 369–77. ©2012 AACR.

Small-Molecule Inhibitors of USP1 Target ID1 Degradation in Leukemic Cells

Inhibitor of DNA binding 1 (ID1) transcription factor is essential for the proliferation and progression of many cancer types, including leukemia. However, the ID1 protein has not yet been therapeutically targeted in leukemia. ID1 is normally polyubiquitinated and degraded by the proteasome. Recently, it has been shown that USP1, a ubiquitin-specific protease, deubiquitinates ID1 and rescues it from proteasome degradation. Inhibition of USP1 therefore offers a new avenue to target ID1 in cancer. Here, using a ubiquitin-rhodamine–based high-throughput screening, we identified small-molecule inhibitors of USP1 and investigated their therapeutic potential for leukemia. These inhibitors blocked the deubiquitinating enzyme activity of USP1 in vitro in a dose-dependent manner with an IC50 in the high nanomolar range. USP1 inhibitors promoted the degradation of ID1 and, concurrently, inhibited the growth of leukemic cell lines in a dose-dependent manner. A known USP1 inhibitor, pimozide, also promoted ID1 degradation and inhibited growth of leukemic cells. In addition, the growth of primary acute myelogenous leukemia (AML) patient-derived leukemic cells was inhibited by a USP1 inhibitor. Collectively, these results indicate that the novel small-molecule inhibitors of USP1 promote ID1 degradation and are cytotoxic to leukemic cells. The identification of USP1 inhibitors therefore opens up a new approach for leukemia therapy. Mol Cancer Ther; 12(12); 2651–62. ©2013 AACR.

FANCD2 activates transcription of TAp63 and suppresses tumorigenesis.

Fanconi anemia (FA) is a rare genetic disorder characterized by an increased susceptibility to squamous cell cancers. Fifteen FA genes are known, and the encoded proteins cooperate in a common DNA repair pathway. A critical step is the monoubiquitination of the FANCD2 protein, and cells from most FA patients are deficient in this step. How monoubiquitinated FANCD2 suppresses squamous cell cancers is unknown. Here we show that Fancd2-deficient mice are prone to Ras-oncogene-driven skin carcinogenesis, while Usp1-deficient mice, expressing elevated cellular levels of Fancd2-Ub, are resistant to skin tumors. Moreover, Fancd2-Ub activates the transcription of the tumor suppressor TAp63, thereby promoting cellular senescence and blocking skin tumorigenesis. For FA patients, the reduction of FANCD2-Ub and TAp63 protein levels may account for their susceptibility to squamous cell neoplasia. Taken together, Usp1 inhibition may be a useful strategy for upregulating TAp63 and preventing or treating squamous cell cancers in the general non-FA population.

MicroRNAs down-regulate homologous recombination in the G1 phase of cycling cells to maintain genomic stability

Homologous recombination (HR)-mediated repair of DNA double-strand break (DSB)s is restricted to the post-replicative phases of the cell cycle. Initiation of HR in the G1 phase blocks non-homologous end joining (NHEJ) impairing DSB repair. Completion of HR in G1 cells can lead to the loss-of-heterozygosity (LOH), which is potentially carcinogenic. We conducted a gain-of-function screen to identify miRNAs that regulate HR-mediated DSB repair, and of these miRNAs, miR-1255b, miR-148b*, and miR-193b* specifically suppress the HR-pathway in the G1 phase. These miRNAs target the transcripts of HR factors, BRCA1, BRCA2, and RAD51, and inhibiting miR-1255b, miR-148b*, and miR-193b* increases expression of BRCA1/BRCA2/RAD51 specifically in the G1-phase leading to impaired DSB repair. Depletion of CtIP, a BRCA1-associated DNA end resection protein, rescues this phenotype. Furthermore, deletion of miR-1255b, miR-148b*, and miR-193b* in independent cohorts of ovarian tumors correlates with significant increase in LOH events/chromosomal aberrations and BRCA1 expression. DOI: http://dx.doi.org/10.7554/eLife.02445.001

Inactivation of Uaf1 causes Defective Homologous Recombination and Early Embryonic Lethality in Mice

ABSTRACT The deubiquitinating enzyme heterodimeric complex USP1-UAF1 regulates the Fanconi anemia (FA) DNA repair pathway. Absence of this complex leads to increased cellular levels of ubiquitinated FANCD2 (FANCD2-Ub) and ubiquitinated PCNA (PCNA-Ub). Mice deficient in the catalytic subunit of the complex, USP1, exhibit an FA-like phenotype and have a cellular deficiency in homologous-recombination (HR) repair. Here, we have characterized mice deficient in the UAF1 subunit. Uaf1+/− mice were small at birth and exhibited reduced fertility, thus resembling Usp1−/− mice. Unexpectedly, homozygous Uaf1−/− embryos died at embryonic day 7.5 (E7.5). These mutant embryos were small and developmentally retarded. As expected, Uaf1 deficiency in mice led to increased levels of cellular Fancd2-Ub and Pcna-Ub. Uaf1+/− murine embryonic fibroblasts (MEFs) exhibited profound chromosome instability, genotoxin hypersensitivity, and a significant defect in homologous-recombination repair. Moreover, Uaf1−/− mouse embryonic stem cells (mESCs) showed chromosome instability, genotoxin hypersensitivity, and impaired Fancd2 focus assembly. Similar to USP1 knockdown, UAF1 knockdown in tumor cells caused suppression of tumor growth in vivo. Taken together, our data demonstrate the important regulatory role of the USP1-UAF1 complex in HR repair through its regulation of the FANCD2-Ub and PCNA-Ub cellular pools.

Reduction of IKKα Expression Promotes Chronic Ultraviolet B Exposure-Induced Skin Inflammation and Carcinogenesis

Ultraviolet B light (UVB) is a common cause of human skin cancer. UVB irradiation induces mutations in the tumor suppressor p53 gene as well as chronic inflammation, which are both essential for UVB carcinogenesis. Inhibitor of nuclear factor kappaB kinase-alpha (IKKalpha) plays an important role in maintaining skin homeostasis, and expression of IKKalpha was found to be down-regulated in human and murine skin squamous cell carcinomas. However, the role of IKKalpha in UVB skin carcinogenesis has not been investigated. Thus, here we performed UVB carcinogenesis experiments on Ikkalpha(+/+) and Ikkalpha(+/-) mice. Ikkalpha(+/-) mice were found to develop a twofold greater number of skin tumors than Ikkalpha(+/+) mice after chronic UVB irradiation. In addition, tumor latency was significantly shorter and tumors were bigger in Ikkalpha(+/-) than in Ikkalpha(+/+) mice. At an early stage of carcinogenesis, an increase in UVB-induced p53 mutations as well as macrophage recruitment and mitogenic activity, and a decrease in UVB-induced apoptosis, were detected in Ikkalpha(+/-) compared with those in Ikkalpha(+/+) skin. Also, reduction of IKKalpha levels in keratinocytes up-regulated the expression of monocyte chemoattractant protein-1 (MCP-1/CCL2), TNFalpha, IL-1, and IL-6, and elevated macrophage migration, which might promote macrophage recruitment and inflammation. Therefore, these findings suggest that reduction of IKKalpha expression orchestrates UVB carcinogen, accelerating tumorigenesis.

PARI Overexpression Promotes Genomic Instability and Pancreatic Tumorigenesis

Treatment options for patients with pancreatic ductal adenocarcinoma (PDAC) remain limited. Therapeutic targets of interest include mutated molecules that predispose to pancreatic cancer such as KRAS and TP53. Here, we show that an element of the homologous recombination pathway of DNA repair, the PARP-binding protein C12orf48/PARI (PARPBP), is overexpressed specifically in pancreatic cancer cells where it is an appealing candidate for targeted therapy. PARI upregulation in pancreatic cancer cells or avian DT40 cells conferred DNA repair deficiency and genomic instability. Significantly, PARI silencing compromised cancer cell proliferation in vitro, leading to cell-cycle alterations associated with S-phase delay, perturbed DNA replication, and activation of the DNA damage response pathway in the absence of DNA damage stimuli. Conversely, PARI overexpression produced tolerance to DNA damage by promoting replication of damaged DNA. In a mouse xenograft model of pancreatic cancer, PARI silencing was sufficient to reduce pancreatic tumor growth in vivo. Taken together, our findings offered a preclinical proof-of-concept for PARI as candidate therapeutic target to treat PDAC.

Role of IKKα in skin squamous cell carcinomas.

Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) are two major types of skin cancer derived from keratinocytes. SCC is a more aggressive type of cancer than BCC in humans. One significant difference between SCC and BCC is that SCC development is generally associated with cell dedifferentiation and morphological changes. When SCC is converted to spindle cell carcinoma, the latest stage of cancer, the tumor cells change to a fibroblastic cell morphology (epithelial-to-mesenchymal transition) and lose their differentiation markers. Recently, several laboratories have reported altered IκB kinase α (IKKα) protein localization, downregulated IKKα, and IKKα gene deletions and mutations in human SCCs of the skin, lung, esophagus, and neck and head. In addition, IKKα reduction promotes chemical carcinogen- and ultraviolet B-induced skin carcinogenesis, and IKKα deletion in keratinocytes causes spontaneous skin SCCs, but not BCCs, in mice. Thus, IKKα emerges as a bona fide skin tumor suppressor. In this article, we will discuss the role of IKKα in skin SCC development.