The base excision repair pathway is largely responsible for the repair of oxidative stress-induced DNA damage. However, it remains unclear how the DNA damage checkpoint is activated by oxidative stress at the molecular level. Here, we provide evidence showing that hydrogen peroxide (H 2 O 2 ) triggers checkpoint kinase 1 (Chk1) phosphorylation in an ATR [ataxia-telangiectasia mutated (ATM) and Rad3-related]-dependent but ATM-independent manner in Xenopus egg extracts. A base excision repair protein, Apurinic/apyrimidinic (AP) endonuclease 2 (APE2, APN2, or APEX2), is required for the generation of replication protein A (RPA)-bound single-stranded DNA, the recruitment of a checkpoint protein complex [ATR, ATR-interacting protein (ATRIP), and Rad9] to damage sites, and H 2 O 2 -induced Chk1 phosphorylation. A conserved proliferating cell nuclear antigen interaction protein box of APE2 is important for the recruitment of APE2 to H 2 O 2 -damaged chromatin. APE2 3′-phosphodiesterase and 3′-5′ exonuclease activity is essential for single-stranded DNA generation in the 3′-5′ direction from single-stranded breaks, referred to as single-stranded break end resection. In addition, APE2 associates with Chk1, and a serine residue (S86) in the Chk1-binding motif of APE2 is essential for Chk1 phosphorylation, indicating a Claspin-like but distinct role for APE2 in ATR-Chk1 signaling. Our data indicate that APE2 plays a vital and previously unexpected role in ATR-Chk1 checkpoint signaling in response to oxidative stress. Thus, our findings shed light on a distinct mechanism of how an ATR-Chk1-dependent DNA damage checkpoint is mediated by APE2 in the oxidative stress response. C ells are constantly challenged by exogenous and endogenous insults that threaten genomic integrity. Excess accumulation of reactive oxygen species leads to oxidative DNA damage, such as DNA strand breaks with 3′-modified termini, which is often the underlying pathology in a variety of diseases including neurodegenerative diseases and cancer (1-6). Cellular responses to DNA damage are mainly coordinated by two distinct DNA damage checkpoint signaling cascades: ATM (ataxia-telangiectasia mutated)-checkpoint kinase 2 (Chk2) and ATR (ATM and Rad3-related)-checkpoint kinase 1 (Chk1) pathways (7-10). ATM is activated by intermolecular autophosphorylation and dimer dissociation in response to double-stranded beaks (DSBs) (11-13). ATR is activated by primed single-stranded DNA (ssDNA) in response to a variety of DNA damage or replication stresses (14,15). Oxidative stress has been demonstrated to activate an ATMdependent DNA damage checkpoint (16-18). However, in previous studies, hyperoxic conditions resulted in the phosphorylation of Chk1 and p53 in an ATR-dependent but ATM-independent fashion (19). Furthermore, it remains unclear which specific DNA structures trigger checkpoint signaling during oxidative stress.To eliminate oxidative DNA damage, base excision repair (BER) has evolved as a major DNA damage repair mechanism (20). In the initial step of BER, o...

Systemic inflammation in breast cancer correlates with poor prognosis, but the molecular underpinnings of this connection are not well understood. In this study, we explored the relationship between HER2 overexpression, inflammation, and expansion of the mammary stem/progenitor and cancer stem-like cell (CSC) population in breast cancer. HER2-positive epithelial cells initiated and sustained an inflammatory milieu needed to promote tumorigenesis. HER2 induced a feedforward activation loop of IL1α and IL6 that stimulated NFκB and STAT3 pathways for generation and maintenance of breast CSC. In mice, Il1a genetic deficiency delayed MMTV-Her2-induced tumorigenesis and reduced inflammatory cytokine expression as well as CSC in primary tumors. In clinical specimens of human breast tumor tissues, tissue microarray analysis revealed a strong positive correlation between IL1α/IL6 expression and CSC-positive phenotype. Pharmacologic blockade of IL1α signaling reduced the CSC population and improved chemotherapeutic efficacy. Our findings suggest new therapeutic or prevention strategies for HER2-positive breast cancers. IL1α signaling driven by HER2 promotes chronic inflammation needed to support cancer stem-like cell maintenance in HER2-positive breast cancers. .

It is postulated that the complexity and heterogeneity in cancer may hinder most efforts that target a single pathway. Thus, discovery of novel therapeutic agents targeting multiple pathways, such as miRNAs, holds promise for future cancer therapy. One such miRNA, miR-489, is downregulated in a majority of breast cancer cells and several drug-resistant breast cancer cell lines, but its role and underlying mechanism for tumor suppression and drug resistance needs further investigation. The current study identifies autophagy as a novel pathway targeted by miR-489 and reports Unc-51 like autophagy activating kinase 1 (ULK1) and lysosomal protein transmembrane 4 beta (LAPTM4B) to be direct targets of miR-489. Furthermore, the data demonstrate autophagy inhibition and LAPTM4B downregulation as a major mechanism responsible for miR-489-mediated doxorubicin sensitization. Finally, miR-489 and LAPTM4B levels were inversely correlated in human tumor clinical specimens, and more importantly, miR-489 expression levels predict overall survival in patients with 8q22 amplification (the region in which LAPTM4B resides). These findings expand the understanding of miR-489-mediated tumor suppression and chemosensitization in and suggest a strategy for using miR-489 as a therapeutic sensitizer in a defined subgroup of resistant breast cancer patients. .

Human epidermal growth factor receptor 2 (HER2 or ErBb2) is a receptor tyrosine kinase overexpressed in 20-30% of breast cancers and associated with poor prognosis and outcome. Dysregulation of several microRNAs (miRNAs) plays a key role in breast cancer progression and metastasis. In this study, we screened and identified miRNAs dysregualted in HER2-positive breast cancer cells. Our molecular study demonstrated that miR-489 was specifically downregulated by the HER2-downstream signaling, especially through the MAPK pathway. Restoration or overexpression of miR-489 in HER2-positive breast cancer cells significantly inhibited cell growth in vitro and decreased the tumorigenecity and tumor growth in xenograft mice. Mechanistically, we found that overexpression of miR-489 led to the decreased levels of HER2 and SHP2 and thus attenuated HER2-downstream signaling. Furthermore, we for the first time demonstrated that HER2 is a direct target of miR-489 and therefore HER2-SHP2-MAPK and miR-489 signaling pathways form a mutually inhibitory loop. Using quantitative real-time PCR analysis and Fluorescent in situ hybridization technique (FISH), we found that miR-489 was expressed at significantly lower level in tumor tissues compared to the adjacent normal tissues. Downregulation of miR-489 in breast cancers was associated with aggressive tumor phenotypes. Overall, our results define a double-negative feedback loop involving miR-489 and the HER2-SHP2-MAPK signaling axis that can regulate breast cancer cell proliferation and tumor progression and might have therapeutic relevance for HER2-positive breast cancer.

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop.Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group 1 . The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle.The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress 2 . Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures 3 . DNA damage can be induced by ultraviolet (UV) irradiation, γ-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin 3, 4 . MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint [4][5][6][7] . UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint 8 . The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy's laboratory and is widely used to induce ATR/ Chk1 checkpoint signaling [9][10][11][12] .Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure.