The life and death of DNA-PK

Double-strand breaks (DSBs) arise endogenously during normal cellular processes and exogenously by genotoxic agents such as ionizing radiation (IR). DSBs are one of the most severe types of DNA damage, which if left unrepaired are lethal to the cell. Several different DNA repair pathways combat DSBs, with nonhomologous end-joining (NHEJ) being one of the most important in mammalian cells. Competent NHEJ catalyses repair of DSBs by joining together and ligating two free DNA ends of little homology (microhomology) or DNA ends of no homology. The core components of mammalian NHEJ are the catalytic subunit of DNA protein kinase (DNA-PK(cs)), Ku subunits Ku70 and Ku80, Artemis, XRCC4 and DNA ligase IV. DNA-PK is a nuclear serine/threonine protein kinase that comprises a catalytic subunit (DNA-PK(cs)), with the Ku subunits acting as the regulatory element. It has been proposed that DNA-PK is a molecular sensor for DNA damage that enhances the signal via phosphorylation of many downstream targets. The crucial role of DNA-PK in the repair of DSBs is highlighted by the hypersensitivity of DNA-PK(-/-) mice to IR and the high levels of unrepaired DSBs after genotoxic insult. Recently, DNA-PK has emerged as a suitable genetic target for molecular therapeutics such as siRNA, antisense and novel inhibitory small molecules. This review encompasses the recent literature regarding the role of DNA-PK in the protection of genomic stability and focuses on how this knowledge has aided the development of specific DNA-PK inhibitors, via both small molecule and directed molecular targeting techniques. This review promotes the inhibition of DNA-PK as a valid approach to enhance the tumor-cell-killing effects of treatments such as IR.     

Premature chromosome condensation reveals DNA-PK independent pathways of chromosome break repair

Cells of higher eukaryotes process double strand breaks (DSBs) in their genome using a non-homologous end joining apparatus that utilizes DNA-PK and other well characterized factors (D-NHEJ). Cells with defects in D-NHEJ, repair the majority of DSBs using a slow-repair pathway which is independent of genes of the RAD52 epistasis group and functions as a backup (B-NHEJ). Recent studies implicate DNA ligase III, PARP-1 and histone H1 in this pathway of NHEJ. The present study investigates the operation of B-NHEJ in the repair of interphase chromosome breaks visualized in irradiated G0 human lymphocytes by premature chromosome condensation (PCC). Chromosome breaks are effectively repaired in human lymphocytes, but repair is significantly compromised after treatment with wortmannin, a DNA-PK inhibitor. Despite slower kinetics, cells exposed to wortmannin rejoin the majority of IR induced chromosome breaks suggesting that B-NHEJ is also functional at the chromosome level. Complementation of D-NHEJ defect in wortmannin-treated lymphocytes by newly made DNA-PK is only possible under conditions of nuclear envelope break down and premature chromosome condensation, suggesting that in interphase cells the shunting of chromosome breaks from D-NHEJ to B-NHEJ is irreversible. The understanding of chromosomal aberration formation allows mechanistic explanations for the carcinogenic potential of D-NHEJ defects.     

PD-L1 deficiency sensitizes tumor cells to DNA-PK inhibition and enhances cGAS-STING activation

Immunotherapies that block PD-L1/PD-1 immune checkpoint proteins represent a landmark breakthrough in cancer treatment. Although the role of PD-L1 in suppressing T cell activity has been extensively studied, its cancer cell-intrinsic functions are not well understood. Herein, we demonstrated that PD-L1 is important for the repair of DNA damage in cancer cells. Mechanically, depletion of PD-L1 led to the downregulation of the critical molecules involved in the homologous recombination (HR) repair pathway, such as ATM and BRCA1, but did not obviously affect the non-homologous end joining (NHEJ) pathway. Notably, PD-L1 silence sensitized cancer cells to chemotherapy agents and the inhibitor of DNA-PK, which is an important kinase for NHEJ. Furthermore, PD-L1 depletion potentiated DNA damage-induced cGAS-STING pathway and induction of IFNβ. The regulation of DNA repair and cGAS-STING pathway by PD-L1 represents its connection with innate immunity that can be exploited to enhance the efficacy of existing immunotherapy. Our findings thus expand the focus of PD-L1 from tumor antigen-specific CD8+ T cells to innate immunity, and support targeting tumor-intrinsic PD-L1 combined with DNA-PK inhibition for tumor eradication, through promoting synthetic lethality and innate immune response.     

NONO phase separation enhances DNA damage repair by accelerating nuclear EGFR-induced DNA-PK activation

Radioresistance is one of the main causes of cancer treatment failure, which leads to relapse and inferior survival outcome of cancer patients. Liquid-liquid phase separation (LLPS) of proteins is known to be involved in various biological processes, whereas its role in the regulation of radiosensitivity remains largely unknown. In this study, we characterized NONO, an RNA/DNA binding protein with LLPS capacity, as an essential regulator of tumor radioresistance. In vitro assay showed that NONO involved in DNA repair via non-homologous end joining (NHEJ) manner. NONO knockout significantly reduced DNA damage repair and sensitized tumor cells to irradiation in vitro and in vivo. NONO overexpression was correlated with an inferior survival outcome in cancer patients. Mechanically, NONO was associated with nuclear EGFR (nEGFR). Both irradiation and EGF treatment induced nEGFR accumulation, thereby increased the association between NONO and nEGFR. However, NONO was not a substrate of EGFR kinase. Furthermore, NONO promoted DNA damage-induced DNA-PK phosphorylation at T2609 by enhancing the interaction between EGFR and DNA-PK. Importantly, NONO protein formed high concentration LLPS droplets in vitro, and recruited EGFR and DNA-PK. Disruption of NONO droplets with LLPS inhibitor significantly reduced the interaction between EGFR and DNA-PK, and suppressed DNA damage-induced phosphorylation of T2609-DNA-PK. Taken together, LLPS of NONO recruits nuclear EGFR and DNA-PK and enhances their interaction, further increases DNA damage-activated pT2609-DNA-PK and promotes NHEJ-mediated DNA repair, finally leads to tumor radioresistance. NONO phase separation-mediated radioresistance may serve as a novel molecular target to sensitize tumor cell to radiotherapy.     

Replication protein A2 phosphorylation after DNA damage by the coordinated action of ataxia telangiectasia-mutated and DNA-dependent protein kinase.

Replication protein A (RPA, also known as human single-stranded DNA-binding protein) is a trimeric, multifunctional protein complex involved in DNA replication, DNA repair, and recombination. Phosphorylation of the RPA2 subunit is observed after exposure of cells to ionizing radiation (IR) and other DNA-damaging agents, which implicates the modified protein in the regulation of DNA replication after DNA damage or in DNA repair. Although ataxia telangiectasia-mutated (ATM) and DNA-dependent protein kinase (DNA-PK) phosphorylate RPA2 in vitro, their role in vivo remains uncertain, and contradictory results have been reported. Here we show that RPA2 phosphorylation is delayed in cells deficient in one of these kinases and completely abolished in wild-type, ATM, or DNA-PK-deficient cells after treatment with wortmannin at a concentration-inhibiting ATM and DNA-PK. Caffeine, an inhibitor of ATM and ATM-Rad3 related (ATR) but not DNA-PK, generates an ataxia-telangiectasia-like response in wild-type cells, prevents completely RPA2 phosphorylation in DNA-PKcs deficient cells, but has no effect on ataxia-telangiectasia cells. These observations rule out ATR and implicate both ATM and DNA-PK in RPA2 phosphorylation after exposure to IR. UCN-01, an inhibitor of protein kinase C, Chk1, and cyclin-dependent kinases, has no effect on IR-induced RPA2 phosphorylation. Because UCN-01 abrogates checkpoint responses, this observation dissociates RPA2 phosphorylation from checkpoint activation. Phosphorylated RPA has a higher affinity for nuclear structures than unphosphorylated RPA suggesting functional alterations in the protein. In an in vitro assay for DNA replication, DNA-PK is the sole kinase phosphorylating RPA2, indicating that processes not reproduced in the in vitro assay are required for RPA2 phosphorylation by ATM. Because RPA2 phosphorylation kinetics are distinct from those of the S phase checkpoint, we propose that DNA-PK and ATM cooperate to phosphorylate RPA after DNA damage to redirect the functions of the protein from DNA replication to DNA repair.     

Anti‐tumor activity of the ATR inhibitor AZD6738 in HER2 positive breast cancer cells

Ataxia telangiectasia and Rad3‐related (ATR) proteins are sensors of DNA damage, which induces homologous recombination (HR)‐dependent repair. ATR is a master regulator of DNA damage repair (DDR), signaling to control DNA replication, DNA repair and apoptosis. Therefore, the ATR pathway might be an attractive target for developing new drugs. This study was designed to investigate the antitumor effects of the ATR inhibitor, AZD6738 and its underlying mechanism in human breast cancer cells. Growth inhibitory effects of AZD6738 against human breast cancer cell lines were studied using a 3‐(4,5‐dimethylthiazol‐2‐yl)?2,5‐diphenyltetrazolium bromide (methyl thiazolyl tetrazolium, MTT) assay. Cell cycle analysis, Western blotting, immunofluorescence and comet assays were also performed to elucidate underlying mechanisms of AZD6738 action. Anti‐proliferative and DDR inhibitory effects of AZD6738 were demonstrated in human breast cancer cell lines. Among 13 cell lines, the IC₅₀ values of nine cell lines were less than 1 μmol/L using MTT assay. Two cell lines, SK‐BR‐3 and BT‐474, were chosen for further evaluation focused on human epidermal growth factor receptor 2 (HER2)‐positive breast cancer cells. Sensitive SK‐BR‐3 but not the less sensitive BT‐474 breast cancer cells showed increased level of apoptosis and S phase arrest and reduced expression levels of phosphorylated check‐point kinase 1 (CHK1) and other repair markers. Decreased functional CHK1 expression induced DNA damage accumulation due to HR inactivation. AZD6738 showed synergistic activity with cisplatin. Understanding the antitumor activity and mechanisms of AZD6738 in HER2‐positive breast cancer cells creates the possibility for future clinical trials targeting DDR in HER2‐positive breast cancer treatment.     

DNA-PK inhibition by NU7441 sensitizes breast cancer cells to ionizing radiation and doxorubicin

DNA-dependent protein kinase (DNA-PK) plays a key role in the repair of DNA double-strand breaks (DSBs) that are probably the most deleterious form of DNA damage. Inhibition of DNA-PK has been considered as an attractive approach to decrease resistance to therapeutically induced DNA DSBs. Ionizing radiation (IR) and doxorubicin, which induce DSBs, are used in the treatment of breast cancer. We determined the cellular concentration of DNA-PK and other DSB-activated kinases: ATM and ATR and the effect of DNA-PK inhibition by NU7441 on DNA repair, cell cycle, and survival after IR or doxorubicin treatment in three human breast cancer cell lines (MCF-7, MDA-MB-231, and T47D) representing different breast cancer subtypes. T47D cells had the highest expression of DNA-PKcs, ATM, and ATR and the most rapid rate of DNA DSB repair. IR caused a 10- to 16-fold increase in DNA-PK activity and two to threefold induction of ATM in all 3 cell lines. NU7441 inhibited IR-induced DNA-PK activity in all cell lines with IC₅₀s in the range 0.17–0.25 μM. NU7441 retarded the repair of DSB and significantly increased the sensitivity of all cell lines to IR (4- to 12-fold) and doxorubicin (3- to 13-fold). The greatest sensitiziation by NU7441 was observed in MDA-MB-231 cells. NU7441 affected the cell cycle distribution in all studied cell lines; increasing accumulation of cells in G2/M phase after DNA damage. Our data indicate that DNA-PK might be an effective target for chemo- and radio-potentiation in breast cancer and suggest that further development of DNA-PK inhibitors for clinical use is warranted.     

DNA Repair by Homologous Recombination, But Not by Nonhomologous End Joining, Is Elevated in Breast Cancer Cells

Aberrant double-stranded break (DSB) repair leads to genomic instability, which is a hallmark of malignant cells. Double-stranded breaks are repaired by two pathways: homologous recombination (HR) and nonhomologous DNA end joining (NHEJ). It is not known whether these repair pathways are affected in sporadic breast tumors. Here, we examined the efficiency of HR and NHEJ repair in a panel of sporadic breast cancer cell lines and tested whether the efficiency of HR or NHEJ correlates with radioresistance. Homologous recombination and NHEJ in breast cancer cells were analyzed using in vivo fluorescent assays. Unexpectedly, our analysis revealed that the efficiency of HR is significantly elevated in breast cancer cells compared with normal mammary epithelial cells. In contrast, the efficiency of NHEJ in breast cancer cells is not different from normal cells. Overall, breast cancer cells were more sensitive to radiation than normal cells, but the levels of resistance did not correlate with either HR or NHEJ efficiency. Thus, we demonstrate that sporadic breast cancers are not associated with a deficiency in DSB repair, but rather with upregulation of the HR pathway. Our finding of elevated HR in sporadic breast cancer cell lines suggests that therapies directed against the components of HR will be highly tumor-specific.     

The Dual PI3K/mTOR Inhibitor NVP-BEZ235 Is a Potent Inhibitor of ATM- and DNA-PKCs-Mediated DNA Damage Responses

Inhibitors of PI3K/Akt signaling are being actively developed for tumor therapy owing to the frequent mutational activation of the PI3K-Akt-mTORC1 pathway in many cancers, including glioblastomas (GBMs). NVP-BEZ235 is a novel and potent dual PI3K/mTOR inhibitor that is currently in phase 1/2 clinical trials for advanced solid tumors. Here, we show that NVP-BEZ235 also potently inhibits ATM and DNA-PKcs, the two major kinases responding to ionizing radiation (IR)-induced DNA double-strand breaks (DSBs). Consequently, NVP-BEZ235 blocks both nonhomologous end joining and homologous recombination DNA repair pathways resulting in significant attenuation of DSB repair. In addition, phosphorylation of ATMtargets and implementation of the G₂/M cell cycle checkpoint are also attenuated by this drug. As a result, NVP-BEZ235 confers an extreme degree of radiosensitization and impairs DSB repair in a panel of GBM cell lines irrespective of their Akt activation status. NVP-BEZ235 also significantly impairs DSB repair in a mouse tumor model thereby validating the efficacy of this drug as a DNA repair inhibitor in vivo. Our results, showing that NVP-BEZ235 is a potent and novel inhibitor of ATM and DNA-PKcs, have important implications for the informed and rational design of clinical trials involving this drug and also reveal the potential utility of NVP-BEZ235 as an effective radiosensitizer for GBMs in the clinic.     

The role of DNA damage and repair in decitabine-mediated apoptosis in multiple myeloma

DNA methyltransferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi) are under investigation for the treatment of cancer, including the plasma cell malignancy multiple myeloma (MM). Evidence exists that DNA damage and repair contribute to the cytotoxicity mediated by the DNMTi decitabine. Here, we investigated the DNA damage response (DDR) induced by decitabine in MM using 4 human MM cell lines and the murine 5T33MM model. In addition, we explored how the HDACi JNJ-26481585 affects this DDR. Decitabine induced DNA damage (gamma-H2AX foci formation), followed by a G0/G1- or G2/M-phase arrest and caspase-mediated apoptosis. JNJ-26481585 enhanced the anti-MM effect of decitabine both in vitro and in vivo. As JNJ-26481585 did not enhance decitabine-mediated gamma-H2AX foci formation, we investigated the DNA repair response towards decitabine and/or JNJ-26481585. Decitabine augmented RAD51 foci formation (marker for homologous recombination (HR)) and/or 53BP1 foci formation (marker for non-homologous end joining (NHEJ)). Interestingly, JNJ-26481585 negatively affected basal or decitabine-induced RAD51 foci formation. Finally, B02 (RAD51 inhibitor) enhanced decitabine-mediated apoptosis. Together, we report that decitabine-induced DNA damage stimulates HR and/or NHEJ. JNJ-26481585 negatively affects RAD51 foci formation, thereby providing an additional explanation for the combinatory effect between decitabine and JNJ-26481585.     

Inhibition of the DNA-dependent protein kinase catalytic subunit radiosensitizes malignant glioma cells by inducing autophagy

DNA-dependent protein kinase (DNA-PK) plays a major role in the repair of DNA double-strand breaks induced by ionizing radiation (IR). Lack of DNA-PK causes defective DNA double-strand break repair and radiosensitization. In general, the cell death induced by IR is considered to be apoptotic. On the other hand, nonapoptotic cell death, autophagy, has recently attracted attention as a novel response of cancer cells to chemotherapy and IR. Autophagy is a protein degradation system characterized by a prominent formation of double-membrane vesicles in the cytoplasm. Little is known, however, regarding the relationship between DNA-PK and IR-induced autophagy. In the present study, we used human malignant glioma M059J and M059K cells to investigate the role of DNA-PK in IR-induced apoptotic and autophagic cell death. Low-dose IR induced massive autophagic cell death in M059J cells that lack the catalytic subunit of DNA-PK (DNA-PKcs). Most M059K cells, the counterpart of M059J cells in which DNA-PKcs are expressed at normal levels, survived, and proliferated although a small portion of the cells underwent apoptosis. Low-dose IR inhibited the phosphorylation of p70(S6K), a molecule downstream of the mammalian target of rapamycin associated with autophagy in M059J cells but not in M059K cells. The treatment of M059K cells with antisense oligonucleotides against DNA-PKcs caused radiation-induced autophagy and radiosensitized the cells. Furthermore, antisense oligonucleotides against DNA-PKcs radiosensitized other malignant glioma cell lines with DNA-PK activity, U373-MG and T98G, by inducing autophagy. The specific inhibition of DNA-PKcs may be promising as a new therapy to radiosensitize malignant glioma cells by inducing autophagy.     

Severe combined immunodeficient cells expressing mutant hRAD54 exhibit a marked DNA double-strand break repair and error-prone chromosome repair defect

DNA double-strand breaks (DSBs) can be induced by a number of endogenous and exogenous agents and are lethal events if left unrepaired. DNA DSBs can be repaired by homologous recombination (HR) and nonhomologous end joining (NHEJ). In mammals and higher eukaryotes, NHEJ is thought to be the primary pathway for repair, but the role for each pathway in DNA DSB repair has not been fully elucidated. To define the relative contributions of HR and NHEJ in mammalian DNA DSB repair, cells defective in both pathways were produced. Double-mutant cells were created by expressing a dominant mutant hRAD54 protein in a DNA-dependent protein kinase (DNA-PK)-deficient severe combined immunodeficient cell line. Double-mutant cells demonstrate an increase in ionizing radiation sensitivity and a decrease in DNA DSB repair as compared with either single mutant, whereas single-mutant hRAD54 cells exhibit a wild-type phenotype. Unexpectedly, DNA-PK-null cells were more resistant to mitomycin-C damage than were wild-type cells. Chromosome aberration analysis reveals numerous incomplete chromatid exchange aberrations in the majority of the double-mutant cells after ionizing radiation exposure. Our findings confirm a role for HR in DSB repair in higher eukaryotes, yet indicate that its role is not evident unless the primary repair pathway, NHEJ, is nonfunctional. Mitomycin-C resistance in DNA-PK-null cells compared with wild-type cells suggests that the HR pathway may be more efficient in cross-link repair in the absence of NHEJ. Lastly, the incorrectly repaired chromatid damage observed in double-mutant cells may result from failed recombination or another error-prone repair process that is apparent in the absence of the two primary repair pathways.     

In normal human fibroblasts variation in DSB repair capacity cannot be ascribed to radiation-induced changes in the localisation, expression or activity of major NHEJ proteins.

BACKGROUND AND PURPOSE: The aim of the present study was to test whether for normal human fibroblasts the variation in double-strand break (DSB) repair capacity results from radiation-induced differences in localisation, expression or activity of major non-homologous end-joining (NHEJ) proteins. MATERIALS AND METHODS: Experiments were performed with 11 normal human fibroblast strains AF01-11. NHEJ proteins were determined by Western blot and DNA-PK activity by pulldown-assay. RESULTS: The four NHEJ proteins tested (Ku70, Ku80, XRCC4 and DNA-PKcs) were found to be localised almost exclusively in the nucleus with no detectable amount in the cytoplasm. This distribution was not altered upon irradiation. In non-irradiated cells the level of these proteins varied with a CV ranging between 16% and 20%, but there was no correlation with the respective cellular DSB repair capacity. Irradiation (3.5 and 15 Gy) did not alter the expression of these proteins and there was also no change in the DNA-PK activity. These results indicate that the variation in DSB repair capacity determined for these fibroblasts can be ascribed to differences neither in the localisation or expression of Ku70, Ku80 and XRCC4 nor in the activity of the DNA-PK complex induced upon irradiation. CONCLUSIONS: For normal human fibroblasts, the level or activity of NHEJ proteins measured prior to or after irradiation cannot be used to predict the DSB repair capacity or cellular radiosensitivity.     

Characterization of LGALS3 (galectin-3) as a player in DNA damage response

DNA damage repair (DDR) is an orchestrated process encompassing the injury detection to its complete resolution. DNA double-strand break lesions are repaired mainly by two distinct mechanisms: the error-free homologous recombination (HR) and the error-prone non-homologous end-joining. Galectin-3 (GAL3) is the unique member of the chimeric galectins subfamily and is reported to be involved in several cancer development and progression related events. Recently our group described a putative protein interaction between GAL3 and BARD1, the main partner of breast and ovarian cancer susceptibility gene product BRCA1, both involved in HR pathway. In this report we characterized GAL3/BARD1 protein interaction and evaluated the role of GAL3 in DDR pathways using GAL3 silenced human cells exposed to different DNA damage agents. In the absence of GAL3 we observed a delayed DDR response activation, as well as a decrease in the G₂/M cell cycle checkpoint arrest associated with HR pathway. Moreover, using a TAP-MS approach we also determined the protein interaction network of GAL3.     

DNA protein kinase-dependent G2 checkpoint revealed following knockdown of ataxia-telangiectasia mutated in human mammary epithelial cells

Members of the phosphatidylinositol 3-kinase-related kinase family, in particular the ataxia-telangiectasia mutated (ATM) kinase and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), regulate cellular responses to DNA double-strand breaks. Increased sensitivity to ionizing radiation (IR) in DNA-PKcs- or ATM-deficient cells emphasizes their important roles in maintaining genome stability. Furthermore, combined knockout of both kinases is synthetically lethal, suggesting functional complementarity. In the current study, using human mammary epithelial cells with ATM levels stably knocked down by >90%, we observed an IR-induced G(2) checkpoint that was only slightly attenuated. In marked contrast, this G(2) checkpoint was significantly attenuated with either DNA-PK inhibitor treatment or RNA interference knockdown of DNA-PKcs, the catalytic subunit of DNA-PK, indicating that DNA-PK contributes to the G(2) checkpoint in these cells. Furthermore, in agreement with the checkpoint attenuation, DNA-PK inhibition in ATM-knockdown cells resulted in reduced signaling of the checkpoint kinase CHK1 as evidenced by reduced CHK1 phosphorylation. Taken together, these results show a DNA-PK-dependent component to the IR-induced G(2) checkpoint, in addition to the well-defined ATM-dependent component. This may have important implications for chemotherapeutic strategies for breast cancers.     

Histone acetylation by CBP and p300 at double-strand break sites facilitates SWI/SNF chromatin remodeling and the recruitment of non-homologous end joining factors

Non-homologous end joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs) generated by ionizing radiation (IR) and anti-cancer drugs. Therefore, inhibiting the activity of proteins involved in this pathway is a promising way of sensitizing cancer cells to both radiotherapy and chemotherapy. In this study, we developed an assay for evaluating NHEJ activity against DSBs in chromosomal DNA in human cells to identify the chromatin modification/remodeling proteins involved in NHEJ. We showed that ablating the activity of the homologous histone acetyltransferases, CBP and p300, using inhibitors or small interfering RNAs-suppressed NHEJ. Ablation of CBP or p300 impaired IR-induced DSB repair and sensitized lung cancer cells to IR and the anti-cancer drug, etoposide, which induces DSBs that are repaired by NHEJ. The CBP/p300 proteins were recruited to sites of DSBs and their ablation suppressed acetylation of lysine 18 within histone H3, and lysines 5, 8, 12, and 16 within histone H4, at the DSB sites. This then suppressed the recruitment of KU70 and KU80, both key proteins for NHEJ, to the DSB sites. Ablation of CBP/p300 also impaired the recruitment of BRM, a catalytic subunit of the SWI/SNF complex involved in chromatin remodeling at DSB sites. These results indicate that CBP and p300 function as histone H3 and H4 acetyltransferases at DSB sites in NHEJ and facilitate chromatin relaxation. Therefore, inhibition CBP and p300 activity may sensitize cancer cells to radiotherapy and chemotherapy.     

Novel cross-talk between IGF-IR and DDR1 regulates IGF-IR trafficking,signaling and biological responses

The insulin-like growth factor-I receptor (IGF-IR), plays a key role in regulating mammalian development and growth, and is frequently deregulated in cancer contributing to tumor initiation and progression. Discoidin domain receptor 1 (DDR1), a collagen receptor tyrosine-kinase, is as well frequently overexpressed in cancer and implicated in cancer progression. Thus, we investigated whether a functional cross-talk between the IGF-IR and DDR1 exists and plays any role in cancer progression.Using human breast cancer cells we found that DDR1 constitutively associated with the IGF-IR. However, this interaction was enhanced by IGF-I stimulation, which promoted rapid DDR1 tyrosine-phosphorylation and co-internalization with the IGF-IR. Significantly, DDR1 was critical for IGF-IR endocytosis and trafficking into early endosomes, IGF-IR protein expression and IGF-I intracellular signaling and biological effects, including cell proliferation, migration and colony formation. These biological responses were inhibited by DDR1 silencing and enhanced by DDR1 overexpression.Experiments in mouse fibroblasts co-transfected with the human IGF-IR and DDR1 gave similar results and indicated that, in the absence of IGF-IR, collagen-dependent phosphorylation of DDR1 is impaired.These results demonstrate a critical role of DDR1 in the regulation of IGF-IR action, and identify DDR1 as a novel important target for breast cancers that overexpress IGF-IR.     

Alternative cyclin D1 splice forms differentially regulate the DNA damage response

The DNA damage response (DDR) activates downstream pathways including cell cycle checkpoints. The cyclin D1 gene is overexpressed or amplified in many human cancers and is required for gastrointestinal, breast, and skin tumors in murine models. A common polymorphism in the human cyclin D1 gene is alternatively spliced, resulting in cyclin D1a and D1b proteins that differ in their carboxyl terminus. Cyclin D1 overexpression enhances DNA damage-induced apoptosis. The role of cyclin D1 and the alternative splice form in regulating the DDR is not well understood. Herein cyclin D1a overexpression enhanced the DDR as characterized by induction of γH2AX phosphorylation, the assembly of DNA repair foci, specific recruitment of DNA repair factors to chromatin, and G(2)-M arrest. Cyclin D1 deletion in fibroblasts or small interfering RNA-mediated reduction of endogenous cyclin D1 in colon cancer cells reduced the 5-fluorouracil-mediated DDR. Mechanistic studies showed that cyclin D1a, like DNA repair factors, elicited the DDR when stably associated with chromatin.