Junran Zhang

The mTOR inhibitor rapamycin suppresses DNA double-strand break repair.

Abstract mTOR (mammalian target of rapamycin) signaling plays a key role in the development of many tumor types. Therefore, mTOR is an attractive target for cancer therapeutics. Although mTOR inhibitors are thought to have radiosensitization activity, the molecular bases remain largely unknown. Here we show that treating MCF7 breast cancer cells with rapamycin (an mTOR inhibitor) results in significant suppression of homologous recombination (HR) and nonhomologous end joining (NHEJ), two major mechanisms required for repairing ionizing radiation-induced DNA DSBs. We observed that rapamycin impaired recruitment of BRCA1 and Rad51 to DNA repair foci, both essential for HR. Moreover, consistent with the suppressive role of rapamycin on both HR and NHEJ, persistent radiation-induced DSBs were detected in cells pretreated with rapamycin. Furthermore, the frequency of chromosome and chromatid breaks was increased in cells treated with rapamycin before and after irradiation. Thus our results show that radiosensitization by mTOR inhibitors occurs via disruption of the major two DNA DSB repair pathways.

The role of RPA2 phosphorylation in homologous recombination in response to replication arrest.

Failure to reactivate stalled or collapsed DNA replication forks is a potential source of genomic instability. Homologous recombination (HR) is a major mechanism for repairing the DNA damage resulting from replication arrest. The single-strand DNA (ssDNA)-binding protein, replication protein A (RPA), plays a major role in multiple processes of DNA metabolism. However, the role of RPA2 hyperphosphorylation, which occurs in response to DNA damage, had been unclear. Here, we show that hyperphosphorylated RPA2 associates with ssDNA and recombinase protein Rad51 in response to replication arrest by hydroxyurea (HU) treatment. In addition, RPA2 hyperphosphorylation is critical for Rad51 recruitment and HR-mediated repair following HU. However, RPA2 hyperphosphorylation is not essential for both ionizing radiation (IR)-induced Rad51 foci formation and I-Sce-I endonuclease-stimulated HR. Moreover, we show that expression of a phosphorylation-deficient mutant of RPA2 leads to increased chromosomal aberrations following HU treatment but not after exposure to IR. Finally, we demonstrate that loss of RPA2 hyperphosphorylation results in a loss of viability when cells are confronted with replication stress whereas cells expressing hyperphosphorylation-defective RPA2 or wild-type RPA2 have a similar sensitivity to IR. Thus, our data suggest that RPA2 hyperphosphorylation plays a critical role in maintenance of genomic stability and cell survival after a DNA replication block via promotion of HR.

Loss of ATF3 Promotes Akt Activation and Prostate Cancer Development in a Pten Knockout Mouse Model

Activating transcription factor 3 (ATF3) responds to diverse cellular stresses, and regulates oncogenic activities (for example, proliferation, survival and migration) through direct transcriptional regulation or protein-protein interactions. Although aberrant ATF3 expression is frequently found in human cancers, the role of ATF3 in tumorigenesis is poorly understood. Here, we demonstrate that ATF3 suppresses the development of prostate cancer induced by knockout of the tumor suppressor Pten in mouse prostates. Whereas the oncogenic stress elicited by Pten loss induced ATF3 expression in prostate epithelium, we found that ATF3 deficiency increased cell proliferation and promoted cell survival, leading to early onset of mouse prostatic intraepithelial neoplasia and the progression of prostate lesions to invasive adenocarcinoma. Importantly, the loss of ATF3 promoted activation of the oncogenic AKT signaling evidenced by high levels of phosphorylated AKT and S6 proteins in ATF3-null prostate lesions. In line with these in vivo results, knockdown of ATF3 expression in human prostate cancer cells by single guided RNA-mediated targeting activated AKT and increased matrix metalloproteinase-9 expression. Our results thus link ATF3 to the AKT signaling, and suggest that ATF3 is a tumor suppressor for the major subset of prostate cancers harboring dysfunctional Pten.

53BP1 promotes microhomology-mediated end-joining in G1-phase cells

Alternative non-homologous end joining (alt-NHEJ) was originally identified as a backup repair mechanism in the absence of classical NHEJ (c-NHEJ) factors but recent studies have demonstrated that alt-NHEJ is active even when c-NHEJ as well as homologous recombination is available. The functions of 53BP1 in NHEJ processes are not well understood. Here, we report that 53BP1 promotes DNA double-strand break (DSB) repair and genomic stability not only in c-NHEJ-proficient but also -deficient human G1-phase cells. Using an array of repair substrates we show that these effects of 53BP1 are correlated with a promotion of microhomology-mediated end-joining (MMEJ), a subtype of alt-NHEJ, in G1-phase. Consistent with a specific role in MMEJ we confirm that 53BP1 status does not affect c-NHEJ. 53BP1 supports sequence deletion during MMEJ consistent with a putative role in facilitating end-resection. Interestingly, promotion of MMEJ by 53BP1 in G1-phase cells is only observed in the presence of functional BRCA1. Depletion of both 53BP1 and BRCA1 increases repair needing microhomology usage and augments loss of DNA sequence, suggesting that MMEJ is a highly regulated DSB repair process. Together, these findings significantly expand our understanding of the cell-cycle-dependent roles of 53BP1 in DSB repair.

The stress-responsive gene ATF3 regulates the histone acetyltransferase Tip60

Tat-interactive protein 60 (Tip60) is a MYST histone acetyltransferase that catalyzes acetylation of the major DNA damage kinase ATM, thereby triggering cellular signaling required for the maintenance of genomic stability upon genotoxic insults. The Tip60 activity is modulated by posttranslational modifications that alter its stability and its interactions with substrates. Here we report that activating transcription factor 3 (ATF3), a common stress mediator and a p53 activator, is a regulator of Tip60. ATF3 directly binds Tip60 at a region adjacent to the catalytic domain to promote the protein acetyltransferase activity. Moreover, the ATF3-Tip60 interaction increases the Tip60 stability by promoting USP7-mediated deubiquitination of Tip60. Consequently, knockdown of ATF3 expression leads to decreased Tip60 expression and suppression of ATM signaling as evidenced by accumulated DNA lesions and increased cell sensitivity to irradiation. Our findings thus reveal a previously unknown function of a common stress mediator in regulating Tip60 function.

RNF126 promotes homologous recombination via regulation of E2F1-mediated BRCA1 expression.

RNF126 is an E3 ubiquitin ligase. The deletion of RNF126 gene was observed in a wide range of human cancers and is correlated with improved disease-free and overall survival. These data highlight the clinical relevance of RNF126 in tumorigenesis and cancer therapy. However, the specific functions of RNF126 remain largely unknown. Homologous recombination (HR)-mediated DNA double-strand break repair is important for tumor suppression and cancer therapy resistance. Here, we demonstrate that RNF126 facilitates HR by promoting the expression of BRCA1, in a manner independent of its E3 ligase activity but depending on E2F1, a well-known transcription factor of BRCA1 promoter. In support of this result, RNF126 promotes transactivation of BRCA1 promoter by directly binding to E2F1. Most importantly, an RNF126 mutant lacking 11 amino acids that is responsible for the interaction with E2F1 has a dominant-negative effect on BRCA1 expression and HR by suppressing E2F1-mediated transactivation of BRCA1 promoter and blocking the enrichment of E2F1 on BRCA1 promoter. Lastly, RNF126 depletion leads to the increased sensitivity to ionizing radiation and poly (ADP-ribose) polymerase inhibition. Collectively, our results suggest a novel role of RNF126 in promoting HR-mediated repair through positive regulation on BRCA1 expression by direct interaction with E2F1. This study not only offers novel insights into our current understanding of the biological functions of RNF126 but also provides a potential therapeutic target for cancer treatment.

Targeting radioresistant breast cancer cells by single agent CHK1 inhibitor via enhancing replication stress

Radiotherapy (RT) remains a standard therapeutic modality for breast cancer patients. However, intrinsic or acquired resistance limits the efficacy of RT. Here, we demonstrate that CHK1 inhibitor AZD7762 alone significantly inhibited the growth of radioresistant breast cancer cells (RBCC). Given the critical role of ATR/CHK1 signaling in suppressing oncogene-induced replication stress (RS), we hypothesize that CHK1 inhibition leads to the specific killing for RBCC due to its abrogation in the suppression of RS induced by oncogenes. In agreement, the expression of oncogenes c-Myc/CDC25A/c-Src/H-ras/E2F1 and DNA damage response (DDR) proteins ATR/CHK1/BRCA1/CtIP were elevated in RBCC. AZD7762 exposure led to significantly higher levels of RS in RBCC, compared to the parental cells. The mechanisms by which CHK1 inhibition led to specific increase of RS in RBCC were related to the interruptions in the replication fork dynamics and the homologous recombination (HR). In summary, RBCC activate oncogenic pathways and thus depend upon mechanisms controlled by CHK1 signaling to maintain RS under control for survival. Our study provided the first example where upregulating RS by CHK1 inhibitor contributes to the specific killing of RBCC, and highlight the importance of the CHK1 as a potential target for treatment of radioresistant cancer cells.

Vitamin D/vitamin D receptor axis regulates DNA repair during oncogene-induced senescence.

Oncogenic Ras expression is associated with activation of the DNA damage response (DDR) pathway, as evidenced by elevated DNA damage, primarily DNA double-strand breaks (DSBs), and activation of DNA damage checkpoints, which in primary human cells leads to entry into senescence. DDR activation is viewed as a physiological barrier against uncontrolled proliferation in oncogenic Ras-expressing cells, and arises in response to genotoxic stress due to the production of reactive oxygen species that damage DNA and to hyper-replication stress. Although oncogene-induced senescence (OIS) is considered a tumor suppressor mechanism, the accumulation of DNA damage in senescent cells is thought to cause genomic instability, eventually allowing secondary hits in the genome that promote tumorigenesis. To date, the molecular mechanisms behind DNA repair defects during OIS remain poorly understood. Here, we show that oncogenic Ras expression in human primary cells results in the downregulation of BRCA1 and 53BP1, two key factors in DNA DSB repair by homologous recombination and non-homologous end joining, respectively. As a consequence, Ras-induced senescent cells are hindered in their ability to recruit BRCA1 and 53BP1 to DNA damage sites. Whereas BRCA1 is downregulated at transcripts levels, 53BP1 loss is caused by activation of cathepsin L-mediated degradation of 53BP1 protein. Moreover, we discovered a marked downregulation of vitamin D receptor (VDR) during OIS, and a role for the vitamin D/VDR axis regulating the levels of these DNA repair factors during OIS. This study reveals a new functional relationship between the oncogene Ras, the vitamin D/VDR axis and the expression of DNA repair factors, in the context of OIS. The observed deficiencies in DNA repair factors in senescent cells could contribute to the genomic instability that allows senescence bypass and tumorigenesis.

Activation of the Constitutive Androstane Receptor Increases the Therapeutic Index of CHOP in Lymphoma Treatment

The constitutive androstane receptor (CAR and NR1i3) is a key regulator of CYP2B6, the enzyme predominantly responsible for the biotransformation of cyclophosphamide (CPA) to its pharmacologically active metabolite, 4-hydroxycyclophosphamide (4-OH-CPA). Previous studies from our laboratory illustrated that CAR activation increases the formation of 4-OH-CPA; however, CPA is rarely used clinically outside of combination therapies. Here, we hypothesize that including a selective human CAR activator with the CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) regimen can improve the efficacy without exacerbating off-target toxicity of this regimen in non-Hodgkin lymphoma treatment. In this study, we have developed a novel multiorgan coculture system containing human primary hepatocytes for hepatic metabolism, lymphoma cells as a model target for CHOP, and cardiomyocytes as a major site of off-target toxicity associated with this regimen. We found that a selective human CAR activator, CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime), altered expression of key drug-metabolizing enzymes and transporters in human hepatocytes, which positively affects the metabolic profile of CHOP. Coadministration of CITCO and CHOP in the coculture model led to significantly enhanced cytotoxicity in lymphoma cells but not in cardiomyocytes. Moreover, the beneficial effects of CITCO were abrogated when CAR knockout HepaRG cells were used in the coculture model. Importantly, synergistic anticancer effects were observed between CITCO and CHOP, in that inclusion of CITCO alongside the CHOP regimen offers comparable antineoplastic activity toward lymphoma cells at significantly reduced drug concentrations, and the decreased CHOP load attenuates cardiotoxicity. Overall, these findings provide a potentially promising novel strategy for facilitating CHOP-based chemotherapy. Mol Cancer Ther; 15(3); 392–401. ©2016 AACR.

Radiosensitization of non-small-cell lung cancer cells and xenografts by the interactive effects of pemetrexed and methoxyamine

BACKGROUND AND PURPOSE The anti-folate pemetrexed is a radiosensitizer. In pre-clinical models, pemetrexed is more effective along with the base-excision-repair inhibitor methoxyamine. We tested whether methoxyamine enhances pemetrexed-mediated radiosensitization of lung adenocarcinoma cells and xenografts. MATERIALS AND METHODS A549 and H1299 cells were evaluated for cell cycle distribution by flow cytometry, radiosensitization by clonogenic assay, and DNA repair by neutral comet assay and repair protein activation. H460 cells were included in some studies. Xenografts in nude mice received drug(s) and/or radiation, and tumor growth was monitored by caliper and in vivo toxicity by animal weight. RESULTS Exposure to pemetrexed/methoxyamine for 24 (H1299, H460) or 48 (A549)hours before irradiation resulted in accumulation of cells near the radiosensitive G1/S border; dose-enhancement factors of 1.62±0.19, 1.97±0.25, and 1.67±0.30, respectively; reduction of mean inactivation dose by 32%, 30%, and 46%, respectively; and significant reductions of SF2 and SF4 (p<0.05). Radiosensitization was associated with rapid DNA double-strand-break rejoining and increased levels of DNA-PKcs. Both tumor-growth rate and tumor-growth delay were significantly improved by adding methoxyamine to pemetrexed pre-irradiation (p<0.0001); no mice lost weight during treatment. CONCLUSIONS Addition of methoxyamine to pemetrexed and fractionated radiotherapy may improve outcome for patients with locally advanced non-squamous non-small-cell lung cancer.