APLF facilitates interstrand DNA crosslink repair and replication fork protection to confer cisplatin resistance.

Replication stress converts the stalled forks into reversed forks, which is an important protection mechanism to prevent fork degradation and collapse into poisonous DNA double-strand breaks (DSBs). Paradoxically, the mechanism also acts in cancer cells to contribute to chemoresistance against various DNA-damaging agents. PARP1 binds to and is activated by stalled forks to facilitate fork reversal. Aprataxin and polynucleotide kinase/phosphatase-like factor (APLF) binds to PARP1 through the poly(ADP-ribose) zinc finger (PBZ) domain and is known to be involved in non-homologous end joining (NHEJ). Here, we identify a novel function of APLF involved in interstrand DNA crosslink (ICL) repair and fork protection. We demonstrate that PARP1 activity facilitates the APLF recruitment to stalled forks, enabling the FANCD2 recruitment to stalled forks. The depletion of APLF sensitizes cells to cisplatin, impairs ICL repair, reduces the FANCD2 recruitment to stalled forks, and results in nascent DNA degradation by MRE11 nucleases. Additionally, cisplatin-resistant cancer cells show high levels of APLF and homologous recombination-related gene expression. The depletion of APLF sensitizes cells to cisplatin and results in fork instability. Our results reveal the novel function of APLF to facilitate ICL repair and fork protection, thereby contributing to cisplatin-resistant phenotypes of cancer cells.

RAD51 paralogs synergize with RAD51 to protect reversed forks from cellular nucleases.

Fork reversal is a conserved mechanism to prevent stalled replication forks from collapsing. Formation and protection of reversed forks are two crucial steps in ensuring fork integrity and stability. Five RAD51 paralogs, namely, RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, which share sequence and structural similarity to the recombinase RAD51, play poorly defined mechanistic roles in these processes. Here, using purified BCDX2 (RAD51BCD-XRCC2) and CX3 (RAD51C-XRCC3) complexes and in vitro reconstituted biochemical systems, we mechanistically dissect their functions in forming and protecting reversed forks. We show that both RAD51 paralog complexes lack fork reversal activities. Whereas CX3 exhibits modest fork protection activity, BCDX2 significantly synergizes with RAD51 to protect DNA against attack by the nucleases MRE11 and EXO1. DNA protection is contingent upon the ability of RAD51 to form a functional nucleoprotein filament on DNA. Collectively, our results provide evidence for a hitherto unknown function of RAD51 paralogs in synergizing with RAD51 nucleoprotein filament to prevent degradation of stressed replication forks.

PARP1 recruits DNA translocases to restrain DNA replication and facilitate DNA repair.

Replication fork reversal which restrains DNA replication progression is an important protective mechanism in response to replication stress. PARP1 is recruited to stalled forks to restrain DNA replication. However, PARP1 has no helicase activity, and the mechanism through which PARP1 participates in DNA replication restraint remains unclear. Here, we found novel protein-protein interactions between PARP1 and DNA translocases, including HLTF, SHPRH, ZRANB3, and SMARCAL1, with HLTF showing the strongest interaction among these DNA translocases. Although HLTF and SHPRH share structural and functional similarity, it remains unclear whether SHPRH contains DNA translocase activity. We further identified the ability of SHPRH to restrain DNA replication upon replication stress, indicating that SHPRH itself could be a DNA translocase or a helper to facilitate DNA translocation. Although hydroxyurea (HU) and MMS induce different types of replication stress, they both induce common DNA replication restraint mechanisms independent of intra-S phase activation. Our results suggest that the PARP1 facilitates DNA translocase recruitment to damaged forks, preventing fork collapse and facilitating DNA repair.

CHK2 activation contributes to the development of oxaliplatin resistance in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC), the most common cancer type, causes high morbidity and mortality. Patients who develop drug resistance to oxaliplatin-based regimens have short overall survival. Thus, identifying molecules involved in the development of oxaliplatin resistance is critical for designing therapeutic strategies. METHODS: A proteomic screen was performed to reveal altered protein kinase phosphorylation in oxaliplatin-resistant (OR) CRC tumour spheroids. The function of CHK2 was characterised using several biochemical techniques and evident using in vitro cell and in vivo tumour models. RESULTS: We revealed that the level of phospho-CHK2(Thr68) was elevated in OR CRC cells and in ~30% of tumour samples from patients with OR CRC. We demonstrated that oxaliplatin activated several phosphatidylinositol 3-kinase-related kinases (PIKKs) and CHK2 downstream effectors and enhanced CHK2/PARP1 interaction to facilitate DNA repair. A phosphorylation mimicking CHK2 mutant, CHK2T68D, but not a kinase-dead CHK2 mutant, CHK2D347A, promoted DNA repair, the CHK2/PARP1 interaction, and cell growth in the presence of oxaliplatin. Finally, we showed that a CHK2 inhibitor, BML-277, reduced protein poly(ADP-ribosyl)ation (PARylation), FANCD2 monoubiquitination, homologous recombination and OR CRC cell growth in vitro and in vivo. CONCLUSION: Our findings suggest that CHK2 activity is critical for modulating oxaliplatin response and that CHK2 is a potential therapeutic target for OR CRC.

SHPRH regulates rRNA transcription by recognizing the histone code in an mTOR-dependent manner.

Many DNA repair proteins have additional functions other than their roles in DNA repair. In addition to catalyzing PCNA polyubiquitylation in response to the stalling of DNA replication, SHPRH has the additional function of facilitating rRNA transcription by localizing to the ribosomal DNA (rDNA) promoter in the nucleoli. SHPRH was recruited to the rDNA promoter using its plant homeodomain (PHD), which interacts with histone H3 when the fourth lysine of H3 is not trimethylated. SHPRH enrichment at the rDNA promoter was inhibited by cell starvation, by treatment with actinomycin D or rapamycin, or by depletion of CHD4. SHPRH also physically interacted with the RNA polymerase I complex. Taken together, we provide evidence that SHPRH functions in rRNA transcription through its interaction with histone H3 in a mammalian target of rapamycin (mTOR)-dependent manner.

Ecological genomics in Xanthomonas: the nature of genetic adaptation with homologous recombination and host shifts.

BACKGROUND: Comparative genomics provides insights into the diversification of bacterial species. Bacterial speciation usually takes place with lasting homologous recombination, which not only acts as a cohering force between diverging lineages but brings advantageous alleles favored by natural selection, and results in ecologically distinct species, e.g., frequent host shift in Xanthomonas pathogenic to various plants. RESULTS: Using whole-genome sequences, we examined the genetic divergence in Xanthomonas campestris that infected Brassicaceae, and X. citri, pathogenic to a wider host range. Genetic differentiation between two incipient races of X. citri pv. mangiferaeindicae was attributable to a DNA fragment introduced by phages. In contrast to most portions of the genome that had nearly equivalent levels of genetic divergence between subspecies as a result of the accumulation of point mutations, 10% of the core genome involving with homologous recombination contributed to the diversification in Xanthomonas, as revealed by the correlation between homologous recombination and genomic divergence. Interestingly, 179 genes were under positive selection; 98 (54.7%) of these genes were involved in homologous recombination, indicating that foreign genetic fragments may have caused the adaptive diversification, especially in lineages with nutritional transitions. Homologous recombination may have provided genetic materials for the natural selection, and host shifts likely triggered ecological adaptation in Xanthomonas. To a certain extent, we observed positive selection nevertheless contributed to ecological divergence beyond host shifting. CONCLUSION: Altogether, mediated with lasting gene flow, species formation in Xanthomonas was likely governed by natural selection that played a key role in helping the deviating populations to explore novel niches (hosts) or respond to environmental cues, subsequently triggering species diversification.

The Yeast Nucleosome Atlas (YNA) database: an integrative gene mining platform for studying chromatin structure and its regulation in yeast.

BACKGROUND: Histone modification and remodeling play crucial roles in regulating gene transcription. These post-translational modifications of histones function in a combinatorial fashion and can be recognized by specific histone-binding proteins, thus regulating gene transcription. Therefore, understanding the combinatorial patterns of the histone code is vital to understanding the associated biological processes. However, most of the datasets regarding histone modification and chromatin regulation are scattered across various studies, and no comprehensive search and query tool has yet been made available to retrieve genes bearing specific histone modification patterns and regulatory proteins. DESCRIPTION: For this reason, we developed the Yeast Nucleosome Atlas database, or the YNA database, which integrates the available experimental data on nucleosome occupancy, histone modifications, the binding occupancy of regulatory proteins, and gene expression data, and provides the genome-wide gene miner to retrieve genes with a specific combination of these chromatin-related datasets. Moreover, the biological significance analyzer, which analyzes the enrichments of histone modifications, binding occupancy, transcription rate, and functionality of the retrieved genes, was constructed to help researchers to gain insight into the correlation among chromatin regulation and transcription. CONCLUSIONS: Compared to previously established genome browsing databases, YNA provides a powerful gene mining and retrieval interface, and is an investigation tool that can assist users to generate testable hypotheses for studying chromatin regulation during transcription. YNA is available online at http://cosbi3.ee.ncku.edu.tw/yna/.

Retinoic acid mediates the expression of glutamate transporter-1 in rat astrocytes through genomic RXR action and non-genomic protein kinase C signaling pathway.

Astrocytic glutamate transporter-1 (GLT-1) is responsible for 90% of forebrain glutamate uptake in the adult CNS. Retinoic acid (RA) is a potent regulator of neural cell differentiation and neuronal maturation in the developing CNS through activation of RA receptors/retinoic X receptors (RXRs) or non-genomic mechanisms. Although rat GLT-1 contains several RXR binding regions, RA-triggered RXR mechanisms regulating GLT-1 expression remain unknown. RA applied at submicromolar concentrations for 24 h significantly reduced GLT-1 mRNA and membrane levels in astrocytes and dibutyryl cAMP (dbcAMP)-primed astrocytes. An RXR agonist reduced astrocytic GLT-1 mRNA expression, whereas an RXR antagonist blocked the effects of RA on the reduction of astrocytic GLT-1 mRNA expression. Electrophoresis motility shift assay indicated that RA-treatment increased astrocytic RXR-DNA binding activity. RA-induced reduction in GLT-1 mRNA expression was also observed in dbcAMP-primed astrocytes. Through lentivirus-mediated astrocytic over-expression of rat GLT-1, levels of GLT-1 in the processes of dbcAMP-treated astrocytes were attenuated by exposure to RA. The protein kinase C inhibitor, Bis I, restored GLT-1 distribution in the processes of RA-treated dbcAMP-primed astrocytes. These results suggest that RA reduces astrocytic GLT-1 levels through both RXR-mediated inhibition at the transcriptional level and triggering activation of protein kinase C which reduces cell surface GLT-1 levels.

BMP2 promotes lung adenocarcinoma metastasis through BMP receptor 2-mediated SMAD1/5 activation.

Bone morphogenetic protein 2 (BMP2) is highly overexpressed in human non-small cell lung cancer (NSCLC) and correlates with tumor stage and metastatic burden. Although several lines of evidence suggest that BMP2 promotes cell migration and invasiveness in vitro, the in vivo role of BMP2 in the metastasis of lung adenocarcinoma cells remains less well understood. Here, we revealed that BMP2 is highly overexpressed in lung adenocarcinoma patients with lymph node metastasis compared with patients without lymph node metastasis. Using an in vivo orthotopic mouse model, we clearly demonstrated that BMP2 promotes lung adenocarcinoma metastasis. The depletion of BMP2 or its receptor BMPR2 significantly reduced cell migration and invasiveness. We further identified that BMP2/BMPR2-mediated cell migration involves the activation of the SMAD1/5/8 signaling pathway, independent of the KRAS signaling pathway. Significantly, the depletion of SMAD1/5/8 or the inhibition of SMAD1/5/8 by LDN193189 inhibitor significantly reduced cell migration. These findings show that BMP2 promotes NSCLC metastasis, indicating that targeting the BMP2 signaling pathway may represent a potential therapeutic strategy for treating patients with metastatic NSCLC.

Polyubiquitination of proliferating cell nuclear antigen by HLTF and SHPRH prevents genomic instability from stalled replication forks.

Chronic stalling of DNA replication forks caused by DNA damage can lead to genomic instability. Cells have evolved lesion bypass pathways such as postreplication repair (PRR) to resolve these arrested forks. In yeast, one branch of PRR involves proliferating cell nuclear antigen (PCNA) polyubiquitination mediated by the Rad5-Ubc13-Mms2 complex that allows bypass of DNA lesion by a template-switching mechanism. Previously, we identified human SHPRH as a functional homologue of yeast Rad5 and revealed the existence of RAD5-like pathway in human cells. Here we report the identification of HLTF as a second RAD5 homologue in human cells. HLTF, like SHPRH, shares a unique domain architecture with Rad5 and promotes lysine 63-linked polyubiquitination of PCNA. Similar to yeast Rad5, HLTF is able to interact with UBC13 and PCNA, as well as SHPRH; and the reduction of either SHPRH or HLTF expression enhances spontaneous mutagenesis. Moreover, Hltf-deficient mouse embryonic fibroblasts show elevated chromosome breaks and fusions after methyl methane sulfonate treatment. Our results suggest that HLTF and SHPRH are functional homologues of yeast Rad5 that cooperatively mediate PCNA polyubiquitination and maintain genomic stability.

The HLTF-PARP1 interaction in the progression and stability of damaged replication forks caused by methyl methanesulfonate.

Human HLTF participates in the lesion-bypass mechanism through the fork reversal structure, known as template switching of post-replication repair. However, the mechanism by which HLTF promotes the replication progression and fork stability of damaged forks remains unclear. Here, we identify a novel protein-protein interaction between HLTF and PARP1. The depletion of HLTF and PARP1 increases chromosome breaks, further reduces the length of replication tracks, and concomitantly increases the number of stalled forks after methyl methanesulfonate treatment according to a DNA fiber analysis. The progression of replication also depends on BARD1 in the presence of MMS treatment. By combining 5-ethynyl-2'-deoxyuridine with a proximity ligation assay, we revealed that the HLTF, PARP1, and BRCA1/BARD1/RAD51 proteins were initially recruited to damaged forks. However, prolonged stalling of damaged forks results in fork collapse. HLTF and PCNA dissociate from the collapsed forks, with increased accumulation of PARP1 and BRCA1/BARD1/RAD51 at the collapsed forks. Our results reveal that HLTF together with PARP1 and BARD1 participates in the stabilization of damaged forks, and the PARP1-BARD1 interaction is further involved in the repair of collapse forks.

Sir3 C-terminal domain involvement in the initiation and spreading of heterochromatin.

Heterochromatin is nucleated at a specific site and subsequently spreads into distal sequences through multiple interactions between modified histones and nonhistone proteins. In the yeast Saccharomyces cerevisiae, these nonhistone proteins include Sir2, Sir3, and Sir4. We have previously shown that loss of the C-terminal Rap1 domain containing Sir3 and Sir4 association sites can be overcome by tethering a 144-amino-acid C-terminal domain (CTD) of Sir3 adjacent to the telomere. Here, we explore the substructure and functions of the CTD. We demonstrate that the CTD is the minimum domain for Sir3 homodimerization, a function that is conserved in related yeasts. However, CTD heterodimers associate at only low efficiencies and correspondingly have low levels of tethered silencing, consistent with an essential role for dimerization in tethered silencing. Six missense alleles were generated, with ctd-Y964A producing the most extreme phenotypes when tethered to the LexA binding sites. Although ctd-Y964A is capable of dimerization, telomere silencing is abrogated, indicating that the CTD serves a second essential function in silencing. Chromatin immunoprecipitation analyses of wild-type and ctd-Y964A mutant cells indicate an association of the CTD with the deacetylated histone tails of H3 and H4 that is necessary for the recruitment of Sir3. The efficiency of spreading depends upon the apparent stoichiometry and stability during the initiation event. The predicted Cdc6 domain III winged-helix structure may well be responsible for dimerization.

YHMI: a web tool to identify histone modifications and histone/chromatin regulators from a gene list in yeast.

Post-translational modifications of histones (e.g. acetylation, methylation, phosphorylation and ubiquitination) play crucial roles in regulating gene expression by altering chromatin structures and creating docking sites for histone/chromatin regulators. However, the combination patterns of histone modifications, regulatory proteins and their corresponding target genes remain incompletely understood. Therefore, it is advantageous to have a tool for the enrichment/depletion analysis of histone modifications and histone/chromatin regulators from a gene list. Many ChIP-chip/ChIP-seq datasets of histone modifications and histone/chromatin regulators in yeast can be found in the literature. Knowing the needs and having the data motivate us to develop a web tool, called Yeast Histone Modifications Identifier (YHMI), which can identify the enriched/depleted histone modifications and the enriched histone/chromatin regulators from a list of yeast genes. Both tables and figures are provided to visualize the identification results. Finally, the high-quality and biological insight of the identification results are demonstrated by two case studies. We believe that YHMI is a valuable tool for yeast biologists to do epigenetics research.

Nm23-H1-stabilized hnRNPA2/B1 promotes internal ribosomal entry site (IRES)-mediated translation of Sp1 in the lung cancer progression.

Our recent studies have indicated that specificity protein-1 (Sp1) accumulates substantially in the early stage of lung cancer but is partially decreased in the late stages, which is an important factor in the progression of the cancer. In this study, we found that Nm23-H1 and hnRNPA2/B1 could be recruited to the 5'UTR of Sp1 mRNA. In investigating the clinical relevance of Nm23-H1/Sp1 levels, we found a positive correlation between lung cancer patients with poor prognosis and low levels of Sp1 and Nm23-H1, suggesting an association between Nm23-H1/Sp1 levels and survival rate. Knockdown of Nm23-H1 inhibits lung cancer growth but increases lung cancer cell malignancy, which could be rescued by overexpression of Sp1, indicating that Nm23-H1-induced Sp1 expression is critical for lung cancer progression. We also found that Nm23-H1 increases the protein stability of hnRNPA2/B1and is thereby co-recruited to the 5'UTR of Sp1 mRNA to regulate cap-independent translational activity. Since the Sp1 level is tightly regulated during lung cancer progression, understanding the molecular mechanisms underlying the regulation by Nm23-H1/hnRNPA2B1 of Sp1 expression in the various stages of lung cancer will be beneficial for lung cancer therapy in the future.

Chronic treatment with cisplatin induces chemoresistance through the TIP60-mediated Fanconi anemia and homologous recombination repair pathways.

The Fanconi anemia pathway in coordination with homologous recombination is essential to repair interstrand crosslinks (ICLs) caused by cisplatin. TIP60 belongs to the MYST family of acetyltransferases and is involved in DNA repair and regulation of gene transcription. Although the physical interaction between the TIP60 and FANCD2 proteins has been identified that is critical for ICL repair, it is still elusive whether TIP60 regulates the expression of FA and HR genes. In this study, we found that the chemoresistant nasopharyngeal carcinoma cells, derived from chronic treatment of cisplatin, show elevated expression of TIP60. Furthermore, TIP60 binds to the promoters of FANCD2 and BRCA1 by using the chromatin immunoprecipitation experiments and promote the expression of FANCD2 and BRCA1. Importantly, the depletion of TIP60 significantly reduces sister chromatid exchange, a measurement of HR efficiency. The similar results were also shown in the FNACD2-, and BRCA1-deficient cells. Additionally, these TIP60-deficient cells encounter more frequent stalled forks, as well as more DNA double-strand breaks resulting from the collapse of stalled forks. Taken together, our results suggest that TIP60 promotes the expression of FA and HR genes that are important for ICL repair and the chemoresistant phenotype under chronic treatment with cisplatin.

Combined Interactions of Plant Homeodomain and Chromodomain Regulate NuA4 Activity at DNA Double-Strand Breaks.

DNA double-strand breaks (DSBs) represent one of the most threatening lesions to the integrity of genomes. In yeast Saccharomyces cerevisiae, NuA4, a histone acetylation complex, is recruited to DSBs, wherein it acetylates histones H2A and H4, presumably relaxing the chromatin and allowing access to repair proteins. Two subunits of NuA4, Yng2 and Eaf3, can interact in vitro with methylated H3K4 and H3K36 via their plant homeodomain (PHD) and chromodomain. However, the roles of the two domains and how they interact in a combinatorial fashion are still poorly characterized. In this study, we generated mutations in the PHD and chromodomain that disrupt their interaction with methylated H3K4 and H3K36. We demonstrate that the combined mutations in both the PHD and chromodomain impair the NuA4 recruitment, reduce H4K12 acetylation at the DSB site, and confer sensitivity to bleomycin that induces DSBs. In addition, the double mutant cells are defective in DSB repair as judged by Southern blot and exhibit prolonged activation of phospho-S129 of H2A. Cells harboring the H3K4R, H3K4R, K36R, or set1Δ set2Δ mutant that disrupts H3K4 and H3K36 methylation also show very similar phenotypes to the PHD and chromodomain double mutant. Our results suggest that multivalent interactions between the PHD, chromodomain, and methylated H3K4 and H3K36 act in a combinatorial manner to recruit NuA4 and regulate the NuA4 activity at the DSB site.

Multiple roles for Saccharomyces cerevisiae histone H2A in telomere position effect, Spt phenotypes and double-strand-break repair.

Telomere position effects on transcription (TPE, or telomeric silencing) are nucleated by association of nonhistone silencing factors with the telomere and propagated in subtelomeric regions through association of silencing factors with the specifically modified histones H3 and H4. However, the function of histone H2A in TPE is unknown. We found that deletion of either the amino or the carboxyltails of H2A substantially reduces TPE. We identified four H2A modification sites necessary for wild-type efficiency of TPE. These "hta1tpe" alleles also act as suppressors of a delta insertion allele of LYS2, suggesting shared elements of chromatin structure at both loci. Interestingly, we observed combinatorial effects of allele pairs, suggesting both interdependent acetylation and deacetylation events in the amino-terminal tail and a regulatory circuit between multiple phosphorylated residues in the carboxyl-terminal tail. Decreases in silencing and viability are observed in most hta1tpe alleles after treatment with low and high concentrations, respectively, of bleomycin, which forms double-strand breaks (DSBs). In the absence of the DSB and telomere-binding protein yKu70, the bleomycin sensitivity of hta1tpe alleles is further enhanced. We also provide data suggesting the presence of a yKu-dependent histone H2A function in TPE. These data indicate that the amino- and carboxyl-terminal tails of H2A are essential for wild-type levels of yKu-mediated TPE and DSB repair.

Downregulation of BRCA1-BRCA2-containing complex subunit 3 sensitizes glioma cells to temozolomide.

We previously found that BRCA1-BRCA2-containing complex subunit 3 (BRCC3) was highly expressed in tumorigenic rat glioma cells. However, the functional role of BRCC3 in human glioma cells remains to be characterized. This study indicated that the upregulation of BRCC3 expression was induced in two human malignant glioblastoma U251 and A172 cell lines following exposure to the alkylating agent, temozolomide (TMZ). Homologous recombination (HR)-dependent DNA repair-associated genes (i.e. BRCA1, BRCA2, RAD51 and FANCD2) were also increased in U251 and A172 cells after treatment with TMZ. BRCC3 gene knockdown through lentivirus-mediated gene knockdown approach not only significantly reduced the clonogenic and migratory abilities of U251 and A172 cells, but also enhanced their sensitization to TMZ. The increase in phosphorylated H2AX foci (γH2AX) formation, an indicator of DNA damage, persisted in TMZ-treated glioma cells with stable knockdown BRCC3 expression, suggesting that BRCC3 gene deficiency is associated with DNA repair impairment. In summary, we demonstrate that by inducing DNA repair, BRCC3 renders glioma cells resistant to TMZ. The findings point to BRCC3 as a potential target for treatment of alkylating drug-resistant glioma.

Sp1-mediated microRNA-182 expression regulates lung cancer progression.

Our recent study indicated that overexpression of Sp1 enhances the proliferation of lung cancer cells, while represses metastasis. In this study, we found that the transcriptional activity of FOXO3 was increased, but its protein levels decreased following Sp1 expression. Sp1 increased expression of miR-182, which was then recruited to the 3'-untranslated region of FOXO3 mRNA to silence its translational activity. Knockdown of miR-182 inhibited lung cancer cells growth, but enhanced the invasive and migratory abilities of these cells through increased N-cadherin expression. Repression of FOXO3 expression in the miR-182 knockdown cells partially reversed this effect, suggesting that miR-182 promotes cancer cell growth and inhibits cancer metastatic activity by regulating the expression of FOXO3. The expression of several cancer metastasis-related genes such as ADAM9, CDH9 and CD44 was increased following miR-182 knockdown. In conclusion, in the early stages of lung cancer progression, Sp1 stimulates miR-182 expression, which in turn decreases FOXO3 expression. This stimulates proliferation and tumor growth. In the late stages, Sp1 and miR-182 decline, thus increasing FOXO3 expression, which leads to lung metastasis.

Chronic treatment with cisplatin induces replication-dependent sister chromatid recombination to confer cisplatin-resistant phenotype in nasopharyngeal carcinoma.

Cisplatin can cause intrastrand and interstrand crosslinks between purine bases and is a chemotherapeutic drug widely used to treat cancer. However, the major barrier to the efficacy of the treatment is drug resistance. Homologous recombination (HR) plays a central role in restoring stalled forks caused by DNA lesions. Here, we report that chronic treatment with cisplatin induces HR to confer cisplatin resistance in nasopharyngeal carcinoma (NPC) cells. A high frequency of sister chromatid exchanges (SCE) occurs in the cisplatin-resistant NPC cells. In addition, several genes in the Fanconi anemia (FA) and template switching (TS) pathways show elevated expression. Significantly, depletion of HR gene BRCA1, TS gene UBC13, or FA gene FANCD2 suppresses SCE and causes cells to accumulate in the S phase, concomitantly with high γH2AX foci formation in the presence of low-dose cisplatin. Consistent with this result, depletion of several genes in the HR, TS, or FA pathway sensitizes the cisplatin-resistant NPC cells to cisplatin. Our results suggest that the enhanced HR, in coordination with the FA and TS pathways, underlies the cisplatin resistance. Targeting the HR, TS, or FA pathways could be a potential therapeutic strategy for treating cisplatin-resistant cancer.