KIAA1549 promotes the development and chemoresistance of colorectal cancer by upregulating ERCC2.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide. Chemotherapy is the mainstay of treatment for patients with CRC in II-IV stages. Resistance to chemotherapy occurs commonly, which results in treatment failure. Therefore, the identification of novel functional biomarkers is essential for recognizing high-risk patients, predicting recurrence, and developing new therapeutic strategies. Herein, we assessed the roles of KIAA1549 in promoting tumor development and chemoresistance in colorectal cancer. As a result, we found that KIAA1549 expression is up-regulation in CRC. Public databases revealed a progressive up-regulation of KIAA1549 expression from adenomas to carcinomas. Functional characterization uncovered that KIAA1549 promotes tumor malignant phenotypes and boosts the chemoresistance of CRC cells in an ERCC2-dependent manner. Inhibition of KIAA1549 and ERCC2 effectively enhanced the sensitivity to chemotherapeutic drugs oxaliplatin and 5-fluorouracil. Our findings suggest that endogenous KIAA1549 might function as a tumor development-promoting role and trigger chemoresistance in colorectal cancer partly by upregulating DNA repair protein ERCC2. Hence, KIAA1549 could be an effective therapeutic target for CRC and inhibition of KIAA1549 combined with chemotherapy might be a potential therapeutic strategy in the future.

Investigation of XPD, miR-145 and miR-770 expression in patients with end-stage renal disease.

BACKGROUND: The effective maintenance of genome integrity and fidelity is vital for the normal function of our tissues and organs, and the prevention of diseases. DNA repair pathways maintain genome stability, and the adequacy of genes acting in these pathways is essential for disease suppression and direct treatment responses. Chronic kidney disease is characterized by high levels of genomic damage. In this study, we examined the expression levels of the xeroderma pigmentosum group D (XPD) gene, which plays a role in the nucleotide excision repair (NER) repair mechanism, and the expression levels of miR-145 and miR-770 genes, which play a role in the regulation of the expression of the XPD gene, in hemodialysis patients with (n = 42) and without malignancy (n = 9) in pre- and post-dialysis conditions. We also evaluated these values with the clinical findings of the patients. METHODS & RESULTS: Gene expression analysis was performed by real-time polymerase chain reaction (qRT-PCR). Compared to the individuals with normal kidney function (2.06 ± 0.32), the XPD gene expression was lower in the pre-dialysis condition both in hemodialysis patients without cancer (1.24 ± 0.18; p = 0.02) and in hemodialysis patients with cancer (0.82 ± 0.114; p = 0.001). On the other hand, we found that miR-145 and miR-770 expression levels were high in both groups. We also found that expression levels were affected by dialysis processes. A statistically significant positive correlation was found between miR-145 and mir770 expression levels in the pre-dialysis group of patients with (r=-0.988. p = 0.0001) and without (r=-0.934. p = 0.0001) malignancy. CONCLUSIONS: Studies on DNA damage repair in the kidney will help develop strategies to protect kidney function against kidney diseases.

Structure of the DNA repair helicase XPD.

The XPD helicase (Rad3 in Saccharomyces cerevisiae) is a component of transcription factor IIH (TFIIH), which functions in transcription initiation and Nucleotide Excision Repair in eukaryotes, catalyzing DNA duplex opening localized to the transcription start site or site of DNA damage, respectively. XPD has a 5' to 3' polarity and the helicase activity is dependent on an iron-sulfur cluster binding domain, a feature that is conserved in related helicases such as FancJ. The xpd gene is the target of mutation in patients with xeroderma pigmentosum, trichothiodystrophy, and Cockayne's syndrome, characterized by a wide spectrum of symptoms ranging from cancer susceptibility to neurological and developmental defects. The 2.25 A crystal structure of XPD from the crenarchaeon Sulfolobus tokodaii, presented here together with detailed biochemical analyses, allows a molecular understanding of the structural basis for helicase activity and explains the phenotypes of xpd mutations in humans.

XPD helicase structures and activities: insights into the cancer and aging phenotypes from XPD mutations.

Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicase activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.

Architecture of the Human and Yeast General Transcription and DNA Repair Factor TFIIH.

TFIIH is essential for both RNA polymerase II transcription and DNA repair, and mutations in TFIIH can result in human disease. Here, we determine the molecular architecture of human and yeast TFIIH by an integrative approach using chemical crosslinking/mass spectrometry (CXMS) data, biochemical analyses, and previously published electron microscopy maps. We identified four new conserved "topological regions" that function as hubs for TFIIH assembly and more than 35 conserved topological features within TFIIH, illuminating a network of interactions involved in TFIIH assembly and regulation of its activities. We show that one of these conserved regions, the p62/Tfb1 Anchor region, directly interacts with the DNA helicase subunit XPD/Rad3 in native TFIIH and is required for the integrity and function of TFIIH. We also reveal the structural basis for defects in patients with xeroderma pigmentosum and trichothiodystrophy, with mutations found at the interface between the p62 Anchor region and the XPD subunit.

The Curcumin Analog PAC Is a Potential Solution for the Treatment of Triple-Negative Breast Cancer by Modulating the Gene Expression of DNA Repair Pathways.

Breast Cancer (BC) is one of the most common and challenging cancers among females worldwide. Conventional treatments for oral cancer rely on the use of radiology and surgery accompanied by chemotherapy. Chemotherapy presents many side effects, and the cells often develop resistance to this chemotherapy. It will be urgent to adopt alternative or complementary treatment strategies that are new and more effective without these negative effects to improve the well-being of patients. A substantial number of epidemiological and experimental studies reported that many compounds are derived from natural products such as curcumin and their analogs, which have a great deal of beneficial anti-BC activity by inducing apoptosis, inhibiting cell proliferation, migration, and metastasis, modulating cancer-related pathways, and sensitizing cells to radiotherapy and chemotherapy. In the present study, we investigated the effect of the curcumin-analog PAC on DNA repair pathways in MCF-7 and MDA-MB-231 human breast-cancer cell lines. These pathways are crucial for genome maintenance and cancer prevention. MCF-7 and MDA-MB-231 cells were exposed to PAC at 10 µM. MTT and LDH assays were conducted to evaluate the effects of PAC on cell proliferation and cytotoxicity. Apoptosis was assessed in breast cancer cell lines using flow cytometry with annexin/Pi assay. The expression of proapoptotic and antiapoptotic genes was determined by RT-PCR to see if PAC is active in programming cell death. Additionally, DNA repair signaling pathways were analyzed by PCR arrays focusing on genes being related and confirmed by quantitative PCR. PAC significantly inhibited breast-cancer cell proliferation in a time-dependent manner, more on MDA-MB-231 triple-negative breast cancer cells. The flow cytometry results showed an increase in apoptotic activity. These data have been established by the gene expression and indicate that PAC-induced apoptosis by an increased Bax and decreased Bcl-2 expression. Moreover, PAC affected multiple genes involved in the DNA repair pathways occurring in both cell lines (MCF-7 and MDA-MB231). In addition, our results suggest that PAC upregulated more than twice 16 genes (ERCC1, ERCC2, PNKP, POLL, MPG, NEIL2, NTHL1, SMUG1, RAD51D, RAD54L, RFC1, TOP3A, XRCC3, XRCC6BP1, FEN1, and TREX1) in MDA-MB-231, 6 genes (ERCC1, LIG1, PNKP, UNG, MPG, and RAD54L) in MCF-7, and 4 genes (ERCC1, PNKP, MPG, and RAD54L) in the two cell lines. In silico analysis of gene-gene interaction shows that there are common genes between MCF-7 and MDA-MB-321 having direct and indirect effects, among them via coexpression, genetic interactions, pathways, predicted and physical interactions, and shared protein domains with predicted associated genes indicating they are more likely to be functionally related. Our data show that PAC increases involvement of multiple genes in a DNA repair pathway, this certainly can open a new perspective in breast-cancer treatment.

TFIIH stabilization recovers the DNA repair and transcription dysfunctions in thermo-sensitive trichothiodystrophy.

Trichothiodystrophy (TTD) is a rare hereditary disease whose prominent feature is brittle hair. Additional clinical signs are physical and neurodevelopmental abnormalities and in about half of the cases hypersensitivity to UV radiation. The photosensitive form of TTD (PS-TTD) is most commonly caused by mutations in the ERCC2/XPD gene encoding a subunit of the transcription/DNA repair complex TFIIH. Here we report novel ERCC2/XPD mutations affecting proper protein folding, which generate thermo-labile forms of XPD associated with thermo-sensitive phenotypes characterized by reversible aggravation of TTD clinical signs during episodes of fever. In patient cells, the newly identified XPD variants result in thermo-instability of the whole TFIIH complex and consequent temperature-dependent defects in DNA repair and transcription. Improving the protein folding process by exposing patient cells to low temperature or to the chemical chaperone glycerol allowed rescue of TFIIH thermo-instability and a concomitant recovery of the complex activities. Besides providing a rationale for the peculiar thermo-sensitive clinical features of these new cases, the present findings demonstrate how variations in the cellular concentration of mutated TFIIH impact the cellular functions of the complex and underlie how both quantitative and qualitative TFIIH alterations contribute to TTD clinical features.

Trichothiodystrophy hair shafts display distinct ultrastructural features.

Hair shafts from three trichothiodystrophy (TTD) patients with mutations in the ERCC2 (XPD) gene were examined by transmission electron microscopy. TTD is a rare, recessive disorder with mutations in several genes in the DNA repair/transcription pathway, including ERCC2. Unlike previous studies, the hair shafts were examined after relaxation of their structure by partial disulphide bond reduction in the presence of sodium dodecyl sulphate, permitting improved visualization. Compared with hair shafts of normal phenotype, TTD cuticle cells displayed aberrant marginal bands and exocuticle layers. Clusters of cells stained differently (light versus dark) in the cortex of aberrant shafts, and the keratin macrofibrils appeared much shorter in the cytoplasm. Considerable heterogeneity in these properties was evident among samples and even along the length of single hair shafts. The results are consistent with not only a paucity of high sulphur components, such as keratin-associated proteins, but also a profound imbalance in protein content and organization.

Adult-Onset Neurodegeneration in Nucleotide Excision Repair Disorders (NERDND ): Time to Move Beyond the Skin.

BACKGROUND: Variants in genes of the nucleotide excision repair (NER) pathway have been associated with heterogeneous clinical presentations ranging from xeroderma pigmentosum to Cockayne syndrome and trichothiodystrophy. NER deficiencies manifest with photosensitivity and skin cancer, but also developmental delay and early-onset neurological degeneration. Adult-onset neurological features have been reported in only a few xeroderma pigmentosum cases, all showing at least mild skin manifestations. OBJECTIVE: The aim of this multicenter study was to investigate the frequency and clinical features of patients with biallelic variants in NER genes who are predominantly presenting with neurological signs. METHODS: In-house exome and genome datasets of 14,303 patients, including 3543 neurological cases, were screened for deleterious variants in NER-related genes. Clinical workup included in-depth neurological and dermatological assessments. RESULTS: We identified 13 patients with variants in ERCC4 (n = 8), ERCC2 (n = 4), or XPA (n = 1), mostly proven biallelic, including five different recurrent and six novel variants. All individuals had adult-onset progressive neurological deterioration with ataxia, dementia, and frequently chorea, neuropathy, and spasticity. Brain magnetic resonance imaging showed profound global brain atrophy in all patients. Dermatological examination did not show any skin cancer or pronounced ultraviolet damage. CONCLUSIONS: We introduce NERDND as adult-onset neurodegeneration (ND ) within the spectrum of autosomal recessive NER disorders (NERD). Our study demonstrates that NERDND is probably an underdiagnosed cause of neurodegeneration in adulthood and should be considered in patients with overlapping cognitive and movement abnormalities. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Ribosomal protein S3 associates with the TFIIH complex and positively regulates nucleotide excision repair.

In mammalian cells, the bulky DNA adducts caused by ultraviolet radiation are mainly repaired via the nucleotide excision repair (NER) pathway; some defects in this pathway lead to a genetic disorder known as xeroderma pigmentosum (XP). Ribosomal protein S3 (rpS3), a constituent of the 40S ribosomal subunit, is a multi-functional protein with various extra-ribosomal functions, including a role in the cellular stress response and DNA repair-related activities. We report that rpS3 associates with transcription factor IIH (TFIIH) via an interaction with the xeroderma pigmentosum complementation group D (XPD) protein and complements its function in the NER pathway. For optimal repair of UV-induced duplex DNA lesions, the strong helicase activity of the TFIIH complex is required for unwinding damaged DNA around the lesion. Here, we show that XP-D cells overexpressing rpS3 showed markedly increased resistance to UV radiation through XPD and rpS3 interaction. Additionally, the knockdown of rpS3 caused reduced NER efficiency in HeLa cells and the overexpression of rpS3 partially restored helicase activity of the TFIIH complex of XP-D cells in vitro. We also present data suggesting that rpS3 is involved in post-excision processing in NER, assisting TFIIH in expediting the repair process by increasing its turnover rate when DNA is damaged. We propose that rpS3 is an accessory protein of the NER pathway and its recruitment to the repair machinery augments repair efficiency upon UV damage by enhancing XPD helicase function and increasing its turnover rate.

Xeroderma Pigmentosum group D suppresses proliferation and promotes apoptosis of HepG2 cells by downregulating ERG expression via the PPARγ pathway.

Xeroderma Pigmentosum group D (XPD) gene has been shown to suppress hepatocellular carcinoma (HCC) progression, but its mechanism remains not fully understood. ETS-related gene (ERG) is generally known as an oncogenic gene. This study aimed to explore whether XPD regulated HCC cell proliferation, apoptosis and cell cycle by inhibiting ERG expression via the PPARγ pathway. The human hepatoma cells (HepG2) were transfected with the XPD overexpression vector (pEGFP-N2/XPD) or empty vector (pEGFP-N2). The PPARγ inhibitor GW9662 was used to determine whether XPD effects were mediated by activation of PPARγ pathway. Cell cycle and apoptosis were ascertained by flow cytometry, and cell viability was measured by MTT assay. Reverse transcription-polymerase chain reaction and Western blot were performed to determine the mRNA and protein levels. Overexpression of XPD significantly enhanced the expression of PPARγ and p-PPARγ, whereas it downregulated that of ERG and cdk7. Furthermore, XPD overexpression notably inhibited proliferation, promoted apoptosis and decreased the percentage of cells in the S + G2 phase of HepG2 cells. However, these effects of XPD overexpression were abrogated by GW9662. Collectively, XPD suppresses proliferation and promotes apoptosis of HepG2 cells by downregulating ERG expression via activation of the PPARγ pathway.

Association of XRCC1 and ERCC2 promoters' methylation with chromatin condensation and sperm DNA fragmentation in idiopathic oligoasthenoteratozoospermic men.

The aim of the study was to investigate whether the promoter methylation of XRCC1 and ERCC2 genes is associated with sperm DNA fragmentation and chromatin condensation in idiopathic oligoasthenoteratozoospermic men. This study involved 77 infertile men with idiopathic oligoasthenoteratozoospermia and 51 normozoospermic controls. The methylight method, TUNEL assay and aniline blue staining were used for the evaluation of XRCC1 and ERCC2 genes' methylation, SDF and sperm chromatin condensation, respectively. SDF (p = .004) and XRCC1 methylation (p = .0056) were found to be significantly higher in men with idiopathic OAT than in the controls, while mature spermatozoa frequency was higher in controls as compared to infertile men (p < .0001). No significant association was found between SDF and methylation of XRCC1 and ERCC2 genes (p = .9277 and p = .8257, respectively). However, compared to the cut-off point obtained by receiver operating characteristic analysis, a significant association was found between SDF and XRCC1 methylation, positive and negative methylation groups, generated according to the cut-off value for XRCC1. XRCC1 methylation was found to have a significant effect on chromatin condensation (p = .0017). No significant difference was detected among ERCC2 methylation, male infertility and SDF. In conclusion, XRCC1 methylation may have a role in sperm chromatin condensation and idiopathic OAT.

Association between Molecular Genetic Markers of DNA Repair and Cell Cycle Control Genes and Response to Platinum-Based Chemotherapy in Ovarian Cancer Patients.

We analyzed associations of polymorphic markers of DNA repair genes (XRCC1, ERCC2), cell cycle control genes (TP53, MDM2, and CDKN1A), methylation of promoter region, and mutation 5382insC of BRCA1 gene in ovarian cancer with effectiveness of platinumbased chemotherapy assessed by the median of progression-free survival time for markers of DNA repair genes and by relapse risk for all studied markers. An increase in the median of progression-free survival time for carriers of the Gln allele (р=0.025) and Gln/Gln genotype (р=0.022) of the Gln399Arg XRCC1 was observed during the 19-months period after chemotherapy. In carriers of C/C genotype of 5382insC mutation of BRCA1 gene (n=6), no relapses were observed (р=0.035), while 17 of 49 patients without this mutation developed relapses. Of 14 patients with BRCA1 gene function inactivation due to promoter methylation or the presence of the C/C genotype of 5382insC, one relapse was observed (p=0.033). Multivariate analysis revealed an association of markers of the XRCC1, TP53, MDM2 genes, BRCA1 gene inactivation, and type of surgery with the risk of relapse during the follow-up period up to 19 months after the end of chemotherapy (р≤0.0007).

Minimotif Miner 4: a million peptide minimotifs and counting.

Minimotif Miner (MnM) is a database and web system for analyzing short functional peptide motifs, termed minimotifs. We present an update to MnM growing the database from ∼300 000 to >1 000 000 minimotif consensus sequences and instances. This growth comes largely from updating data from existing databases and annotation of articles with high-throughput approaches analyzing different types of post-translational modifications. Another update is mapping human proteins and their minimotifs to know human variants from the dbSNP, build 150. Now MnM 4 can be used to generate mechanistic hypotheses about how human genetic variation affect minimotifs and outcomes. One example of the utility of the combined minimotif/SNP tool identifies a loss of function missense SNP in a ubiquitylation minimotif encoded in the excision repair cross-complementing 2 (ERCC2) nucleotide excision repair gene. This SNP reaches genome wide significance for many types of cancer and the variant identified with MnM 4 reveals a more detailed mechanistic hypothesis concerning the role of ERCC2 in cancer. Other updates to the web system include a new architecture with migration of the web system and database to Docker containers for better performance and management. Weblinks:minimotifminer.org and mnm.engr.uconn.edu.

GTF2E2 Mutations Destabilize the General Transcription Factor Complex TFIIE in Individuals with DNA Repair-Proficient Trichothiodystrophy.

The general transcription factor IIE (TFIIE) is essential for transcription initiation by RNA polymerase II (RNA pol II) via direct interaction with the basal transcription/DNA repair factor IIH (TFIIH). TFIIH harbors mutations in two rare genetic disorders, the cancer-prone xeroderma pigmentosum (XP) and the cancer-free, multisystem developmental disorder trichothiodystrophy (TTD). The phenotypic complexity resulting from mutations affecting TFIIH has been attributed to the nucleotide excision repair (NER) defect as well as to impaired transcription. Here, we report two unrelated children showing clinical features typical of TTD who harbor different homozygous missense mutations in GTF2E2 (c.448G>C [p.Ala150Pro] and c.559G>T [p.Asp187Tyr]) encoding the beta subunit of transcription factor IIE (TFIIEß). Repair of ultraviolet-induced DNA damage was normal in the GTF2E2 mutated cells, indicating that TFIIE was not involved in NER. We found decreased protein levels of the two TFIIE subunits (TFIIEα and TFIIEß) as well as decreased phosphorylation of TFIIEα in cells from both children. Interestingly, decreased phosphorylation of TFIIEα was also seen in TTD cells with mutations in ERCC2, which encodes the XPD subunit of TFIIH, but not in XP cells with ERCC2 mutations. Our findings support the theory that TTD is caused by transcriptional impairments that are distinct from the NER disorder XP.

Lack of association between functional polymorphism of DNA repair genes (XRCC1, XPD) and clinical response in Indian chronic myeloid leukemia patients.

The resistance for the tyrosine kinase inhibitors in chronic myeloid leukemia (CML) occurs mainly due to BCR/ABL1 dependent and independent mechanisms. The defective DNA repair due to functional polymorphisms in DNA repair genes, might act as an etiological factor for leukemia progression. The study was carried out to understand the role of DNA repair genes (XRCC1, XPD) polymorphisms in Imatinib mesylate (IM) resistant CML patients. The study was carried out in total 87 CML patients (43 nonresponders-cases and 44 responders) who were treated with Imatinib. The treatment and follow-up was done according to European LeukemiaNet guidelines. The genotyping of selected SNPs were studied using RFLP and confirmed with Sanger sequencing (20%). The statistical analysis was performed using online tools (Socscistatistics and GraphPad InStat software). In our study no significant association was inferred between genotypes of DNA repair genes (XRCC1; rs1799782, rs25487, and XPD; rs13181) and complete cytogenetic response as well as molecular response. However there might be a possibility of association between XRCC1 Arg399Gln genotype AA/GA and cytogenetic response though it is statistically insignificant (p > 0.05). Though none of the genotypes of the DNA repair genes showed association with IM response, near association between XRCC1Arg399Gln genotype and cytogenetic response observed in our study. Hence, large sample size should be studied to establish the association of SNPs of DNA repair genes and IM response. Our study is a novel and important to explain the role of DNA repair genes polymorphisms in IM resistance.

Low-power laser alters mRNA levels from DNA repair genes in acute lung injury induced by sepsis in Wistar rats.

Acute lung injury (ALI) is defined as respiratory failure syndrome, in which the pathogenesis could occur from sepsis making it a life-threatening disease by uncontrolled hyperinflammatory responses. A possible treatment for ALI is the use of low-power infrared lasers (LPIL), whose therapeutical effects depend on wavelength, power, fluence, and emission mode. The evaluation mRNA levels of repair gene related to oxidative damage after exposure to LPIL could provide important information about the modulation of genes as treatment for ALI. Thus, the aim of this study was to evaluate the mRNA levels from OGG1, APEX1, ERCC2, and ERCC1 genes in lung tissue from Wistar rats affected by ALI and after exposure to LPIL (808 nm; 100 mW). Adult male Wistar rats (n = 30) were randomized into six groups (n = 5, for each group): control, 10 J/cm2 (2 J), 20 J/cm2 (5 J), ALI, ALI + LPIL 10 J/cm2 and ALI + LPIL 20 J/cm2. ALI was induced by intraperitoneal E. coli lipopolysaccharide injection (10 mg/kg). Lungs were removed, and samples were withdrawn for total RNA extraction, cDNA synthesis, and mRNA levels were evaluated by RT-qPCR. Data normality was verified by Kolmogorov-Smirnov, comparisons among groups were by Student's t test, Mann-Whitney test, one-way ANOVA, Kruskal-Wallis followed by post-tests. Data showed that OGG1 (0.39 ± 0.10), ERCC2 (0.67 ± 0.24), and ERCC1 (0.60 ± 0.19) mRNA levels are reduced in ALI group when compared with the control group (1.00 ± 0.07, 1.03 ± 0.25, 1.01 ± 0.16, respectively) and, after LPIL, mRNA relative levels from DNA repair genes are altered when compared to non-exposed ALI group. Our research shows that ALI alter mRNA levels from genes related to base and nucleotide excision repair genes, suggesting that DNA repair is part of cell response to sepsis, and that photobiomodulation could modulate the mRNA levels from these genes in lung tissue.

A phase II trial of irinotecan in patients with advanced or recurrent endometrial cancer and correlation with biomarker analysis.

OBJECTIVE: Chemotherapy for advanced or recurrent endometrial cancer requires further development. Irinotecan hydrochloride (CPT-11) suppresses tumor growth in several endometrial cancer strains. The present study evaluated the anti-tumor activity and toxicity of CPT-11 in patients with advanced or recurrent endometrial cancer. METHODS: Enrolled patients had advanced endometrial cancer with measurable lesions and received 2 pretreatment regimens. A 90-minute intravenous infusion of CPT-11 (100 mg/m2) was given on days 1, 8, and 15 of a 4-week cycle, aiming for an effect with ≤2 cycles. Treatment was continued until the primary disease worsened or severe toxicity occurred. The primary endpoint was response rate, and the secondary endpoints were progression-free survival, overall survival, and adverse events. Antitumor effect and adverse events were evaluated according to RECIST version 1.1 and NCI-CTC AE version 3.0, respectively. RESULTS: Twenty-two patients were registered (11 endometrioid carcinomas and 11 serous carcinomas). The median duration of the treatment-free interval (TFI) was 7.5 months, and the median number of administered cycles per patient was 4. Response rate was 36.4% (complete response: 1 patient, partial response: 7 patients). Clinical benefit rate, including stable disease, was 77.3%. Median progression-free and overall survival was 4.4 and 18.4 months, respectively. Observed adverse events included grade 4 hematotoxicity (neutropenia and thrombocytopenia), and grade 2 or 3 non-hematotoxicity (diarrhea). All adverse events were manageable. Biomarker predictors of therapeutic effectiveness were not observed. CONCLUSION: As a single agent, CPT-11 has anti-tumor activity for advanced or recurrent endometrial cancer and has manageable adverse events.

Analysis of the conserved NER helicases (XPB and XPD) and UV-induced DNA damage in Hydra.

BACKGROUND: Nucleotide excision repair (NER) pathway is an evolutionarily conserved mechanism of genome maintenance. It detects and repairs distortions in DNA double helix. Xeroderma Pigmentosum group B (XPB) and group D (XPD) are important helicases in NER and are also critical subunits of TFIIH complex. We have studied XPB and XPD for the first time from the basal metazoan Hydra which exhibits lack of organismal senescence. METHODS: In silico analysis of proteins was performed using MEGA 6.0, Clustal Omega, Swiss Model, etc. Gene expression was studied by in situ hybridization and qRT-PCR. Repair of CPDs was studied by DNA blot assay. Interactions between proteins were determined by co- immunoprecipitation. HyXPB and HyXPD were cloned in pET28b, overexpressed and helicase activity of purified proteins was checked. RESULTS: In silico analysis revealed presence of seven classical helicase motifs in HyXPB and HyXPD. Both proteins revealed polarity-dependent helicase activity. Hydra repairs most of the thymine dimers induced by UVC (500 J/m2) by 72 h post-UV exposure. HyXPB and HyXPD transcripts, localized all over the body column, remained unaltered post-UV exposure indicating their constitutive expression. In spite of high levels of sequence conservation, XPB and XPD failed to rescue defects in human XPB- and XPD-deficient cell lines. This was due to their inability to get incorporated into the TFIIH multiprotein complex. CONCLUSIONS: Present results along with our earlier work on DNA repair proteins in Hydra bring out the utility of Hydra as model system to study evolution of DNA repair mechanisms in metazoans.

Whole exome sequencing of breast cancer (TNBC) cases from India: association of MSH6 and BRIP1 variants with TNBC risk and oxidative DNA damage.

Whole exome sequencing in triple negative breast cancer cases (n = 8) and targeted sequencing in healthy controls (n = 48) revealed BRIP1 rs552752779 (MAF: 75% vs. 6.25%, OR 45.00, 95% CI 9.43-243.32), ERBB2 rs527779103 (MAF: 62.5% vs. 7.29%, OR 21.19, 95% CI 5.11-94.32), ERCC2 rs121913016 (MAF: 56.25% vs. 7.29%, OR 16.34, 95% CI 4.02-70.41), MSH6 rs2020912 (MAF: 56.25% vs. 1.04%, OR 122.13, 95% CI 12.29-2985.48) as risk factors for triple negative breast cancer. Construction of classification and regression tree followed by smart pruning identified MSH6 and BRIP1 variants as the major determinants of TNBC (Triple Negative Breast Cancer) risk. Except for ERBB2, all other genes regulate DNA repair and chromosomal integrity. In TNBC cases, two likely pathogenic variations i.e. NCOR1 rs562300336 and PIM1 rs746748226 were observed at frequencies of 18.75% and 12.5%, respectively. Among the 24 variants of unknown significance, MMP9 rs199676062, SYNE1 rs368709678, AURKA rs373550419, ABCC4 rs11568694 have variant allele frequency ≥ 62.5%. These genes regulate metastasis, nuclear modeling, cell cycle and cellular detoxification, respectively. To conclude, aberrations in DNA mismatch repair, nucleotide excision repair or BRCA1 associated genome surveillance mechanism contribute towards triple negative breast cancer.