To obtain tissue-specific mRNA samples from three medicinal leech species, H. medicinalis, H. verbаna, and H. orientalis, we isolated salivary cells and muscles from the cryosections of the anterior body parts using laser microdissection (Fig. 2a). Then, we constructed two cDNA libraries with and without normalization for each mRNA sample using the oligo-dT primer and sequenced them on the Ion Torrent PGM (Supplementary Table 8). Four read datasets corresponding to the constructed cDNA libraries were used for the de novo assembly of a combined transcriptome for each medicinal leech species using the Trinity RNA assembler [20] (Supplementary Table 9). We used the combined transcriptomes to map non-normalized tissue-specific reads. Read mapping was necessary to perform consecutive differential expression analysis.
Nastia Muntean Sets 1 10 1l
The sample preparation method is critical to the resultant repertoire of the identified proteins because the SCS consists of both low- and high-molecular-weight components [9] and contains proteinase inhibitors, glycoprotein complexes, and lipids. The latter may form complexes with proteins [26]. Therefore, we combined several sample preparation methods and several mass spectrometry techniques to cover the broadest repertoire of the SCS proteins. Proteomic datasets obtained by different sample preparation methods and mass spectrometry techniques were combined to create a final list of the identified proteins for each medicinal leech species.
To annotate a draft genome, three sets of so-called hints, sequences in the genome that exhibit features of specific gene structures, such as exons, introns, etc., were generated (for details, see Supplementary Methods). The first set of hints was generated using sequences from the H. robusta protein coding genes. The second set of hints was generated using contigs corresponding to the de novo transcriptome assembly (see below). The third set of hints was generated using the cDNA reads (see below). All sets of hints were combined, and AUGUSTUS software [93] (version 3.7.1) was used for annotation of the draft genome.
On the basis of meta-analyses of multiple SCCHN data sets, we nominated Thyroid hormone Receptor Interactor 13 (TRIP13 or HPV16E1BP) as an oncogene. TRIP13 is the mouse orthologue of pachytene checkpoint 2 (Pch2), a checkpoint for synapsis before DSB repair and recombination in yeast and Caenorhabditis elegans7,8. In mice, TRIP13 mediates DSB repair9,10; however, the role of TRIP13 in humans has not been investigated. In this study, we investigated a mechanism by which TRIP13 promotes treatment resistance. Overexpression of TRIP13 in non-malignant cells leads to malignant transformation. High expression of TRIP13 in SCCHN promoted aggressive tumor growth, treatment resistance and enhanced repair of DNA damage. TRIP13-binding partners, including DNA-PKcs complex proteins mediating NHEJ, were identified using mass spectrometry. Repair-deficient reporter systems revealed that TRIP13 promotes NHEJ. Overexpression of TRIP13 sensitized SCCHN to an inhibitor of DNA-PKcs and impairment of TRIP13 ATPase activity diminishes its DSB repair efficiency. These findings define a mechanism of treatment resistance in SCCHN and emphasize the importance of targeting NHEJ to overcome treatment failure.
Recent sequencing studies emphasized the limited number of mutations present in SCCHN11. Therefore, we performed meta-analysis of multiple SCCHN data sets (Fig. 1a, left to right: Cromer Head-Neck, accession GSE2379 (ref. 12), Estilo Head-Neck, accession GSE13601 (ref. 13), Ye Head-Neck, accession GSE9844 (ref. 14), Kuriakose Head-Neck, accession GDS2520 (ref. 15), Ginos Head-Neck16, Toruner Head-Neck, accession GSE3524 (ref. 17), all available from Oncomine, Compendia Bioscience, Ann Arbor, MI, USA), to nominate potential novel oncogenes. TRIP13 was significantly upregulated in all six data sets interrogated (Fig. 1a). The Cancer Genome Atlas data set showed highly increased TRIP13 copy number (approximately four copies) in 14% (40/290) of SCCHN patients (Fig. 1b). TRIP13 was nominated as an oncogene and is not mutated in SCCHN (Supplementary Fig. 1A, left panel). TRIP13 copy number and gene expression frequency are increased in multiple cancers (Supplementary Fig. 1A, middle and right panels). Compared with immortalized non-malignant keratinocytes, fluorescence in situ hybridization (FISH) showed increased TRIP13 copy number in SCCHN cells and tissue (Fig. 1c, Supplementary Fig. 1B,C).
Chemotherapy and radiation are current treatment options for advanced SCCHN31. Resistance to treatment is correlated with recurrence and morbidity, underscoring the importance of developing new treatment strategies. Here we report that TRIP13 sensitizes SCCHN to a DNA-PKcs inhibitor. TRIP13 was designated as an oncogene by meta-analysis of several gene expression data sets and functionally validated as an oncogene by malignant transformation of non-malignant cells, tumour growth and invasion assays. Several NHEJ proteins were identified as binding partners of TRIP13, suggesting a role for TRIP13 in NHEJ, which was verified by multiple robust approaches. Consistent with the correlation between NHEJ and treatment resistance27, SCCHN cells overexpressing TRIP13 are resistant to chemotherapy and radiation but responsive to NHEJ inhibitors. Our studies define a mechanism of treatment resistance in cancer and emphasize the importance of targeting NHEJ to overcome treatment resistance in SCCHN overexpressing TRIP13.
Meta-analysis of TRIP13 in multiple cancers was performed using Oncomine ( ) for SCCHN data sets and was graphed. Data sets used for this study were Cromer-Head-Neck12, Estilo-Head-Neck13, Ye-Head-Neck14, Kuriakose-Head-Neck15, Ginos-Head-Neck16 and Toruner-Head-Neck17. 2ff7e9595c
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