(A) Images and analyses of DNA tail moments of RNAi-treated Hep3B and SNU449 cells by Comet assay. homologous recombination (HR) repair genes in human HCC samples and functionally intersects with those involved in replication stress response and HR repair in yeasts. In support, NS-high HCCs are more reliant around the replicative/oxidative stress response pathways, whereas NS-low HCCs depend more around the mTOR pathway. Perturbation studies showed NS function in protecting human HCC cells from replication- and drug-induced DNA damage. Notably, NS depletion in HCC cells increases the amounts of physical DNA damage and cytosolic dsDNA, leading to a reactive increase of cytokines and PD-L1. This study shows that NS provides an essential mechanism for HCC to adapt to high genomic stress for oncogenic maintenance and propagation. NS deficiency sensitizes HCC cells to chemotherapy but also triggers tumor immune responses. NS expression is usually upregulated in hepatic precursor cells and regenerating adult hepatocytes12, 16, NS depletion sensitizes MHCC97 cells to UV and serum starvation-induced apoptosis17, and high NS expression correlates with poor survival18 and sorafenib resistance in HCC patients19. Our current data show that NS correlates with tumor grade and uniquely predicts both overall survival (OS) and progression-free survival (PFS) in HCC patients. In addition, NS protects HCCs from drug-induced DNA damage, and NS-high HCCs are more addicted to stress response pathways than NS-low HCCs. Notably, NS depletion increases the amount of physical DNA damage and cytoplasmic accumulation of fragmented dsDNA, triggering innate immune responses in HCC cells. These results raise NS Valpromide as a new HCC end result predictor and a potential therapeutic target for sensitizing HCCs to standard chemotherapy as well as immunotherapy. MATERIALS AND METHODS Animal care and procedures Animals were housed by the Program for Animal Resources at the TAMHSC-Houston campus and dealt with in accordance with the principles explained by the Guideline for the Care and Use of Laboratory Animals. All procedures were approved by the IACUC. TCGA and Gene Set Enrichment Assay (GSEA) analyses Processed RNA-seq gene expression data for HCC were acquired through the GDC data portal (https://portal.gdc.malignancy.gov/). Other data Serpine1 were acquired through cBioPortal. Sample size determination followed the same TCGA recommendations explained previously20. For GSEA, HCC RNA-seq data were run against the HALLMARK and Canonical gene units in the Molecular Signatures Database (MsigDB). Spearman correlation coefficients between each of the genes and NS were determined by using pre-ranked gene set enrichment analysis. To compare NS expression between subtypes of cancers, normality was Valpromide tested by the Kolmogorov-Smirnov test. For data not normally distributed, variances between groups were sufficiently much like use non-normal statistical assessments (a rank-sum test for two groups or a Kruskal-Wallis H test for multiple groups) to determine the statistical significance. For survival analysis, patients Valpromide were stratified into high-NS (highest 33% of samples) and low-NS (bottom 66% of samples). Molecular and chemical intervention of HCC cells Hep3B and SNU449 cells were purchased from ATCC (Hep3B: Cat# HB-8064, RRID:CVCL_0326; SNU449: Cat#CRL-2234, RRID:CVCL_0454). HuH7 cells were obtained from David Moore (Baylor College of Medicine). All three cell lines were confirmed for their identity by Short Tandem Repeat (STR) fingerprinting. Mycoplasma was tested on 5/25/18. Cells were passaged every 2C4 days up to 10 passages after thawing. For RNAi knockdown, cells were treated with oligofectamine-complexed siRNA duplex (50nM) for 24 hours. For drug treatment, cells were treated with 5-FU (10g/ml), oxaliplatin (OXP, 10M), doxorubicin (DRB, 5M), or olaparib (200M). Supplemental Data More details of the above and other methods. RESULTS High NS expression correlates with high tumor grades and poor prognosis and survival in HCC patients To establish the clinical relevance of NS in HCC, we first compared its total transcript levels between human HCC (n=369) and adjacent nontumor tissues (ANT, n=64). Based on the TCGA HCC cohorts, there is a significant increase of NS in HCC compared to ANT (Fig.1A). From a pan-cancer perspective, HCC is usually ranked among the top tertile in NS expression (Fig.S1A). Comparing against numerous clinicopathological parameters, NS expression shows a significant increase in WHO grade 3 tumors (G3, poorly differentiated) compared to G1 (well differentiated) and G2 (moderately differentiated) (more in the S2 (aggressive, Myc/Akt-activated) than in the S1 (aggressive, TGF–activated) or S3 (differentiated) subtype based on the Hoshidas classification22, more in the strong cluster 2 (C2, advanced tumors with more vascular invasion and extrahepatic metastasis) than Valpromide in the C1 subtype based on the hepatoblastoma classification23, and more in the A (proliferation/unfavorable) than in the B subclass by the NCI proliferation classification24 (Fig.1D). Finally, univariate log-rank analyses.