Accurate tumor clonal structures require single-cell analysis.

Tumor clonal structure is closely related to future progression, which has been mainly investigated as mutation abundance clustering in bulk samples. With relatively limited studies at single-cell resolution, a systematic comparison of the two approaches is still lacking. Here, using bulk and single-cell mutational data from the liver and colorectal cancers, we checked whether co-mutations determined by single-cell analysis had corresponding bulk variant allele frequency (VAF) peaks. While bulk analysis suggested the absence of subclonal peaks and, possibly, neutral evolution in some cases, the single-cell analysis identified coexisting subclones. The overlaps of bulk VAF ranges for co-mutations from different subclones made it difficult to separate them. Complex subclonal structures and dynamic evolution could be hidden under the seemingly clonal neutral pattern at the bulk level, suggesting single-cell analysis is necessary to avoid underestimation of tumor heterogeneity.

BRD4 inhibition induces synthetic lethality in ARID2-deficient hepatocellular carcinoma by increasing DNA damage.

Hepatocellular carcinoma (HCC) has emerged as the third cause of cancer-related death owing to lacking effective systemic therapies. Genomic DNA sequencing revealed the high frequency of loss-of-function mutations in ARID2, which encodes a subunit of SWI/SNF chromatin remodeling complex, however, the therapeutic strategy for the HCC patients with ARID2 mutations is still completely unclear. In this study, we first performed a high-throughput screening approach using a compound library consisting of 2 180 FDA-approved drugs and other compounds, to elicit the potential drugs for synthetic lethality to target ARID2-deficient HCC cells. Interestingly, JQ1, a selective inhibitor of bromodomain protein BRD4, uniquely suppressed the growth of ARID2- deficient HCC cells. Next JQ1 is further confirmed to predominantly induce cell lethality upon ARID2 depletion through exacerbating DNA damage, especially double strand breaks (DSBs). Functional assays demonstrated that both BRD4 inhibition and ARID2 deficiency synergistically impede two main DNA damage repair pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ), through attenuating the transcription of BRCA1, RAD51, and 53BP1, which encode the core molecules responsible for DSB repair. Mechanistically, both ARID2 and BRD4 exert a synergistic effect for maintaining transcriptional enhancer-promoter loops of these genes within chromatin conformation. However, as both ARID2 and BRD4 are disrupted, the expression of these DNA repair-related genes in response to DNA damage are hindered, resulting in DSB accumulation and cell apoptosis. Taken together, this study discloses that BRD4 inhibition may induce synthetic lethality in ARID2-deficient HCC cells, which might provide a potential therapeutic strategy for HCC patients with ARID2 mutations.

Clonal evolution in liver cancer at single-cell and single-variant resolution.

Genetic heterogeneity of tumor is closely related to its clonal evolution, phenotypic diversity and treatment resistance, and such heterogeneity has only been characterized at single-cell sub-chromosomal scale in liver cancer. Here we reconstructed the single-variant resolution clonal evolution in human liver cancer based on single-cell mutational profiles. The results indicated that key genetic events occurred early during tumorigenesis, and an early metastasis followed by independent evolution was observed in primary liver tumor and intrahepatic metastatic portal vein tumor thrombus. By parallel single-cell RNA-Seq, the transcriptomic phenotype of HCC was found to be related with genetic heterogeneity. For the first time we reconstructed the single-cell and single-variant clonal evolution in human liver cancer, and dissection of both genetic and phenotypic heterogeneity will facilitate better understanding of their relationship.

LZP is required for hepatic triacylglycerol transportation through maintaining apolipoprotein B stability.

The conserved zona pellucida (ZP) domain is found in hundreds of extracellular proteins that are expressed in various organs and play a variety of roles as structural components, receptors and tumor suppressors. A liver-specific zona pellucida domain-containing protein (LZP), also named OIT3, has been shown to be mainly expressed in human and mouse hepatocytes; however, the physiological function of LZP in the liver remains unclear. Here, we show that Lzp deletion inhibited very low-density lipoprotein (VLDL) secretion, leading to hepatic TG accumulation and lower serum TG levels in mice. The apolipoprotein B (apoB) levels were significantly decreased in the liver, serum, and VLDL particles of LZP-deficient mice. In the presence of LZP, which is localized to the endoplasmic reticulum (ER) and Golgi apparatus, the ER-associated degradation (ERAD) of apoB was attenuated; in contrast, in the absence of LZP, apoB was ubiquitinated by AMFR, a known E3 ubiquitin ligase specific for apoB, and was subsequently degraded, leading to lower hepatic apoB levels and inhibited VLDL secretion. Interestingly, hepatic LZP levels were elevated in mice challenged with a high-fat diet and humans with simple hepatic steatosis, suggesting that LZP contributes to the physiological regulation of hepatic TG homeostasis. In general, our data establish an essential role for LZP in hepatic TG transportation and VLDL secretion by preventing the AMFR-mediated ubiquitination and degradation of apoB and therefore provide insight into the molecular function of LZP in hepatic lipid metabolism.

Single-cell RNA-Seq analysis reveals dynamic trajectories during mouse liver development.

BACKGROUND: The differentiation and maturation trajectories of fetal liver stem/progenitor cells (LSPCs) are not fully understood at single-cell resolution, and a priori knowledge of limited biomarkers could restrict trajectory tracking. RESULTS: We employed marker-free single-cell RNA-Seq to characterize comprehensive transcriptional profiles of 507 cells randomly selected from seven stages between embryonic day 11.5 and postnatal day 2.5 during mouse liver development, and also 52 Epcam-positive cholangiocytes from postnatal day 3.25 mouse livers. LSPCs in developing mouse livers were identified via marker-free transcriptomic profiling. Single-cell resolution dynamic developmental trajectories of LSPCs exhibited contiguous but discrete genetic control through transcription factors and signaling pathways. The gene expression profiles of cholangiocytes were more close to that of embryonic day 11.5 rather than other later staged LSPCs, cuing the fate decision stage of LSPCs. Our marker-free approach also allows systematic assessment and prediction of isolation biomarkers for LSPCs. CONCLUSIONS: Our data provide not only a valuable resource but also novel insights into the fate decision and transcriptional control of self-renewal, differentiation and maturation of LSPCs.

Structural diversity of anti-pancreatic cancer capsimycins identified in mangrove-derived Streptomyces xiamenensis 318 and post-modification via a novel cytochrome P450 monooxygenase.

Polycyclic tetramate macrolactams (PTMs) were identified as distinct secondary metabolites of the mangrove-derived Streptomyces xiamenensis 318. Together with three known compounds-ikarugamycin (1), capsimycin (2) and capsimycin B (3)-two new compounds, capsimycin C (4) with trans-diols and capsimycin D (5) with trans-configurations at C-13/C-14, have been identified. The absolute configurations of the tert/tert-diols moiety was determined in 4 by NMR spectroscopic analysis, CD spectral comparisons and semi-synthetic method. The post-modification mechanism of the carbocyclic ring at C-14/C-13 of compound 1 in the biosynthesis of an important intermediate 3 was investigated. A putative cytochrome P450 superfamily gene, SXIM_40690 (ikaD), which was proximally localized to the ikarugamycin biosynthetic pathway, was characterized. In vivo gene inactivation and complementation experiment confirmed that IkaD catalysed the epoxide-ring formation reaction and further hydroxylation of ethyl side chain to form capsimycin G (3'). Binding affinities and kinetic parameters for the interactions between ikarugamycin (1) and capsimycin B (3) with IkaD were measured with Surface Plasmon Resonance. The intermediate compound 3' was isolated and identified as 30-hydroxyl-capsimycin B. The caspimycins 2 and 3, were transferred to methoxyl derivatives, 6 and 7, under acidic and heating conditions. Compounds 1-3 exhibited anti-proliferative activities against pancreatic carcinoma with IC50 values of 1.30-3.37 µM.

Chromatin accessibility contributes to simultaneous mutations of cancer genes.

Somatic mutations of many cancer genes tend to co-occur (termed co-mutations) in certain patterns during tumor initiation and progression. However, the genetic and epigenetic mechanisms that contribute to the co-mutations of these cancer genes have yet to be explored. Here, we systematically investigated the association between the somatic co-mutations of cancer genes and high-order chromatin conformation. Significantly, somatic point co-mutations in protein-coding genes were closely associated with high-order spatial chromatin folding. We propose that these regions be termed Spatial Co-mutation Hotspots (SCHs) and report their occurrence in different cancer types. The conserved mutational signatures and DNA sequences flanking these point co-mutations, as well as CTCF-binding sites, are also enriched within the SCH regions. The genetic alterations that are harboured in the same SCHs tend to disrupt cancer driver genes involved in multiple signalling pathways. The present work demonstrates that high-order spatial chromatin organisation may contribute to the somatic co-mutations of certain cancer genes during tumor development.

Malignancy of Cancers and Synthetic Lethal Interactions Associated With Mutations of Cancer Driver Genes.

The mutation status of cancer driver genes may correlate with different degrees of malignancy of cancers. The doubling time and multidrug resistance are 2 phenotypes that reflect the degree of malignancy of cancer cells. Because most of cancer driver genes are hard to target, identification of their synthetic lethal partners might be a viable approach to treatment of the cancers with the relevant mutations.The genome-wide screening for synthetic lethal partners is costly and labor intensive. Thus, a computational approach facilitating identification of candidate genes for a focus synthetic lethal RNAi screening will accelerate novel anticancer drug discovery.We used several publicly available cancer cell lines and tumor tissue genomic data in this study.We compared the doubling time and multidrug resistance between the NCI-60 cell lines with mutations in some cancer driver genes and those without the mutations. We identified some candidate synthetic lethal genes to the cancer driver genes APC, KRAS, BRAF, PIK3CA, and TP53 by comparison of their gene phenotype values in cancer cell lines with the relevant mutations and wild-type background. Further, we experimentally validated some of the synthetic lethal relationships we predicted.We reported that mutations in some cancer driver genes mutations in some cancer driver genes such as APC, KRAS, or PIK3CA might correlate with cancer proliferation or drug resistance. We identified 40, 21, 5, 43, and 18 potential synthetic lethal genes to APC, KRAS, BRAF, PIK3CA, and TP53, respectively. We found that some of the potential synthetic lethal genes show significantly higher expression in the cancers with mutations of their synthetic lethal partners and the wild-type counterparts. Further, our experiments confirmed several synthetic lethal relationships that are novel findings by our methods.We experimentally validated a part of the synthetic lethal relationships we predicted. We plan to perform further experiments to validate the other synthetic lethal relationships predicted by this study.Our computational methods achieve to identify candidate synthetic lethal partners to cancer driver genes for further experimental screening with multiple lines of evidences, and therefore contribute to development of anticancer drugs.