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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.

Background As a rare subtype of prostate carcinoma, basal cell carcinoma (BCC) has not been studied extensively and thus lacks systematic molecular characterization. Methods Here, we applied single-cell genomic amplification and RNA-Seq to a specimen of human prostate BCC (CK34βE12+ /P63+ /PAP- /PSA- ). The mutational landscape was obtained via whole exome sequencing of the amplification mixture of 49 single cells, and the transcriptomes of 69 single cells were also obtained. Results The five putative driver genes mutated in BCC are CASC5, NUTM1, PTPRC, KMT2C, and TBX3, and the top three nucleotide substitutions are C>T, T>C, and C>A, similar to common prostate cancer. The distribution of the variant allele frequency values indicated that these single cells are from the same tumor clone. The 69 single cells were clustered into tumor, stromal, and immune cells based on their global transcriptomic profiles. The tumor cells specifically express basal cell markers like KRT5, KRT14, and KRT23 and epithelial markers EPCAM, CDH1, and CD24. The transcription factor covariance network analysis showed that the BCC tumor cells have distinct regulatory networks. By comparison with current prostate cancer datasets, we found that some of the bulk samples exhibit basal cell signatures. Interestingly, at single-cell resolution the gene expression patterns of prostate BCC tumor cells show uniqueness compared with that of common prostate cancer-derived circulating tumor cells. Conclusions This study, for the first time, discloses the comprehensive mutational and transcriptomic landscapes of prostate BCC, which lays a foundation for the understanding of its tumorigenesis mechanism and provides new insights into prostate cancers in general.

Recently, some reports show that Ligand of Numb Protein-X 1 (LNX1) could be a suppressor gene in gliomas, while our current research has firstly shown that PDZ domain containing ring finger 4 (PDZRN4), another member of LNX family, could also be a potential suppressor in hepatocellular carcinoma (HCC). PDZRN4, also named LNX4 (Ligand of Numb Protein-X 4), is a member of the LNX family. We recently found that PDZRN4, but not LNX1, was down-regulated in HCC samples, and the role of PDZRN4 in the progression of HCC had not been studied before. To address this question, firstly, we evaluated the expression of PDZRN4 in HCC samples and adjacent non-cancerous tissues. Semi-quantitative polymerase chain reaction showed that PDZRN4 was down-regulated in 24/36 (66.7%) HCC samples separately. In addition, our research shows that PDZRN4 is silenced in all of the 12 HCC cell lines tested. Subsequently, cell-based functional assay exhibited that ectopic expression of PDZRN4 inhibits the proliferation, plate colony formation and anchorage-independent colony formation of HCC cells. Collectively, our results showed that PDZRN4 might be a potential tumour suppressor gene and had anti-proliferative effect on HCC cell proliferation, which would be of great significance to the researches on HCC.

Oncogene activation or inactivation of tumor suppressor genes are crucial to tumor initiation and progression. DNA copy number amplification is one of many mechanisms that activate oncogenes in many tumors, including hepatocellular carcinoma (HCC). Although it has been known that some oncogenes such as c-myc amplification is involved in HCC pathogenesis, more oncogenes with DNA copy amplification contribute to HCC initiation and progression remain to be characterized. Here, we identified NOXIN as a novel potential oncogene with DNA copy number amplification by Single Nucleotide Polymorphism microarray-based genome-wide DNA copy number analysis of 43 human HCC samples. We identified the focal DNA gain and amplification region containing NOXIN on chromosome 11q14.1 and NOXIN overexpression significantly associated with HCC progression. We then assessed the role of NOXIN in HCC cells. NOXIN overexpression promoted cellular proliferation, colony formation, cellular migration and in vivo tumorigenicity, whereas NOXIN knockdown attenuated these effects. Interestingly, NOXIN overexpression accelerated the G1-S phase transition by enhancing DNA synthesis. Furthermore, we found that NOXIN interacts with DNA polymerase α, suggesting that NOXIN may promote de novo DNA synthesis by promoting DNA polymerase-primase complex formation. These collective data indicated that NOXIN overexpression, as a result of genomic DNA gain or amplification, promotes HCC tumorigenesis by accelerating DNA synthesis and cell cycle progression, where NOXIN functions as a cofactor of DNA polymerase-primase complex by associating with DNA polymerase α.

Cancer stem-like cells represent a population of tumour-initiating cells that lead to the relapse and metastasis of cancer. Conventional anti-cancer therapeutic drugs are usually ineffective in eliminating the cancer stem-like cells. Therefore, new drugs or therapeutic methods effectively targeting cancer stem-like cells are in urgent need to successfully cure cancer. Gamboge is a natural anti-cancer medicine whose pharmacological effects are different from those of conventional chemotherapeutical drugs and they can kill some kinds of cancer cells selectively. In this study, we identified a new gamboge derivative, Compound 2 (C2), which presents eminent suppression effects on cancer cells. Interestingly, when compared with cisplatin (CDDP), C2 effectively suppresses the growth of both cancer stem-like cells and non-cancer stem-like cells derived from head and neck squamous cell carcinoma (HNSCC), inhibiting the formation of tumour spheres and colony in vitro, resulting in the loss of expression of multiple cancer stem cell (CSC)-related molecules in HNSCC. Treating with C2 effectively inhibited the growth of HNSCC in BALB/C nude mice. Further investigation found that C2 notably inhibits the activation of epithelial growth factor receptor and the phosphorylation of its downstream protein kinase homo sapiens v-akt murine thymoma viral oncogene homolog (AKT) in HNSCC, resulting in down-regulation of multiple CSC-related molecules in HNSCC. Our study has demonstrated that C2 effectively inhibits the stem-like property of cancer stem-like cells in HNSCC and may be a hopeful targeting drug in cancer therapy.

The polycomb group protein enhancer of zeste homologue 2 (EZH2), which has histone methyltransferase (HMT) activity, is overexpressed in malignant tumours. However, the role of EZH2 in colorectal cancer (CRC) invasion is little known. Here we investigated the clinical significance, biological effects, and mechanisms of EZH2 signalling. Knockdown of EZH2 significantly reduced cell invasion and secretion of matrix metalloproteinases 2/9 (MMP2/9) in in vitro studies. Knockdown of EZH2 dramatically increased overall survival and decreased metastasis of lung in in vivo studies. Conversely, overexpression of EZH2 significantly increased lung metastasis and shortened overall survival when compared with control tumours. EZH2-induced CRC cell invasion may depend on down-regulation of vitamin D receptor (VDR), which is considered to be a marker of CRC invasion. EZH2 regulates the histone trimethylation of lysine 27 (H3K27me3) in the VDR promoter. Moreover, we found that STAT3 directly binds to the EZH2 promoter and regulates VDR down-regulation in CRC cells. Significant inverse correlations were observed between the expression of EZH2 and pSTAT3 and that of VDR in CRC tissues compared with normal tissue in patients. We show the role of EZH2 in CRC metastasis and identify VDR as a target gene of EZH2. EZH2 expression may be directly regulated by STAT3, and STAT3 may play an important role in EZH2-mediated VDR down-regulation in CRC. This pathway may provide potential targets in aggressive CRC.

DNA methyltransferase inhibitors (MTIs) have recently emerged as promising chemotherapeutic or preventive agents for cancer, despite their poorly characterized mechanisms of action. The present study shows that DNA methylation is integral to the regulation of SH2-containing protein tyrosine phosphatase 1 (SHP1) expression, but not for regulation of suppressors of cytokine signalling (SOCS)1 or SOCS3 in colorectal cancer (CRC) cells. SHP1 expression correlates with down-regulation of Janus kinase/signal transducers and activators of transcription (JAK2/STAT3/STAT5) signalling, which is mediated in part by tyrosine dephosphorylation events and modulation of the proteasome pathway. Up-regulation of SHP1 expression was achieved using a DNA MTI, 5-aza-2'-deoxycytidine (5-aza-dc), which also generated significant down-regulation of JAK2/STAT3/STAT5 signalling. We demonstrate that 5-aza-dc suppresses growth of CRC cells, and induces G2 cell cycle arrest and apoptosis through regulation of downstream targets of JAK2/STAT3/STAT5 signalling including Bcl-2, p16(ink4a), p21(waf1/cip1) and p27(kip1). Although 5-aza-dc did not significantly inhibit cell invasion, 5-aza-dc did down-regulate expression of focal adhesion kinase and vascular endothelial growth factor in CRC cells. Our results demonstrate that 5-aza-dc can induce SHP1 expression and inhibit JAK2/STAT3/STAT5 signalling. This study represents the first evidence towards establishing a mechanistic link between inhibition of JAK2/STAT3/STAT5 signalling and the anticancer action of 5-aza-dc in CRC cells that may lead to the use of MTIs as a therapeutic intervention for human colorectal cancer.

The tegument proteins of schistosome have attracted the most attention in studies of host-parasite interplay, while the host proteins acting at the host-parasite interface remained largely elusive. Here, we undertook a high-throughput proteomic approach to characterize the schistosome-adsorbed host proteins. Fifty five distinct host proteins were confidently identified in S. japonicum samples, including cercaria, schistosomula, adults, eggs, and miracidia, together with tegument and eggshell preparations, of which 23 and 38 host proteins were identified in adult worms and eggs, respectively. Among the schistosome-adsorbed host proteins, host neutrophil elastases were found in the granuloma initiated by schistosome egg deposition, implying that the host innate immune molecules could participate in the granuloma formation for fighting against schistosome invasion, except for the adaptive immune system. In addition, some host proteins, such as proteinase inhibitor and superoxide dismutase, might be utilized by schistosome to counteract or attenuate the host attacks. These parasite-adsorbed host proteins will provide new insights into the host immune responses against schistosome infection, the evasive behavior of the adult worms, and the granuloma formation, which could render an in-depth understanding for the host-parasite interplay.

Overexpression of type 1 insulin-like growth factor receptor (IGF1R) contributes to the progression and metastasis of liver cancer, implying that IGF1R gene is a suitable target of RNA interference (RNAi) for liver cancer therapy. To investigate the possible regulation of IGF1R by P53, we examined the level of IGF1R expression in liver cancer cell lines in response to adriamycin. Levels of IGF1R mRNA and protein in cell lines with wild-type P53 decreased dramatically after P53 induction, but no such reduction of IGF1R was observed in cell lines with mutated P53. Inhibition of wild-type P53 in HEPG2 cells by small interfering RNA (siRNA) significantly upregulated the expression of IGF1R. IGF1R inhibition by siRNA in Huh7 cells with mutated P53 significantly depressed cell proliferation. To investigate the sensitivity of cancer cells to adriamycin after inhibition of IGF1R, we depressed IGF1R expression using siRNA, and then added adriamycin at an IC50 dose. After a further 48 h incubation with adriamycin, proliferation was significantly depressed in the cells treated with siRNA targeting IGF1R, in comparison with siRNA targeting scramble. Furthermore, both TUNEL and pro-caspase-3 expression assay showed a significant increase in apoptosis after combined treatment with adriamycin and siRNA targeting IGF1R. Our results demonstrate that IGF1R is downregulated by P53, and that siRNA targeting of IGF1R increases liver cancer cells sensitivity to adriamycin and promotes apoptosis. siRNA targeting of IGF1R could be potentially useful for increasing sensitivity to anti-cancer drugs, especially in drug-resistant cells with mutated P53.

Purpose: To analyze the gene expression pattern in rat hepatic fibrogenesis and further assess the role of some key genes during the pathological process. Methods: Hepatic fibrosis was induced by intraperitoneal injection of dimethylnitrosamine or carbon tetrachloride (CCl4) injection subcutaneously in rats, and identification of the hepatic fibrosis related genes with cDNA microarray was performed. After some key genes up-regulated during the development of hepatic fibrosis were screened and confirmed, their effects on the function of the activated rat hepatic stellate cells (HSC) were assessed using the small interfering RNA (siRNA) technique. Results: Using an Atlas rat cDNA array, a number of differentially expressed genes in fibrotic liver tissues were identified compared with non-diseased control. A total of 15 genes predominantly associated with the mitogen-activated protein kinase (MAPK) signal transduction pathway were upregulated in the fibrotic liver. Immunohistochemical study revealed that the expressions of both extracellular signal-regulated kinases (ERK) and ribosomal protein S6 kinase (RSK), two of the key genes in the MAPK pathway, were remarkably induced, which was closely correlated to that of collagen types I and III during the development of hepatic fibrosis. Transfection of siRNA targeting ERK1 mRNA (siERK1) into HSC led to a 66% and 72% reduction of ERK1 mRNA and protein expression, respectively. Furthermore, siERK1 exerted the inhibition of the proliferation of HSC, accompanied by the induction of HSC apoptosis and reduction of collagen types I and III. In addition, siERK1 abolished the effect of platelet-derived growth factor-BB on the proliferation of HSC. Conclusions: The present study provided strong evidence for the participation of the MAPK pathway in the pathogenesis of hepatic fibrosis. Selective targeting of ERK1 inhibitors to HSC might present as a novel strategy for the treatment of hepatic fibrosis.

At present, little is known about signal transduction mechanisms in schistosomes, which cause the disease of schistosomiasis. The mitogen-activated protein kinase (MAPK) signaling pathways, which are evolutionarily conserved from yeast to Homo sapiens, play key roles in multiple cellular processes. Here, we reconstructed the hypothetical MAPK signaling pathways in Schistosoma japonicum and compared the schistosome pathways with those of model eukaryote species. We identified 60 homologous components in the S. japoncium MAPK signaling pathways. Among these, 27 were predicted to be full-length sequences. Phylogenetic analysis of these proteins confirmed the evolutionary conservation of the MAPK signaling pathways. Remarkably, we identified S. japonicum homologues of GTP-binding protein β and α-I subunits in the yeast mating pathway, which might be involved in the regulation of different life stages and female sexual maturation processes as well in schistosomes. In addition, several pathway member genes, including ERK, JNK, Sja-DSP, MRAS and RAS, were determined through quantitative PCR analysis to be expressed in a stage-specific manner, with ERK, JNK and their inhibitor Sja-DSP markedly upregulated in adult female schistosomes.

Human hepatocellular carcinoma (HCC) is one of the most common tumors worldwide, in which the genetic mechanisms of oncogenesis are still unclear. To investigate whether the genomic DNA copy number alterations may contribute to primary HCC, the cDNA microarray-based comparative genomic hybridization (CGH) analysis was here performed in 41 primary HCC infected by hepatitis B virus and 12 HCC cell lines. The resulting data showed that, on average, 7.25% of genome-wide DNA copy numbers was significantly altered in those samples (4.61±2.49% gained and 2.64±1.78% lost). Gains involving 1q, 6p, 8q and 9p were frequently observed in these cases; and whilst, losses involving Ip, 16q and 19p occurred in most patients. To address the correlation between the alteration of genomic DNA copy numbers and transcriptional expression, the same cDNA microarray was further applied in 20 HCC specimens and all available cell lines to figure out the gene expression profiles of those samples. Interestingly, the genomic DNA copy number alterations of most genes appeared not to be in generally parallel with the corresponding transcriptional expression. However, the transcriptional deregulation of a few genes, such as osteopontin (SPP1), transgelin 2 (TAGLN2) and PEG10, could be ascribed partially to their genomic aberrations, although the many alternative mechanisms could be involved in the deregulation of these genes. In general, this work would provide new insights into the genetic mechanisms in hepatocarcinogenesis associated with hepatitis B virus through the comprehensive survey on correlation between genomic DNA copy number alterations and transcriptional expression.

The categorization of genes by structural distinctions relevant to biological characteristics is very important for understanding of gene functions and predicting functional implications of uncharacterized genes. It was absolutely necessary to deploy an effective and efficient strategy to deal with the complexity of the large olfactomedin-like (OLF) gene family sharing sequence similarity but playing diversified roles in many important biological processes, as the simple highest-hit homology analysis gave incomprehensive results and led to inappropriate annotation for some uncharacterized OLF members. In light of evolutionary information that may facilitate the classification of the OLF family and proper association of novel OLF genes with characterized homologs, we performed phylogenetic analysis on all 116 OLF proteins currently available, including two novel members cloned by our group. The OLF family segregated into seven subfamilies and members with similar domain compositions or functional properties all fell into relevant subfamilies. Furthermore, our Northern blot analysis and previous studies revealed that the typical human OLF members in each subfamily exhibited tissue-specific expression patterns, which in turn supported the segregation of the OLF subfamilies with functional divergence. Interestingly, the phylogenetic tree topology for the OLF domains alone was almost identical with that of the full-length tree representing the unique phylogenetic feature of full-length OLF proteins and their particular domain compositions. Moreover, each of the major functional domains of OLF proteins kept the same phylogenetic feature in defining similar topology of the tree. It indicates that the OLF domain and the various domains in flanking non-OLF regions have coevolved and are likely to be functionally interdependent. Expanded by a plausible gene duplication and domain couplings scenario, the OLF family comprises seven evolutionarily and functionally distinct subfamilies, in which each member shares similar structural and functional characteristics including the composition of coevolved and interdependent domains. The phylogenetically classified and preliminarily assessed subfamily framework may greatly facilitate the studying on the OLF proteins. Furthermore, it also demonstrated a feasible and reliable strategy to categorize novel genes and predict the functional implications of uncharacterized proteins based on the comprehensive phylogenetic classification of the subfamilies and their relevance to preliminary functional characteristics.

The known members of inhibitor of growth (ING) gene family are considered as candidate tumor suppressor genes. ING4, a novel member of ING family, is recently reported to interact with tumor suppressor p53, p300 (a major component of histone acetyl transferase complexes), and p65(RelA) subunit of NF-κB. In this study, we investigated the cellular behaviors of HepG2 cells with exogenous ING4. Interestingly, the overexpression of ING4 negatively regulated the cell growth with significant G2/M arrest of cell cycle, and moreover, enhanced the cell apoptosis triggered by serum starvation in HepG2 cells. Furthermore, the exogenous ING4 could upregulate endogenous p21 and Bax in HepG2 cells, not in p53-deficient Saos-2 cells, suggesting that G2/M arrest induced by ING4 could be mediated by the increased p21 expression in a p53-dependent manner, although there is no significant increase of p53 expression in HepG2 cells. Moreover, HepG2 cells with exogenous ING4 could significantly increase cell death, as exposed to some DNA-damage agents, such as etoposide and doxorubicin, implying that ING4 could enhance chemosensitivity to certain DNA-damage agents in HepG2 cells.