SAP deletion promotes malignant insulinoma progression by inducing CXCL12 secretion from CAFs via the CXCR4/p38/ERK signalling pathway.

Malignant insulinoma is an extremely rare type of functioning pancreatic neuroendocrine tumour with a high degree of malignancy and a high incidence of metastasis. However, it is still unclear how malignant insulinomas develop and metastasize. Serum amyloid P component (SAP), a member of the pentraxin protein family, is an acute-phase protein secreted by liver cells. The role of SAP in insulinoma and the related mechanism are still unknown. To determine the effect of SAP on insulinoma, we crossed Rip1-Tag2 mice, which spontaneously develop insulinoma, and SAP knockout (KO) mice to generate Rip1-Tag2;SAP-/- mice. We found that SAP deletion significantly promoted the growth, invasion and metastasis of malignant insulinoma through C-X-C motif chemokine ligand 12 (CXCL12) secreted by cancer-associated fibroblasts (CAFs). Further study showed that SAP deletion promoted CXCL12 secretion by CAFs through the CXCR4/p38/ERK signalling pathway. These findings reveal a novel role and mechanism of SAP in malignant insulinoma and provide direct evidence that SAP may be a therapeutic agent for this disease.

NF-κB Activator 1 downregulation in macrophages activates STAT3 to promote adenoma-adenocarcinoma transition and immunosuppression in colorectal cancer.

BACKGROUND: Adenoma-adenocarcinoma transition is a key feature of colorectal cancer (CRC) occurrence and is closely regulated by tumor-associated macrophages (TAMs) and CD8+ T cells. Here, we investigated the effect of the NF-κB activator 1 (Act1) downregulation of macrophages in the adenoma-adenocarcinoma transition. METHODS: This study used spontaneous adenoma-developing ApcMin/+, macrophage-specific Act1-knockdown (anti-Act1), and ApcMin/+; anti-Act1 (AA) mice. Histological analysis was performed on CRC tissues of patients and mice. CRC patients' data retrieved from the TCGA dataset were analyzed. Primary cell isolation, co-culture system, RNA-seq, and fluorescence-activated cell sorting (FACS) were used. RESULTS: By TCGA and TISIDB analysis, the downregulation of Act1 expression in tumor tissues of CRC patients negatively correlated with accumulated CD68+ macrophages in the tumor. Relative expression of EMT markers in the tumor enriched ACT1lowCD68+ macrophages of CRC patients. AA mice showed adenoma-adenocarcinoma transition, TAMs recruitment, and CD8+ T cell infiltration in the tumor. Macrophages depletion in AA mice reversed adenocarcinoma, reduced tumor amounts, and suppressed CD8+ T cell infiltration. Besides, macrophage depletion or anti-CD8a effectively inhibited metastatic nodules in the lung metastasis mouse model of anti-Act1 mice. CRC cells induced activation of IL-6/STAT3 and IFN-γ/NF-κB signaling and the expressions of CXCL9/10, IL-6, and PD-L1 in anti-Act1 macrophages. Anti-Act1 macrophages facilitated epithelial-mesenchymal-transition and CRC cells' migration via CXCL9/10-CXCR3-axis. Furthermore, anti-Act1 macrophages promoted exhaustive PD1+ Tim3+ CD8+ T cell formation. Anti-PD-L1 treatment repressed adenoma-adenocarcinoma transition in AA mice. Silencing STAT3 in anti-Act1 macrophages reduced CXCL9/10 and PD-L1 expression and correspondingly inhibited epithelial-mesenchymal-transition and CRC cells' migration. CONCLUSIONS: Act1 downregulation in macrophages activates STAT3 that promotes adenoma-adenocarcinoma transition via CXCL9/10-CXCR3-axis in CRC cells and PD-1/PD-L1-axis in CD8+ T cells.

CYD0281, a Bcl-2 BH4 domain antagonist, inhibits tumor angiogenesis and breast cancer tumor growth.

BACKGROUND: B-cell lymphoma 2 (Bcl-2) family proteins are key regulators of apoptosis, which possess four conserved Bcl-2 homologies (BH) domains. Among the BH domains, the BH3 domain is considered as a potent 'death domain' while the BH4 domain is required for anti-apoptotic activity. Bcl-2 can be converted to a pro-apoptotic molecule through the removal or mutation of the BH4 domain. Bcl-2 is considered as an inducer of angiogenesis, which can promote tumor vascular network formation and further afford nutrients and oxygen to promote tumor progression. However, whether disrupting the function of the BH4 domain to convert Bcl-2 into a pro-apoptotic molecule could make Bcl-2 possess the potential for anti-angiogenic therapy remains to be defined. METHODS: CYD0281 was designed and synthesized according to the lead structure of BDA-366, and its function on inducing a conformational change of Bcl-2 was further evaluated via immunoprecipitation (IP) and immunofluorescence (IF) assays. Moreover, the function of CYD0281 on apoptosis of endothelial cells was analyzed via cell viability, flow cytometry, and western blotting assays. Additionally, the role of CYD0281 on angiogenesis in vitro was determined via endothelial cell migration and tube formation assays and rat aortic ring assay. Chick embryo chorioallantoic membrane (CAM) and yolk sac membrane (YSM) models, breast cancer cell xenograft tumor on CAM and in mouse models as well as the Matrigel plug angiogenesis assay were used to explore the effects of CYD0281 on angiogenesis in vivo. RESULTS: We identified a novel potent small-molecule Bcl-2-BH4 domain antagonist, CYD0281, which exhibited significant anti-angiogenic effects both in vitro and in vivo, and further inhibited breast cancer tumor growth. CYD0281 was found to induce conformational changes in Bcl-2 through the exposure of the BH3 domain and convert Bcl-2 from an anti-apoptotic molecule into a cell death inducer, thereby resulting in the apoptosis of vascular endothelial cells. CONCLUSIONS: This study has revealed CYD0281 as a novel Bcl-2-BH4 antagonist that induces conformational changes of Bcl-2 to convert to a pro-apoptotic molecule. Our findings indicate that CYD0281 plays a crucial role in anti-angiogenesis and may be further developed as a potential anti-tumor drug candidate for breast cancer. This work also provides a potential anti-angiogenic strategy for breast cancer treatment.

LFA-1 knockout inhibited the tumor growth and is correlated with treg cells.

Cancer immunotherapy has been proven to be clinically effective in multiple types of cancers. Lymphocyte function-associated antigen 1 (LFA-1), a member of the integrin family of adhesion molecules, is expressed mainly on αß T cells. LFA-1 is associated with tumor immune responses, but its exact mechanism remains unknown. Here, two kinds of mice tumor model of LFA-1 knockout (LFA-1-/-) mice bearing subcutaneous tumor and Apc Min/+;LFA-1-/- mice were used to confirm that LFA-1 knockout resulted in inhibition of tumor growth. Furthermore, it also demonstrated that the numbers of regulatory T cells (Treg cells) in the spleen, blood, mesenteric lymph nodes were decreased in LFA-1-/- mice, and the numbers of Treg cells in mesenteric lymph nodes were also decreased in Apc Min/+;LFA-1-/- mice compared with Apc Min/+ mice. LFA-1 inhibitor (BIRT377) was administered to subcutaneous tumor-bearing LFA-1+/+ mice, and the results showed that the tumor growth was inhibited and the number of Treg cells was reduced. The analysis of TIMER tumor database indicated that LFA-1 expression is positively associated with Treg cells and TNM stage. Conclusively, this suggests that LFA-1 knockout would inhibit tumor growth and is correlated with Treg cells. LFA-1 may be one potential target for cancer immunotherapy. Video Abstract.

Postponing tumor onset and tumor progression can be achieved by alteration of local tumor immunity.

BACKGROUND: It has been known for years that the same genetic defects drive breast cancer formation, yet, the onset of breast cancer in different individuals among the same population differs greatly in their life spans with unknown mechanisms. METHODS: We used a MMTV-PyMT mouse model with different genetic backgrounds (FVB/NJ vs. C57BL/6J) to generate different cancer onset phenotypes, then profiled and analyzed the gene expression of three tumor stages in both Fvb.B6 and Fvb mice to explore the underlying mechanisms. RESULTS: We found that in contrast with the FVB/N-Tg (MMTV-PyMT) 634Mul mice (Fvb mice), mammary tumor initiation was significantly delayed and tumor progression was significantly suppressed in the Fvb.B6 mice (generated by crossing FVB/NJ with C57BL/6J mice). Transcriptome sequencing and analysis revealed that the differentially expressed genes were enriched in immune-related pathways. Flow cytometry analysis showed a higher proportion of matured dendritic cells in the Fvb.B6 mice. The plasma levels of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) were significantly reduced in the Fvb.B6 mice. IL-6 also impaired the maturation of bone marrow dendritic cells (BMDCs) of the Fvb mice in vitro. CONCLUSION: All these findings suggest that immunity levels (characterized by a reduced IL-6 level and intact DC maturation in Fvb.B6 mice) are the key factors affecting tumor onset in a murine mammary cancer model.

MMP12 knockout prevents weight and muscle loss in tumor-bearing mice.

BACKGROUND: Colorectal cancer is a malignant gastrointestinal cancer, in which some advanced patients would develop cancer cachexia (CAC). CAC is defined as a multi-factorial syndrome characterized by weight loss and muscle loss (with or without fat mass), leading to progressive dysfunction, thereby increasing morbidity and mortality. ApcMin/+ mice develop spontaneous intestinal adenoma, which provides an established model of colorectal cancer for CAC study. Upon studying the ApcMin/+ mouse model, we observed a marked decrease in weight gain beginning around week 15. Such a reduction in weight gain was rescued when ApcMin/+ mice were crossed with MMP12-/- mice, indicating that MMP12 has a role in age-related ApcMin/+-associated weight loss. As a control, the weight of MMP12-/- mice on a weekly basis, their weight were not significantly different from those of WT mice. METHODS: ApcMin/+; MMP12-/- mice were obtained by crossing ApcMin/+ mice with MMP12 knockout (MMP12 -/-) mice. Histological scores were assessed using hematoxylin-eosin (H&E) staining. MMP12 expression was confirmed by immunohistochemistry and immunofluorescence staining. ELISA, protein microarrays and quantitative Polymerase Chain Reaction (qPCR) were used to investigate whether tumor could up-regulate IL-6. Cell-based assays and western blot were used to verify the regulatory relationship between IL-6 and MMP12. Fluorescence intensity was measured to determine whether MMP12 is associated with insulin and insulin-like growth factor 1 (IGF-1) in vitro. MMP12 inhibitors were used to explore whether MMP12 could affect the body weight of ApcMin/+ mice. RESULTS: MMP12 knockout led to weight gain and expansion of muscle fiber cross-sectional area (all mice had C57BL/6 background) in ApcMin/+ mice, while inhibiting MMP12 could suppress weight loss in ApcMin/+ mice. MMP12 was up-regulated in muscle tissues and peritoneal macrophages of ApcMin/+ mice. IL-6 in tumor cells and colorectal cancer patients is up-regulation. IL-6 stimulated MMP12 secretion of macrophage. CONCLUSIONS: MMP12 is essential for controlling body weight of Apc Min/+ mice. Our study shows that it exists the crosstalk between cancer cells and macrophages in muscle tissues that tumor cells secrete IL-6 inducing macrophages to up-regulate MMP12. This study may provide a new perspective of MMP12 in the treatment for weight loss induced by CAC.

R5, a neutralizing antibody to Robo1, suppresses breast cancer growth and metastasis by inhibiting angiogenesis via down-regulating filamin A.

Breast cancer (BC) is one of the most common cancers among women in both developed and developing countries with a rising incidence. Using the MMTV-PyMT transgenic mouse model and xenografted breast cancer model, we found that R5, a neutralizing antibody to Robo1, significantly inhibited BC growth and metastasis. Angiogenesis is involved in the growth and metastasis of BC. Interestingly, R5 significantly decreases microvessel density in BC tissues, and inhibits blood vessel formation and development in in vivo chick embryo chorioallantoic membrane (CAM), yolk sac membrane (YSM) and Matrigel plug models. To investigate whether its anti-breast cancer efficacy is ascribed to its direct antiangiogenic properties, xenografted breast cancer model on CAM was established. Furthermore, R5 significantly reduces the tube formation of the vascular plexus on xenografted breast tumor on CAM. R5 also suppresses the migration and the tubular structure formation of human umbilical vein endothelial cells (HUVECs) by down-regulating the expression of filamin A (FLNA). These findings show that R5 has the potential to be a promising agent for the treatment of BC by suppressing the tumor-induced angiogenesis.

Expansion of cancer stem cell pool initiates lung cancer recurrence before angiogenesis.

Angiogenesis is essential in the early stage of solid tumor recurrence, but how a suspensive tumor is reactivated before angiogenesis is mostly unknown. Herein, we stumble across an interesting phenomenon that s.c. xenografting human lung cancer tissues can awaken the s.c. suspensive tumor in nude mice. We further found that a high level of insulin-like growth factor 1 (IGF1) was mainly responsible for triggering the transition from suspensive tumor to progressive tumor in this model. The s.c. suspensive tumor is characterized with growth arrest, avascularity, and a steady-state level of proliferating and apoptotic cells. Intriguingly, CD133+ lung cancer stem cells (LCSCs) are highly enriched in suspensive tumor compared with progressive tumor. Mechanistically, high IGF1 initiates LCSCs self-renewal from asymmetry to symmetry via the activation of a PI3K/Akt/ß-catenin axis. Next, the expansion of LCSC pool promotes angiogenesis by increasing the production of CXCL1 and PlGF in CD133+ LCSCs, which results in lung cancer recurrence. Clinically, a high level of serum IGF1 in lung cancer patients after orthotopic lung cancer resection as an unfavorable factor is strongly correlated with the high rate of recurrence and indicates an adverse progression-free survival. Vice versa, blocking IGF1 or CXCL1/PlGF with neutralizing antibodies can prevent the reactivation of a suspensive tumor induced by IGF1 stimulation in the mouse model. Collectively, the expansion of LCSC pool before angiogenesis induced by IGF1 is a key checkpoint during the initiation of cancer relapse, and targeting serum IGF1 may be a promising treatment for preventing recurrence in lung cancer patients.

Decylubiquinone suppresses breast cancer growth and metastasis by inhibiting angiogenesis via the ROS/p53/ BAI1 signaling pathway.

Breast cancer is one of the most common cancers worldwide with a rising incidence, and is the leading cause of cancer-related death among females. Angiogenesis plays an important role in breast cancer growth and metastasis. In this study, we identify decylubiquinone (DUb), a coenzyme Q10 analog, as a promising anti-breast cancer agent through suppressing tumor-induced angiogenesis. We screened a library comprising FDA-approved drugs and found that DUb significantly inhibits blood vessel formation using in vivo chick embryo chorioallantoic membrane (CAM) and yolk sac membrane (YSM) models. DUb was further identified to inhibit angiogenesis in the rat aortic ring and Matrigel plug assay. Moreover, DUb was found to suppress breast cancer growth and metastasis in the MMTV-PyMT transgenic mouse and human xenograft tumor models. To explore whether the anticancer efficacy of DUb was directly corrected with tumor-induced angiogenesis, the MDA-MB-231 breast cancer assay on the CAM was performed. Interestingly, DUb significantly inhibits the angiogenesis of breast cancer on the CAM. Brain angiogenesis inhibitor 1 (BAI1), a member of the G protein-coupled receptor (GPCR) adhesion subfamily, has an important effect on the inhibition of angiogenesis. Further studies demonstrate that DUb suppresses the formation of tubular structures by regulating the reactive oxygen species (ROS)/p53/BAI1 signaling pathway. These results uncover a novel finding that DUb has the potential to be an effective agent for the treatment of breast cancer by inhibiting tumor-induced angiogenesis.

Knocking out matrix metalloproteinase 12 causes the accumulation of M2 macrophages in intestinal tumor microenvironment of mice.

MMP12 is mainly secreted by macrophages, is involved in macrophage development, and decomposes the extracellular matrix. Herein, we investigated whether macrophages would change in the intestinal tumor microenvironment after MMP12 knockout. ApcMin/+;MMP12-/-mice were obtained by crossbreeding ApcMin/+ mice with MMP12 knockout mice (MMP12-/- mice). The data showed that the number and volume of intestinal tumors were significantly increased in ApcMin/+;MMP12-/- mice compared with ApcMin/+ mice. Additionally, the tumor biomarkers CA19-9, CEA, and ß-catenin appeared relatively early in intestinal tumors in ApcMin/+;MMP12-/- mice. The results demonstrated that knocking out MMP12 accelerated the tumor growth and pathological process. On further investigation of its mechanism, the proportions of M2 macrophages in the spleen and among peritoneal macrophages were significantly up-regulated in ApcMin/+;MMP12-/- mice. Expression of M2 macrophage-related genes was up-regulated in tumor and peritoneal macrophages. The M2-related cytokine levels of IL-4 and IL-13 were increased in the serum of ApcMin/+;MMP12-/-mice. In vitro, bone marrow-derived M2 macrophages were obtained by treating bone marrow cells with IL-4 and IL-13, and these M2 macrophages secreted cytokines being changed. This finding reveals the crucial role of MMP12 in macrophage development and provides a new target for the control of macrophage polarization. Knocking out MMP12 causes intestinal M2 macrophage accumulation in tumor microenvironment, promoting the growth of intestinal tumors in ApcMin/+ mice.

Overexpression of MUC13, a Poor Prognostic Predictor, Promotes Cell Growth by Activating Wnt Signaling in Hepatocellular Carcinoma.

Recently RNA sequencing revealed high mucin 13 (MUC13) expression in hepatocellular carcinoma (HCC) tissues. To understand the clinicopathologic significance of MUC13 in HCC, quantitative PCR and immunohistochemistry were used to detect its expression in paired tumor tissues and nontumor tissues. The oncoprotein role of MUC13 was determined by in vitro and in vivo assays. Overexpression of MUC13 was detected in 74 of 168 primary HCC cases (44%) and was significantly associated with tumor size (P = 0.027), stage (P = 0.006), encapsulation (P = 0.044), venous invasion (P = 0.024), and poor outcome (P = 0.004). Functional studies demonstrated MUC13 had strong oncogenic activity by promoting cell growth, colony formation, cell migration, and tumor formation in nude mice. The pro-oncogenic effect of MUC13 were effectively inhibited by RNA interference. MUC13 promoted cellular G1/S phase transition by activating Wnt signaling. Mechanistically, MUC13 bound to ß-catenin and increased its phosphorylation at Ser552 and Ser675 sites, which subsequently promoted nuclear translocation of ß-catenin and up-regulation of its downstream target genes Axin2, c-Myc, and CyclinD1. Knockdown of AKT with shRNA in MUC13-overexpressing cells nullified the elevated phosphorylation of ß-catenin by MUC13. In clinical HCC samples, nuclear translocation of ß-catenin was significantly associated with MUC13 overexpression (P = 0.001). Overexpression of MUC13 plays a critical role in the development and progression of HCC by activating Wnt signaling.

Homology-independent multiallelic disruption via CRISPR/Cas9-based knock-in yields distinct functional outcomes in human cells.

BACKGROUND: Cultured human cells are pivotal models to study human gene functions, but introducing complete loss of function in diploid or aneuploid cells has been a challenge. The recently developed CRISPR/Cas9-mediated homology-independent knock-in approach permits targeted insertion of large DNA at high efficiency, providing a tool for insertional disruption of a selected gene. Pioneer studies have showed promising results, but the current methodology is still suboptimal and functional outcomes have not been well examined. Taking advantage of the promoterless fluorescence reporter systems established in our previous study, here, we further investigated potentials of this new insertional gene disruption approach and examined its functional outcomes. RESULTS: Exemplified by using hyperploid LO2 cells, we demonstrated that simultaneous knock-in of dual fluorescence reporters through CRISPR/Cas9-induced homology-independent DNA repair permitted one-step generation of cells carrying complete disruption of target genes at multiple alleles. Through knocking-in at coding exons, we generated stable single-cell clones carrying complete disruption of ULK1 gene at all four alleles, lacking intact FAT10 in all three alleles, or devoid of intact CtIP at both alleles. We have confirmed the depletion of ULK1 and FAT10 transcripts as well as corresponding proteins in the obtained cell clones. Moreover, consistent with previous reports, we observed impaired mitophagy in ULK1-/- cells and attenuated cytokine-induced cell death in FAT10-/- clones. However, our analysis showed that single-cell clones carrying complete disruption of CtIP gene at both alleles preserved in-frame aberrant CtIP transcripts and produced proteins. Strikingly, the CtIP-disrupted clones raised through another two distinct targeting strategies also produced varied but in-frame aberrant CtIP transcripts. Sequencing analysis suggested that diverse DNA processing and alternative RNA splicing were involved in generating these in-frame aberrant CtIP transcripts, and some infrequent events were biasedly enriched among the CtIP-disrupted cell clones. CONCLUSION: Multiallelic gene disruption could be readily introduced through CRISPR/Cas9-induced homology-independent knock-in of dual fluorescence reporters followed by direct tracing and cell isolation. Robust cellular mechanisms exist to spare essential genes from loss-of-function modifications, by generating partially functional transcripts through diverse DNA and RNA processing mechanisms.

Myeloid-derived suppressor cells accumulate among myeloid cells contributing to tumor growth in matrix metalloproteinase 12 knockout mice.

Myeloid-derived suppressor cells (MDSCs) are found frequently in patients and mice bearing tumors, which derived from immature myeloid cells. In healthy individuals, immature myeloid cells formed in the bone marrow differentiating to dendritic cells, macrophages and neutrophils. However, it is unclear whether some gene deficiency will lead to MDSCs accumulation in mice without bearing tumor. Here, we observed that MDSCs accumulated in the bone marrow of matrix metalloproteinase 12 knockout mice (MMP12-/- mice) compared with wild type mice (MMP12+/+ mice). And the number of CD4+ cells dramatically decreased, regulatory T cells was up-regulation and MDSCs function were determined. The results suggested that immune surveillance have been impaired in MMP12-/- transgenic mice. After intravenous administration of B16 murine melanoma cells, MMP12-/- mice developed more metastatic pulmonary nodules than MMP12+/+ mice. Meanwhile, more MDSCs appeared in the tumors of MMP12-/- mice compared with those of MMP12+/+ mice. Mechanistically, we performed a MDSC blocking assay, finding that blockade of MDSCs resulted in reducing growth of tumors in MMP12-/- mice. Furthermore, we ascertained that macrophages in MMP12-/- mice abundantly secrete IL-1ß in bone marrow which induce the accumulation of MDSCs in the bone marrow. Together, these results demonstrated that the macrophages in MMP12-/- mice could crosstalk with myeloid cells through IL-1ß, inducing MDSCs accumulation, then contributing to tumor growth. It has revealed that the critical roles of macrophage in myeloid cells differentiation.

Glipizide suppresses embryonic vasculogenesis and angiogenesis through targeting natriuretic peptide receptor A.

Glipizide, a second-generation sulfonylurea, has been widely used for the treatment of type 2 diabetes. However, it is controversial whether or not glipizide would affect angiogenesis or vasculogenesis. In the present study, we used early chick embryo model to investigate the effect of glipizide on angiogenesis and vasculogenesis, which are the two major processes for embryonic vasculature formation as well as tumor neovascularization. We found that Glipizide suppressed both angiogenesis in yolk-sac membrane (YSM) and blood island formation during developmental vasculogenesis. Glipizide did not affect either the process of epithelial to mesenchymal transition (EMT) or mesoderm cell migration. In addition, it did not interfere with separation of smooth muscle cell progenitors from hemangioblasts. Moreover, natriuretic peptide receptor A (NPRA) has been identified as the putative target for glipizide׳s inhibitory effect on vasculogenesis. When NPRA was overexpressed or activated, blood island formation was reduced. NPRA signaling may play a crucial role in the effect of glipizide on vasculogenesis during early embryonic development.

Activation of Slit2-Robo1 signaling promotes liver fibrosis.

BACKGROUND & AIMS: The secretory protein Slit2 and its receptor Robo1 are believed to regulate cell growth and migration. Here, we aimed to determine whether Slit2-Robo1 signaling mediates the pathogenesis of liver fibrosis. METHODS: Serum levels of Slit2 in patients with liver fibrosis were determined by ELISA. Liver fibrosis was induced in wild-type (WT), Slit2 transgenic (Slit2-Tg) and Robo1(+/-)Robo2(+/-) double heterozygotes (Robo1/2(+/-)) mice by carbon tetrachloride (CCl4). The functional contributions of Slit2-Robo1 signaling in liver fibrosis and activation of hepatic stellate cells (HSCs) were investigated using primary mouse HSCs and human HSC cell line LX-2. RESULTS: Significantly increased serum Slit2 levels and hepatic expression of Slit2 and Robo1 were observed in patients with liver fibrosis. Compared to WT mice, Slit2-Tg mice were much more vulnerable to CCl4-induced liver injury and more readily develop liver fibrosis. Development of hepatic fibrosis in Slit2-Tg mice was associated with a stronger hepatic expression of collagen I and α-smooth muscle actin (α-SMA). However, liver injury and hepatic expression of collagen I and α-SMA were attenuated in CCl4-treated Robo1/2(+/-) mice in response to CCl4 exposure. In vitro, Robo1 neutralizing antibody R5 and Robo1 siRNA downregulated phosphorylation of Smad2, Smad3, PI3K, and AKT in HSCs independent of TGF-ß1. R5 and Robo1 siRNA also inhibited the expression of α-SMA by HSCs. Finally, the protective effect of R5 on the CCl4-induced liver injury and fibrosis was further verified in mice. CONCLUSIONS: Slit2-Robo1 signaling promotes liver injury and fibrosis through activation of HSCs.

Characterization of 26 deletion CNVs reveals the frequent occurrence of micro-mutations within the breakpoint-flanking regions and frequent repair of double-strand breaks by templated insertions derived from remote genomic regions.

Copy number variations (CNVs) have increasingly been reported to cause, or predispose to, human disease. However, a large fraction of these CNVs have not been accurately characterized at the single-base-pair level, thereby hampering a better understanding of the mutational mechanisms underlying CNV formation. Here, employing a composite pipeline method derived from various inference-based programs, we have characterized 26 deletion CNVs [including three novel pathogenic CNVs involving an autosomal gene (EXT2) causing hereditary osteochondromas and an X-linked gene (CLCN5) causing Dent disease, as well as 23 CNVs previously identified by inference from a cohort of Canadian autism spectrum disorder families] to the single-base-pair level of accuracy from whole-genome sequencing data. We found that breakpoint-flanking micro-mutations (within 22 bp of the breakpoint) are present in a significant fraction (5/26; 19%) of the deletion CNVs. This analysis also provided evidence that a recently described error-prone form of DNA repair (i.e., repair of DNA double-strand breaks by templated nucleotide sequence insertions derived from distant regions of the genome) not only causes human genetic disease but also impacts on human genome evolution. Our findings illustrate the importance of precise CNV breakpoint delineation for understanding the underlying mutational mechanisms and have implications for primer design in relation to the detection of deletion CNVs in clinical diagnosis.

Maelstrom promotes hepatocellular carcinoma metastasis by inducing epithelial-mesenchymal transition by way of Akt/GSK-3ß/Snail signaling.

UNLABELLED: Amplification of 1q is one of the most frequent chromosomal alterations in human hepatocellular carcinoma (HCC). In this study we identified and characterized a novel oncogene, Maelstrom (MAEL), at 1q24. Amplification and overexpression of MAEL was frequently detected in HCCs and significantly associated with HCC recurrence (P = 0.031) and poor outcome (P = 0.001). Functional study demonstrated that MAEL promoted cell growth, cell migration, and tumor formation in nude mice, all of which were effectively inhibited when MAEL was silenced with short hairpin RNA (shRNAs). Further study found that MAEL enhanced AKT activity with subsequent GSK-3ß phosphorylation and Snail stabilization, finally inducing epithelial-mesenchymal transition (EMT) and promoting tumor invasion and metastasis. In addition, MAEL up-regulated various stemness-related genes, multidrug resistance genes, and cancer stem cell (CSC) surface markers at the messenger RNA (mRNA) level. Functional study demonstrated that overexpression of MAEL increased self-renewal, chemoresistance, and tumor metastasis. CONCLUSION: MAEL is an oncogene that plays an important role in the development and progression of HCC by inducing EMT and enhancing the stemness of HCC.

Slit2 promotes tumor growth and invasion in chemically induced skin carcinogenesis.

Slit, a neuronal guidance cue, binds to Roundabout (Robo) receptors to modulate neuronal, leukocytic, and endothelial migration. Slit has been reported to have an important effect on tumor growth and metastasis. In the current study, we evaluated the role of Slit2 in skin tumor growth and invasion in mice using a two-step chemical carcinogenesis protocol. We found that Slit2 expression correlated with the loss of basement membrane in the samples of human skin squamous cell carcinoma at different stages of disease progression. Slit2-Tg mice developed significantly more skin tumors than wild-type mice. Furthermore, the skin tumors that occurred in Slit2-Tg mice were significantly larger than those in the wild-type mice 10 weeks after 7,12-dimethylbenz[a]anthracene initiation until the end of the experiment. We also found that pathological development of the wild-type mice was delayed compared with that of Slit2-Tg mice. To further investigate the mechanism of increasing tumors in Slit2-Tg mice, we analyzed the expression of 5-bromo-2'-deoxyuridine (BrdU) in mouse skin lesions and found that the number of BrdU-positive cells and microvessel density in skin lesions were significantly higher in Slit2-Tg mice than in wild-type mice. Histological staining of PAS and type IV collagen and the colocalization of Slit2 and type IV collagen demonstrated varying degrees of loss of the basement membrane in the skin lesions from Slit2-Tg mice that were at the stage of carcinoma in situ. However, the basement membrane was well defined in the wild-type mice. In addition, MMP2, but not MMP9, was upregulated in the skin tissue of Slit2-Tg mice. Interruption of Slit2-Robo1 signaling by the antibody R5 significantly repressed the invasive capability of the squamous cell carcinoma cell line A431. Taken together, our findings reveal that Slit2 promotes DMBA/TPA-induced skin tumorigenesis by increasing cell proliferation, microvessel density, and invasive behavior of cutaneous squamous cell carcinoma, along with loss of basement membrane, by upregulation of MMP2 expression.

microRNA-146 up-regulation predicts the prognosis of non-small cell lung cancer by miRNA in situ hybridization.

Non-small cell lung cancer (NSCLC) accounts for approximately 70% of all lung cancer-related deaths worldwide. Prognostic markers are essential for the early detection of lung cancer in patients. In this study, we first identified microRNA146 (miR-146) expression in cancer cell lines using miRNA in situ hybridization (MISH) and confirmed the accuracy of MISH using q-RT-PCR. In addition, two different systems, BCIP/NBT and ELF, were used to detect the signal for a comparative analysis of the specificity of MISH. Compared to the BCIP/NBT system, the ELF detection system was more effective for MISH. Furthermore we detected the expression of miR-146 in NSCLC tissues (43 cases) and normal tissues (32 cases). Based on our results, we can conclude that miR-146 is more highly expressed in cancer tissue than normal tissue (t-test, P<0.05) and that miR-146 can predict the prognosis of NSCLC by MISH. Our findings preliminary demonstrate that MISH can be applied as a molecular diagnostic tool to determine the expression and localization of miRNAs in cancer tissues and that miR-146, determined by MISH, predicts the prognosis of NSCLC patients.

The 60 nm gold nanoparticles improve qPCR amplification efficiency through specific palindromic sequences (GGATCC or ACCGGT) in primers.

BACKGROUND: Polymerase chain reaction (PCR) technology and quantitative real-time PCR (qPCR) technology are widely used in clinical diagnosis and research, but amplification efficiency and sensitivity are still key problems for researchers. An increasing number of reports show that gold nanoparticles (AuNPs) can be used to improve the sensitivity and amplification efficiency of PCR. Here, we found that 60 nm gold nanoparticles with a positive charge (60 nm- Au+) can enhance the amplification efficiency of qPCR. METHODS: Mouse DNA was extracted by the alkaline lysis method. Primer 5.0 software was used to design primers and mutation primers, and the DNA fragments were obtained by the method of synthesizing plasmids. The qPCR was applied to amplify target gene fragments. RESULTS: The amplification efficiency of qPCR was improved by about 1.828 times in the experimental group with 60 nm- Au+ compared with the control group without 60 nm- Au+. The primer pair contains a specific palindromic sequence (GGATCC or ACCGGT). And 60 nm Au+ did not enhance the amplification efficiency of qPCR when the above primer was mutated. CONCLUSIONS: The primers contain special palindrome sequences (GGATCC or ACCGGT) with 60 nm- Au+ can enhance the amplification efficiency of qPCR. Therefore, it suggests a more in-depth understanding of the mechanism and function of gold nanoparticles and primer sequences. This study has presented some implications for gold nanoparticles application in the development of qPCR technology.