Functional synergy of a human-specific and an ape-specific metabolic regulator in human neocortex development.

Metabolism has recently emerged as a major target of genes implicated in the evolutionary expansion of human neocortex. One such gene is the human-specific gene ARHGAP11B. During human neocortex development, ARHGAP11B increases the abundance of basal radial glia, key progenitors for neocortex expansion, by stimulating glutaminolysis (glutamine-to-glutamate-to-alpha-ketoglutarate) in mitochondria. Here we show that the ape-specific protein GLUD2 (glutamate dehydrogenase 2), which also operates in mitochondria and converts glutamate-to-αKG, enhances ARHGAP11B's ability to increase basal radial glia abundance. ARHGAP11B + GLUD2 double-transgenic bRG show increased production of aspartate, a metabolite essential for cell proliferation, from glutamate via alpha-ketoglutarate and the TCA cycle. Hence, during human evolution, a human-specific gene exploited the existence of another gene that emerged during ape evolution, to increase, via concerted changes in metabolism, progenitor abundance and neocortex size.

Apolipoprotein E2 inhibits mitochondrial apoptosis in pancreatic cancer cells through ERK1/2/CREB/BCL-2 signaling.

BACKGROUND: Apolipoprotein E2 (ApoE2) is a pleiotropic protein that influences several aspects of cancer metabolism and development. Evading apoptosis is a vital factor for facilitating cancer cell growth. However, the role and mechanism of ApoE2 in regulating cell apoptosis of pancreatic cancer remain unclear. METHODS: In this study, we firstly detected the mRNA and protein expressions of ApoE2 in PANC-1 and Capan-2 cells by real-time polymerase chain reaction and Western blotting. We then performed TUNEL and flow cytometric analyses to explore the role of recombinant human ApoE2, pCMV6-ApoE2 and siApoE2 in the apoptosis of PANC-1 and Capan-2 cells. Furthermore, we investigated the molecular mechanism through which ApoE2 affected apoptosis in PANC-1 cells using immunofluorescence, immunoprecipitation, Western blotting and co-immunoprecipitation analysis. RESULTS: ApoE2 phosphorylated ERK1/2 and inhibited pancreatic cancer cell apoptosis. In addition, our data showed that ApoE2/ERK1/2 altered the expression and mitochondrial localization of BCL-2 via activating CREB. ApoE2/ERK1/2/CREB also increased the total BCL-2/BAX ratio, inhibited the opening of the mitochondrial permeability transition pore and the depolarization of mitochondrial transmembrane potential, blocked the leakage of cytochrome-c and the formation of the apoptosome, and consequently, suppressed mitochondrial apoptosis. CONCLUSIONS: ApoE2 regulates the mitochondrial localization and expression of BCL-2 through the activation of the ERK1/2/CREB signaling cascade to evade the mitochondrial apoptosis of pancreatic cancer cells. ApoE2 may be a distinct prognostic marker and a potential therapeutic target for pancreatic cancer.

Unveiling the improved targeting migration of mesenchymal stem cells with CXC chemokine receptor 3-modification using intravital NIR-II photoacoustic imaging.

BACKGROUND: Therapy with genetically modified mesenchymal stem cells (MSCs) has clinical translation promise. Optimizing the targeting migratory ability of MSCs relies on accurate imaging of the distribution and extravasation kinetics of MSCs, and the corresponding imaging results could be used to predict therapeutic outcomes and guide the optimization of the treatment program. Among the different imaging modalities, second near-infrared (NIR-II) optical-resolution photoacoustic microscopy (OR-PAM) has merits, including a fine resolution, a deep penetration, a high sensitivity, and a large signal-to-background ratio. It would be an ideal candidate for precise monitoring of MSCs, although it has not been tested for this purpose so far. RESULTS: Penetrating peptide-decorated conjugated polymer nanoparticles (TAT-CPNPs) with strong NIR-II absorbance were used to label chemokine-receptor genetically modified MSCs, which were subsequently evaluated under intravital NIR-II OR-PAM regarding their targeting migratory ability. Based on the upregulation of chemokine (C-X-C motif) ligand 10 in the inflamed ears of contact hypersensitivity mice, MSCs with overexpression of corresponding receptor, chemokine (C-X-C motif) receptor 3 (Cxcr3) were successfully generated (MSCCxcr3). TAT-CPNPs labeling enabled NIR-II photoacoustic imaging to discern MSCCxcr3 covered by 1.2 cm of chicken breast tissue. Longitudinal OR-PAM imaging revealed enhanced inflammation-targeting migration of MSCCxcr3 over time attributed to Cxcr3 gene modification, which was further validated by histological analysis. CONCLUSIONS: TAT-CPNPs-assisted NIR-II PA imaging is promising for monitoring distribution and extravasation kinetics of MSCs, which would greatly facilitate optimizing MSC-based therapy.

Dual and opposing roles of the androgen receptor in VETC-dependent and invasion-dependent metastasis of hepatocellular carcinoma.

BACKGROUND & AIMS: Contradictory roles of the androgen receptor (AR) in hepatocellular carcinoma (HCC) metastasis have been reported. We have shown that VETC (vessels encapsulating tumor clusters) mediates invasion-independent metastasis, whereas VETC- HCCs metastasize in an invasion-dependent manner. Herein, we aimed to reveal the roles of AR in HCC metastasis. METHODS: Mouse xenograft models, clinical samples, and cell models were used. RESULTS: AR expression was significantly lower in HCCs with a VETC pattern, portal vein tumor thrombus, endothelium-coated microemboli or high recurrence rates. Overexpressing AR in VETC+ hepatoma cells suppressed VETC formation and intrahepatic metastasis but promoted pulmonary metastasis of mouse xenografts. AR decreased the transcription of Angiopoietin-2 (Angpt2), a factor essential for VETC formation, by binding to the Angpt2 promoter. The roles of AR in inhibiting VETC formation and intrahepatic metastasis were attenuated by restoring Angpt2 expression, suggesting that AR may repress VETC-dependent intrahepatic metastasis by inhibiting Angpt2 expression and VETC formation. On the other hand, AR upregulated Rac1 expression, promoted lamellipodia formation and increased cell migration/invasion. A Rac1 inhibitor abrogated the AR-mediated promotion of migration/invasion and pulmonary metastasis of VETC+ hepatoma cells, but did not affect the AR-mediated inhibition of intrahepatic metastasis. Furthermore, an AR inhibitor decreased Rac1 expression and attenuated both intrahepatic and pulmonary metastasis of VETC- xenografts, an effect which was abrogated by restoring Rac1 expression. These data indicate that AR may facilitate the lung metastasis of VETC+ HCCs and both the liver/lung metastases of VETC- HCCs by upregulating Rac1 expression and then promoting migration/invasion. CONCLUSION: AR plays dual and opposing roles in VETC-dependent and invasion-dependent metastasis, which highlights the complex functions of AR and the importance of individualized cancer therapy. LAY SUMMARY: In this study, we uncovered the dual and opposing roles of the androgen receptor in VETC (vessels encapsulating tumor clusters)-dependent and invasion-dependent metastasis of hepatocellular carcinoma (HCC). We elucidated the underlying mechanisms of these processes, which provided novel insights into the complex regulatory network of the androgen receptor in HCC metastasis and may have important implications for precision medicine.

Intravital NIR-II three-dimensional photoacoustic imaging of biomineralized copper sulfide nanoprobes.

Photoacoustic (PA) imaging with functional nanoprobes in the second near-infrared region (NIR-II, 1000-1700 nm) has aroused much interest due to its deep tissue penetration and high maximum laser permissible exposure. However, most NIR-II PA imaging is performed using the two-dimensional (2D) imaging modality, which impedes the comprehension of the in vivo biodistribution, angiography and molecular-targeted performance of NIR-II nanoprobes (NPs). Herein, we report the systematic monitoring of biomineralized copper sulfide (CuS) NPs, typical NIR-II NPs, in mouse models by employing NIR-II three-dimensional (3D) PA imaging. The advanced imaging modality provides dynamic information about the 3D biodistribution and metabolic pathway of CuS NPs. We also achieved contrast-enhanced 3D PA imaging of abdominal and cerebral vessels at a high signal-to-background ratio. Moreover, the tumor-targeted CuS NPs conjugated with the bombesin peptide endowed NIR-II 3D PA with superior performance in imaging orthotopic tumors both deep in the prostate and in the brain beneath the intact scalp and skull. Our results highlight the potential of NIR-II 3D PA imaging for the evaluation of the in vivo behavior of NPs, thus providing a promising strategy for screening NPs in clinical translational studies.

Contrast-Enhanced Ultrasound for Biliary Ischemia: A Possible New Clinical Indication.

OBJECTIVES: Biliary perfusion is considered to contribute to biliary diseases, but routine imaging methods are insufficient to show it. This research investigated the ability of contrast-enhanced ultrasound (CEUS) for biliary perfusion in a biliary ischemia model. METHODS: This research consisted of 2 parts. First, to determine whether CEUS enhancement of the tiny biliary wall represents biliary perfusion, a vascular tracer was used as a reference to evaluate the consistency with the enhancement of the biliary wall on CEUS and the staining by the vascular tracer under the conditions of occluded and recovered biliary perfusion. In the second part, the ability of CEUS for biliary ischemia was further evaluated with microvascular density measurement as a reference. The enhancement patterns were assigned CEUS scores, in which higher scores meant more decreased enhancement, and the diagnostic ability of CEUS was assessed by a receiver operating characteristic curve analysis. RESULTS: The biliary wall was unstained by the vascular tracer and nonenhanced on CEUS when biliary perfusion was interrupted and was stained blue and enhanced after recovery. The biliary wall in the ischemia surgery group showed lower microvascular density measurements (P < .001), decreased enhancement levels (P < .001), and higher CEUS scores (P < .001). When a CEUS score of 3 or higher (obvious decrease of the biliary wall to hypoenhancement or nonenhancement in the arterial phase or rapid wash-out to nonenhancement in the portal venous phase) was applied, CEUS had sensitivity of 87.8%, specificity of 98.3%, accuracy of 93.8%, and an area under the receiver operating characteristic curve of 0.98. CONCLUSIONS: Contrast enhancement of the biliary wall on CEUS represents biliary perfusion and has reasonably good diagnostic performance for biliary ischemia in an experimental animal setting.

Clinical and Contrast-enhanced Ultrasound Characteristics of Epithelioid and Classic Hepatic Angiomyolipoma: Comparison With Alpha-fetoprotein-negative Hepatocellular Carcinoma.

Hepatic angiomyolipoma (HAML) comprises epithelioid angiomyolipoma (EAML) and classic hepatic angiomyolipoma (CAML). The imaging appearance of HAML varies widely, and EAML is more easily misdiagnosed as hepatocellular carcinoma (HCC) than as CAML. The clinical and contrast-enhanced ultrasound (CEUS) features of CAML, EAML and HCC with negative alpha-fetoprotein protein expression (HCC[AFP-]) were retrospectively reviewed. The hyper-vascular type was more commonly found in CAML and EAML lesions than in HCC lesions. Most lesions were hyper-enhanced in the arterial phase. CAMLs showed prolonged hyper-enhancement or iso-enhancement during the portal and late phases on CEUS, making them easily distinguishable from HCC(AFP-). Some EAML lesions (41.7%) were hypo-echoic, similar to HCC(AFP-). However, the hypo-enhancement of EAML lesions occurred later than that of HCC(AFP-) lesions. Thus, our findings may be useful in distinguishing among these lesions to improve diagnostic accuracy.

Optical-resolution photoacoustic microscopy for monitoring vascular normalization during anti-angiogenic therapy.

Monitoring the changes in tumor vascularity is important for anti-angiogenic therapy assessment with therapeutic implications. However, monitoring vascularity is quite challenging due to the lack of appropriate imaging techniques. Here, we describe a non-invasive imaging technique using optical-resolution photoacoustic microscopy (OR-PAM) to track vascular changes in prostate cancer treated with an anti-angiogenic agent, DC101, on a mouse ear xenograft model. Approximately 1-3 days after the initial therapy, OR-PAM imaging detected tumor vascular changes such as reduced vessel tortuosity, decreased vessel diameter and homogenized intratumoral vessel distribution. These observations indicated vessel normalization, which was pathologically validated as increased fractional pericyte coverage, functional perfusion and drug delivery of the vessels. After four DC101 interventions, OR-PAM imaging eventually revealed intratumoral vessel regression. Therefore, OR-PAM imaging of the vasculature offers a promising method to study anti-angiogenic drug mechanisms of action in vivo and holds potential in monitoring and guiding anti-angiogenic therapy.

In silico mining and FISH mapping of a chromosome-specific satellite DNA in Capsicum annuum L.

BACKGROUND: A large proportion of eukaryote nuclear genomes is composed of repetitive DNA. Tracing the dynamics of repetitive elements in the genomes of related taxa can reveal important information about their phylogenic relationships as well as traits that have become distinct to a lineage. OBJECTIVE: Study the genomic abundance and chromosomal location of repetitive DNA in Capsicum annuum L. to understand the repeat dynamics. METHOD: We quantified repeated DNA content in the C. annuum genome using the RepeatExplorer pipeline. RESULTS: About 42% of the C. annuum genome dataset comprised repetitive elements. Of these, 0.011, 0.98, 3.09, and 0.024% represented high and low confidence satellite repeats, putative long-terminal repeats (LTRs), and rDNA sequences, respectively. One novel high confidence 167-bp satellite repeat with a genomic proportion of 0.011%, Ca167TR, was identified. Furthermore, FISH with Ca167TR on metaphase chromosomes of C. annuum revealed signals in the subtelomeric regions of the short and long arms of chromosome 3 and 4, respectively. CONCLUSION: Further understanding of the origin and associated functions of Ca167TR and other repeats in Capsicum will give us insights into the genomic relationships and functions of the genome.

Pre-labelled oligo probe-FISH karyotype analyses of four Araliaceae species using rDNA and telomeric repeat.

BACKGROUND: The family Araliaceae contains many medicinal species including ginseng of which the whole genome sequencing analyses have been going on these days. OBJECTIVE: To characterize the chromosomal distribution of 5S and 45S rDNAs and telomeric repeat in four ginseng related species of Aralia elata (Miq.) Seem., Dendropanax morbiferus H. Lév., Eleutherococcus sessiliflorus (Rupr. Et Maxim.) Seem., Kalopanax septemlobus (Thunb. ex A.Murr.) Koidz. METHOD: Pre-labelled oligoprobe (PLOP)-fluorescence in situ hybridization (FISH) was carried out. RESULTS: The chromosome number of A. elata was 2n = 24, whereas that of the other three species of D. morbiferus, E. sessiliflorus, and K. septemlobus was 2n = 48, corresponding to diploid and tetraploid, respectively, based on the basic chromosome number x = 12 in Araliaceae. In all four species, one pair of 5S signals were detected in the proximal regions of the short arms of chromosome 3, whereas in K. septemlobus, the 5S rDNA signals localized in the subtelomeric region of short arm of chromosome 3, while all the 45S rDNA signals localized at the paracentromeric region of the short arm of chromosome 1. And the telomeric repeat signals were detected at the telomeric region of both short and long arms of most chromosomes. CONCLUSION: The PLOP-FISH was very effective compared with conventional FISH method. These results provide useful comparative cytogenetic information to better understand the genome structure of each species and will be useful to trace the history of ginseng genomic constitution.

MicroRNAs miR-125b and miR-100 suppress metastasis of hepatocellular carcinoma by disrupting the formation of vessels that encapsulate tumour clusters.

We have previously shown that vessels that encapsulated tumour cluster (VETC), a prevalent vascular pattern in hepatocellular carcinoma (HCC), facilitates the entry of the whole tumour cluster into the bloodstream in an invasion-independent manner, and that angiopoietin 2 (Angpt2), the levels of which are increased in HCC cells, is essential for VETC formation. However, the mechanisms underlying VETC formation remains unclear. Herein, we characterized miR-125b and miR-100 as novel VETC suppressors by using human HCC specimens, and cell and animal models. We showed that reduced expression of either miR-125b or miR-100 in human HCC tissues was significantly associated with the presence of VETC, venous invasion of tumour cells, and the occurrence of endothelium-coated microemboli. To confirm the role of miR-125b and miR-100 in VETC formation and HCC metastasis, cell lines with stable miR-125b and miR-100 expression were established by using human VETC-2 cells and mouse Hepa1-6 cells, the hepatoma cells that developed xenografts with VETC patterns. Our results showed that expression of miR-125b or miR-100 in VETC-2 and Hepa1-6 cells dramatically reduced VETC formation in xenografts, and consequently inhibited in vivo metastasis, suggesting that miR-125b and miR-100 may attenuate metastasis by repressing VETC formation. Further investigation revealed that miR-125b directly suppressed the expression of Angpt2 by binding to its 3'-untranslated region, whereas miR-100 reduced the protein level of Angpt2 by targeting mechanistic target of rapamycin (MTOR) and blocking the MTOR-p70S6K signalling pathway. Moreover, the suppressive effect of miR-125b and miR-100 on VETC formation was abrogated by injecting Angpt2-expressing viruses into xenografts. Taken together, our findings imply that miR-125b and miR-100 negatively regulate Angpt2 expression through different mechanisms, in turn inhibit VETC formation, and consequently abrogate the VETC-dependent metastasis of hepatoma cells. This study uncovers new regulatory mechanisms of VETC formation, identifies novel functions of miR-125b and miR-100, and provides new targets for antimetastasis therapy of HCC. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A novel vascular pattern promotes metastasis of hepatocellular carcinoma in an epithelial-mesenchymal transition-independent manner.

UNLABELLED: Early metastasis is responsible for frequent relapse and high mortality of hepatocellular carcinoma (HCC), but its underlying mechanisms remain unclear. Epithelial-mesenchymal transition (EMT) has been considered a key event in metastasis. Based on histological examination of serial HCC sections and three-dimensional reconstruction, we found a novel and prevalent vascular pattern, vessels that encapsulated tumor clusters (VETC) and formed cobweb-like networks. The presence of VETC (VETC(+) ) predicted higher metastasis and recurrence rates of HCC. Using clinical samples and mouse xenograft models, we further showed that VETC was composed of functional vessels with blood perfusion and induced by tumor cells at the early stage of HCC. Subsequent investigations revealed that HCC cell-derived angiopoietin-2 was a prerequisite for VETC formation and that the VETC pattern was a critical factor promoting HCC metastasis as knockdown of angiopoietin-2 abolished this vascular pattern and consequently attenuated in vivo tumor metastasis. Interestingly, abrogation of EMT by knockdown of Snail or Slug significantly diminished in vivo metastasis of VETC(-) xenografts but did not affect that of VETC(+) ones, although silencing of Snail or Slug substantially reduced the in vitro migration of both VETC(+) and VETC(-) HCC cells. In contrast to human VETC(-) cases, EMT signatures were rarely observed in VETC(+) cases with metastatic potential. Further analysis revealed that VETC provided an efficient metastasis mode by facilitating the release of whole tumor clusters into the bloodstream. CONCLUSION: Our findings identify a novel metastasis mechanism that relies on vascular pattern but is independent of EMT, which may provide new targets for antimetastasis therapy and offer a basis for selecting patients who may benefit from certain molecularly targeted drugs.

Downregulation of microRNA-100 enhances the ICMT-Rac1 signaling and promotes metastasis of hepatocellular carcinoma cells.

Metastasis is responsible for rapid recurrence of hepatocellular carcinoma (HCC) and poor survival of HCC patients. Here we showed that miR-100 downregulation in HCC tissues was significantly associated with venous invasion, advanced TNM stage, tumor nodule without complete capsule, poorer cell differentiation, and shorter recurrence-free survival. Both gain- and loss-of-function studies showed that miR-100 dramatically suppressed the ability of HCC cells to migrate and to invade through Matrigel in vitro. Analyses using mouse orthotopic xenograft model further revealed that xenografts of miR-100-stable-expressing HCC cells displayed a significant reduction in pulmonary metastasis, compared with control group. Subsequent investigations revealed that miR-100 directly inhibited the expression of isoprenylcysteine carboxyl methyltransferase (ICMT) and ras-related C3 botulinum toxin substrate 1 (Rac1) by binding to their 3'-UTRs, and in turn suppressed lamellipodia formation and matrix metallopeptidase 2 (MMP2) activation. Furthermore, knockdown of ICMT and Rac1 phenocopied the anti-metastasis effect of miR-100, whereas overexpression of the constitutively active Rac1 (Q61L) antagonized the function of miR-100. Taken together, miR-100 represses metastasis of HCC cells by abrogating the ICMT-Rac1 signaling. Downregulation of miR-100 contributes to HCC metastasis and the restoration of miR-100 is a potential strategy for cancer therapy.

MicroRNA-29b suppresses tumor angiogenesis, invasion, and metastasis by regulating matrix metalloproteinase 2 expression.

UNLABELLED: Hepatocellular carcinoma (HCC) is a highly vascularized tumor with frequent intrahepatic metastasis. Active angiogenesis and metastasis are responsible for rapid recurrence and poor survival of HCC. We previously found that microRNA-29b (miR-29b) down-regulation was significantly associated with poor recurrence-free survival of HCC patients. Therefore, the role of miR-29b in tumor angiogenesis, invasion, and metastasis was further investigated in this study using in vitro capillary tube formation and transwell assays, in vivo subcutaneous and orthotopic xenograft mouse models, and Matrigel plug assay, and human HCC samples. Both gain- and loss-of-function studies showed that miR-29b dramatically suppressed the ability of HCC cells to promote capillary tube formation of endothelial cells and to invade extracellular matrix gel in vitro. Using mouse models, we revealed that tumors derived from miR-29b-expressed HCC cells displayed significant reduction in microvessel density and in intrahepatic metastatic capacity compared with those from the control group. Subsequent investigations revealed that matrix metalloproteinase-2 (MMP-2) was a direct target of miR-29b. The blocking of MMP-2 by neutralizing antibody or RNA interference phenocopied the antiangiogenesis and antiinvasion effects of miR-29b, whereas introduction of MMP-2 antagonized the function of miR-29b. We further disclosed that miR-29b exerted its antiangiogenesis function, at least partly, by suppressing MMP-2 expression in tumor cells and, in turn, impairing vascular endothelial growth factor receptor 2-signaling in endothelial cells. Consistently, in human HCC tissues and mouse xenograft tumors miR-29b level was inversely correlated with MMP-2 expression, as well as tumor angiogenesis, venous invasion, and metastasis. CONCLUSION: miR-29b deregulation contributes to angiogenesis, invasion, and metastasis of HCC. Restoration of miR-29b represents a promising new strategy in anti-HCC therapy.