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.

COL11A1-Driven Epithelial-Mesenchymal Transition and Stemness of Pancreatic Cancer Cells Induce Cell Migration and Invasion by Modulating the AKT/GSK-3ß/Snail Pathway.

BACKGROUND: Collagen type XI α1 (COL11A1) is associated with tumorigenesis and development in many human malignancies. Previous reports indicate that COL11A1 may be a significant diagnostic marker for pancreatic ductal adenocarcinoma (PDAC); however, its biological role in PDAC progression remains unclear. In this study, we investigated the influence of COL11A1 on the invasion and migration abilities of pancreatic cancer cells and explored its potential molecular mechanisms. METHODS: Cell migration and invasion were assessed using Transwell assays in pancreatic cancer cells transfected with siCOL11A1 and pCNV3-COL11A1 plasmids. The protein and mRNA expression levels of N-cadherin, E-cadherin, Vimentin, cluster of differentiation (CD)-24, CD44, serine-threonine kinase (AKT), glycogen synthase kinase (GSK)-3ß, phospho (p)-AKTSer473, p-GSK-3ßSer9, and Snail were analyzed using Western blotting and real-time polymerase chain reaction (PCR). The effect of COL11A1 on cell stemness was tested using flow cytometry and clone formation assays. RESULTS: These results demonstrated that COL11A1 significantly promoted the invasion and migration abilities of PDAC cells. Furthermore, COL11A1 facilitated the occurrence of epithelial-mesenchymal transition (EMT) and cell stemness by upregulating the expression levels of p-AKTSer473, p-GSK-3ßSer9, and Snail. CONCLUSIONS: This study suggests that the activation of the AKT/GSK-3ß/Snail signaling pathway induced by COL11A1 plays a major role in the progression of PDAC. Therefore, COL11A1 could serve as a potential target for PDAC treatment.

Intracolic ultrasound molecular imaging: a novel method for assessing colonic tumor necrosis factor-α expression in inflammatory bowel disease.

BACKGROUND: While anti-tumor necrosis factor alpha (TNF-α) therapy has been proven effective in inflammatory bowel disease (IBD), approximately 40% of patients lose the response. Transmembrane TNF-α (mTNF-α) expression in the intestinal mucosa is correlated with therapeutic efficacy, and quantification of mTNF-α expression is significant for predicting response. However, conventional intravenous application of microbubbles is unable to assess mTNF-α expression in intestinal mucosa. Herein, we proposed intracolic ultrasound molecular imaging with TNF-α-targeted microbubbles (MBTNF-α) to quantitatively detect mTNF-α expression in the intestinal mucosa. METHODS: MBTNF-α was synthesized via a biotin-streptavidin bridging method. TNF-α-targeted ultrasound imaging was performed by intracolic application of MBTNF-α to detect mTNF-α expression in surgical specimens from a murine model and patients with IBD. Linear regression analyses were performed to confirm the accuracy of quantitative targeted ultrasound imaging. RESULTS: On quantitative TNF-α-targeted ultrasound images, a greater signal intensity was observed in the mouse colons with colitis ([1.96 ± 0.45] × 106 a.u.) compared to that of the controls ([0.56 ± 0.21] × 106 a.u., P < 0.001). Targeted US signal intensities and inflammatory lesions were topographically coupled in mouse colons. Linear regression analyses in specimens of mice and patients demonstrated significant correlations between the targeted ultrasound signal intensity and mTNF-α expression (both P < 0.001). Furthermore, TNF-α-targeted ultrasound imaging qualitatively distinguished the varying inflammatory severity in intestinal specimens from IBD patients. CONCLUSION: Intracolic ultrasound molecular imaging with MBTNF-α enables quantitative assessment of mTNF-α expression. It may be a potential tool for facilitating the implementation of personalized medicine in IBD.

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.

Photoacoustic visualization of the fluence rate dependence of photodynamic therapy.

This study investigates the fluence rate effect, an essential modulating mechanism of photodynamic therapy (PDT), by using photoacoustic imaging method. To the best of our knowledge, this is the first time that the fluence rate dependence is investigated at a microscopic scale, as opposed to previous studies that are based on tumor growth/necrosis or animal surviving rate. This micro-scale examination enables subtle biological responses, including the vascular damage and the self-healing response, to be studied. Our results reveal the correlations between fluence rate and PDT efficacy/self-healing magnitude, indicating that vascular injuries induced by high fluence rates are more likely to recover and by low fluence rates (≤126 mW/cm2) are more likely to be permanent. There exists a turning point of fluence rate (314 mW/cm2), above which PDT practically produces no permanent therapeutic effect and damaged vessels can fully recover. These findings have practical significance in clinical setting. For cancer-related diseases, the 'effective fluence rate' is useful to provoke permanent destruction of tumor vasculature. Likewise, the 'non effective range' can be applied when PDT is used in applications such as opening the blood brain barrier to avoid permanent brain damage.

Simple structural indocyanine green-loaded microbubbles for dual-modality imaging and multi-synergistic photothermal therapy in prostate cancer.

As problems with the overuse of radical prostate cancer (PCa) treatment are increasingly exposed, focal therapy represents the direction of low- or intermediate-risk PCa management in the future. However, inaccurate diagnosis and low controllability of focal therapy hinder its clinical translation. In this study, we develop simple structural cyclic arginine-glycine-aspartic (cRGD) peptide-modified and indocyanine green (ICG)-loaded microbubbles (cRGD-ICG-MBs) for ultrasound-photoacoustic imaging and multi-synergistic photothermal therapy (PTT) to address the above problems. Precise PCa diagnosis is achieved by molecular ultrasound imaging. cRGD-targeting and low-frequency ultrasound with an amplitude of 500kPa convert MBs into nanoparticles for enhanced ICG delivery. Alow frequency2500 kPa amplitude ultrasound enables temporary vasculature destruction, which minimizes heat loss during PTT. Specifically, ICG in the tumor region is 14-fold higher than the control, resulting in satisfactory PTT. Our study highlights that this theranostic strategy possesses considerable clinical translational potential, especially in mini-invasive and individualized PCa therapy.

Targeted imaging of orthotopic prostate cancer by using clinical transformable photoacoustic molecular probe.

BACKGROUND: To obtain high-yield histological samples by targeted prostate cancer (PCa) biopsy is the current trend compared with transrectal ultrasound (TRUS)-guided systematic histological biopsy, which is regarded as the gold standard for prostate cancer (PCa) diagnosis. In this paper, we present a targeted PCa imaging strategy using a real-time molecular photoacoustic imaging system integrated with a handheld US probe (PAI/US) and synthesized an integrin αvß3 targeted probe based on ICG (cRGD-ICG). METHODS: To prepare cRGD-ICG, ICG-NHS was linked to cRGD through carboxyl-co-reaction. In vitro PA imaging ability of cRGD-ICG was tested. Orthotopic PCa-bearing rats were used as animal models. After injected with either cRGD-ICG or non-targeted probe, rats were implemented with PA imaging to confirm the specific accumulation of cRGD-ICG at tumor region. Moreover, pathological frozen slices were made to observe distribution of the probe in prostate tissue ex vivo. RESULTS: A small molecular PAI probe was synthesized and exhibited excellent targeted imaging ability in vitro. In vivo photoacoustic imaging was carried out after intravenous injection of cRGD-ICG in orthotopic PCa-bearing rats under the facilitation of the PAI/US system. Maximum molecular photoacoustic signals were observed in the tumor area in vivo after the probe injection, which showed 3.8-fold higher signal enhancement than that in the control group (P < 0.05). Significantly higher cRGD-ICG accumulation was observed under confocal microscopy in the tumor region than in normal prostate tissue. CONCLUSIONS: All our results showed that the comprehensive strategy provided a high-yield and reliable method for PCa diagnosis and targeted prostate biopsy, with great clinical translation potential.

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.

Investigating the Accuracy of Ultrasound-Ultrasound Fusion Imaging for Evaluating the Ablation Effect via Special Phantom-Simulated Liver Tumors.

The goal of this study was to investigate the accuracy of ultrasound-ultrasound (US-US) fusion imaging for evaluating the ablation effect via phantom-simulated liver tumors. Twenty special phantom models were established, ablated and divided into a complete ablation group (n = 10) and an incomplete ablation group (n = 10). US-US fusion imaging was performed to evaluate the ablation effect. Gross specimens were observed as a standard reference. In this US-US fusion imaging study, the registration success rate was 100% (20/20), and the assessment time was 3.8 ± 0.9 min. The accuracy rate of the evaluation was 100% (20/20). There was no significant difference in the residual pseudo-tumoral area between the evaluation with US-US fusion imaging and gross specimen observation (p = 0.811), and the measurement error was 1.1 ± 0.6 mm. In conclusion, the feasibility and accuracy of US-US fusion imaging when evaluating the ablation effect can be investigated with this phantom-simulated liver tumor ablation model in an ideal state.

Linear array-based real-time photoacoustic imaging system with a compact coaxial excitation handheld probe for noninvasive sentinel lymph node mapping.

We developed a linear ultrasound array-based real-time photoacoustic imaging system with a compact coaxial excitation handheld photoacoustic imaging probe for guiding sentinel lymph node (SLN) needle biopsy. Compared with previous studies, our system and probe have the following advantages: (1) the imaging probe is quite compact and user-friendly; (2) laser illumination and ultrasonic detection are achieved coaxially, enabling high signal-to-noise ratio; and (3) GPU-based image reconstruction enables real-time imaging and displaying at a frame rate of 20 Hz. With the system and probe, clear visualization of the SLN at the depth of 2 cm (~human SLN depth) was demonstrated on a living rat. A fine needle was pushed towards the SLN based on the guidance of real-time photoacoustic imaging. The proposed photoacoustic imaging system and probe was shown to have great potential to be used in clinics for guiding SLN needle biopsy, which may reduce the high morbidity rate related to the current gold standard clinical SLN biopsy procedure.

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.