High Levels Of Angiogenesis Research Articles

Downregulation of UBB potentiates SP1/VEGFA-dependent angiogenesis in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) presents a unique profile characterized by high levels of angiogenesis and robust vascularization. Understanding the underlying mechanisms driving this heterogeneity is essential for developing effective therapeutic strategies. This study revealed that ubiquitin B (UBB) is downregulated in ccRCC, which adversely affects the survival of ccRCC patients. UBB exerts regulatory control over vascular endothelial growth factor A (VEGFA) by directly interacting with specificity protein 1 (SP1), consequently exerting significant influence on angiogenic processes. Subsequently, we validated that DNA methyltransferase 3 alpha (DNMT3A) is located in the promoter of UBB to epigenetically inhibit UBB transcription. Additionally, we found that an unharmonious UBB/VEGFA ratio mediates pazopanib resistance in ccRCC. These findings underscore the critical involvement of UBB in antiangiogenic therapy and unveil a novel therapeutic strategy for ccRCC.

High‐grade bladder cancer cells secrete extracellular vesicles containing miRNA‐146a‐5p and promotes angiogenesis

AbstractRecurrence is one of the major issues in bladder cancer (BCa). Novel technologies, such as the detection of microRNAs carried by extracellular vesicles (EVs) in urine, have been proposed as biomarkers for detecting recurrence in BCa. Although the usefulness of microRNAs in body fluids from cancer patients has been reported, it is also known that they play essential roles in cancer progression. We previously proposed miR‐146a‐5p as a prognostic marker in BCa, since its urinary expression was associated with grade and tumour depth. However, the specific mechanisms of miR‐146a‐5p remain unclear. Here, we show the proangiogenic effects of miR‐146a‐5p secreted by high‐grade BCa cells. The urinary miR‐146a‐5p level was higher in patients with high‐grade BCa than in those with low‐grade BCa. Similarly, tumours generated by miR‐146a‐overexpressing BCa cells in mice grew rapidly with high levels of angiogenesis. BCa‐derived EV treatment promoted the proliferation of endothelial cells via the inhibition of the demethylase TET2 and the subsequent increase in its downstream target c‐Myc. These findings demonstrate that secreted miR‐146a‐5p contributes to cancer progression by promoting angiogenesis. Therefore, miRNAs in EVs may become not only a diagnostic tool but also a target molecule for therapy.

Tumor Microenvironment: Necroptosis Switches the Subtype of Liver Cancer While Necrosis Promotes Tumor Recurrence and Progression.

Liver cancer is a heterogeneous group of solid tumors that include mainly epithelial tumors. As with other solid carcinomas, tumor development results from an accumulation of genetic and epigenetic alterations. Hepatocellular carcinoma and intrahepatic cholangiocarcinoma, derived from malignant transformation of hepatocytes and cholangiocytes, respectively, are 2 primary types of liver cancers. However, it has been shown that the same kind of cell can give rise to different types of cancer, depending on manner of cell death in the tumor microenvironment. In a recent animal study, hepatocytes gave rise to both hepatocellular carcinoma and intrahepatic cholangiocarcinoma. Oncogenically activated hepatocytes were shown to give rise to intrahepatic cholangiocarcinoma or hepatocellular carcinoma depending on cell death type of neighboring cells. Hepatocytes within the necroptotic microenvironment gave rise to intrahepatic cholangiocarcinoma; however, hepatocytes harboring the same oncogenic driver gave rise to hepatocellular carcinoma within the apoptotic microenvironment. The hepatic cytokine microenvironment structured by the necroptosis can also switch hepatocellular carcinoma to intrahepatic cholangiocarcinoma independently of the oncogenic drivers. Cell death by necrosis in damaged livers can also lead to development of carcinoma. Cancer cells are known to be resistant to apoptosis as a result of p53 mutation. Therefore, necrosis is the primary cell death pathway in cancer therapy. Necrosis is associated with high levels of angiogenesis, tumor-associated macrophages, and increased inflammation in the tumor microenvironment. Patients with hepatocellular carcinoma or intrahepatic cholangiocarcinoma characterized by necrosis and tumor-associated macrophages have reduced overall survival and recurrence-free survival. Cytotoxicity from anticancer therapy can also lead to accelerated necrosis. The content of cells undergoing necrosis triggers cytokine secretion, which designs cancer progression via inflammatory and noninflammatory pathways. Thus, the tumor microenvironment and manner of cell death (necrosis, apoptosis, or necroptosis) are crucial factors in the development of primary liver cancers and tumor progression.

Fusion 3D gross sampling method to overcome heterogeneity in clear cell renal cell carcinoma (ccRCC) and grading angiogenic versus immune signatures.

e16565 Background: Grading tissue signatures in ccRCC is a potential tool to improve patients’ selection for anti-angiogenic drugs and immunotherapies. After the molecular audit to the TCGA public platforms, we aimed to grade angiogenic and immune signatures in ccRCC using a new 3D next-generation gross sampling method to overcome intratumoral heterogeneity. Methods: 100 consecutive advanced ccRCCs (≥pT3a) were sampled. Paraffin-embedded blocks were obtained after mapping the position of each sample to the whole tumor, to allow the reconstruction of the entire 3D tumor mass ( fusion 3D). Multisite tumor sampling was performed to analyze the whole tumor. TCGA platforms were assessed for angiogenic and immune molecular signatures: CD31 and CD34 to evaluate the absolute count and density of vessels, while E1L3N and sp263 clones for PD-L1 expression in tumor cells (TCs). The digital analysis was performed with image processing: comparing each tissue block to whole 3D assessment, the coefficient of variation (CV) was the statistical measure of the dispersion of data points in the data series around the mean. CV < 0.2 defined the homogeneity of the assessment. Results: 656 gross photographs representative of the 3D tumoral masses were collected and 4231 paraffin blocks and tissue sections were stored. Matching gross photographs with tissue samples was performed. 6324 tissue cores were evaluated after combining standard routine sampling plus the 3D multisite sampling and tissue microarray cores. Only 18% of cores displayed homogeneous profile of angiogenesis (CV < 0.2) with two distinct patterns: homogeneous high level of angiogenesis (pattern A) (10% of cases) or homogeneous low level of angiogenesis (pattern B) (8% of cases). The heterogeneous profile of angiogenesis was more frequent than the homogeneous one and was characterized by zones with high and low density of angiogenesis (82% of cases) (pattern C). On the other hand, the homogeneity of PD-L1 expression was more frequently observed both as diffuse absence ( < 1% of TCs, grade 0) or high expression (≥50% of TCs, grade 2) compared to low PD-L1 expression (1-49% of TCs, grade 1) (60% plus 7% versus 33% of cases, respectively). After the comparison of grading angiogenic versus immune signatures, we observed that cases with low PD-L1 expression (grade 0/1) usually expressed high density of angiogenesis (pattern A). Conclusions: Grading angiogenic (pattern A, B and C) versus immune (grade 0, 1 and 2) signatures in ccRCCs can be performed using commonly available tissue vascular (CD31 or CD34) and immune (PD-L1) antibodies. We promoted a simple assay to perform fusion 3D gross sampling to reduce at minimum the bias of heterogeneity in RCCs analyses. Both angiogenic versus immune signature by using the grading systems may help treatment decision-making and response assessment in ccRCC patients.

VEGF receptor targeted imaging of angiogenic response to limb ischemia in diabetic vs. non-diabetic Yucatan minipigs

BackgroundNew therapies to treat diabetic peripheral artery disease (PAD) require target-specific non-invasive imaging modalities to follow efficacy. As a translational study, we performed targeted imaging of receptors for vascular endothelial growth factor (VEGF) in response to anterior femoral artery occlusion (FAO) in Yucatan minipigs and compare the normal response to response in diabetic Yucatan minipigs.MethodsEleven Yucatan minipigs, 6 non-diabetic (non-D) and 5 purpose bred diabetic (D) (Sinclair, Auxvasse MO), underwent intravascular total occlusion of the anterior femoral artery (FA). At days 1 and 28, pigs underwent SPECT/CT 201Tl hindlimb perfusion imaging and at day 7 were injected with [99mTc]DOTA-PEG-scVEGF (scV/Tc) tracer targeting VEGF receptor, and underwent biopsies of the hindlimb muscles for gamma counting and histology, followed by imaging. One day after the final scan, pigs underwent contrast angiography of the lower extremities. Counts from scans were converted to percentage injected activity (%IA).ResultsPerfusion was lower in the occluded hindlimb compared to non-occluded on day 1 in both the D and non-D pigs. At day 7, scV/Tc count ratio of counts from ROIs drawn in proximal gastrocnemius muscle for the occluded over non-occluded limb was significantly higher in non-D vs. D pigs (1.32 ± 0.06 vs. 1.04 ± 0.13, P = 0.02) reflecting higher level of angiogenesis. Perfusion increased between days 1 and 28 in the muscles in the occluded limb for the non-diabetic pigs while the diabetic pig showed no increase (+ 0.13 ± 0.08 %IA vs. − 0.13 ± 0.11, P = 0.003). The anterior FA showed poor contrast filling beyond occluder and qualitatively fewer bridging collaterals compared to non-D pigs at 28 days.ConclusionVEGF receptor targeted imaging showed the effects of diabetes to suppress angiogenesis in response to occlusion of the anterior femoral artery of purpose bred diabetic Yucatan minipigs and indicates potential applicability as a marker to follow efficacy of novel therapies to improve blood flow by stimulating angiogenesis in diabetic PAD.

The Relationship between KIT Copy Number Variation, Protein Expression, and Angiogenesis in Sporadic Breast Cancer.

KIT is a protooncogene that encodes for the KIT oncoprotein, which is a transmembrane tyrosine kinase growth factor receptor that holds a critical role in a variety of normal physiological and pathological processes including angiogenesis. KIT has been shown to be involved in tumorigenesis, contributing to the development of gastrointestinal carcinoma and leukemia. A link between KIT overexpression and breast cancer development has previously been reported. In the current study, we explored KIT gene expression and exonic copy number variants (CNV) and the relationship with angiogenesis (CD34) and the clinicopathological features of breast cancer. MLPA technique was used to determine the CNV in 64 breast cancer tumor samples from patients diagnosed with primary sporadic breast cancer. Results were confirmed by quantitative PCR. Expression of KIT and CD34 was determined using immunohistochemistry (IHC). Our results show that 28.1% of the tumor samples from patients with primary sporadic breast cancer had CNV in the KIT gene. Among the breast tumor samples, 54.7% showed positive KIT expression. The expression of the CD34 angiogenesis marker was reported in 43.8% of the tumor samples as low, 42.2% as moderate and 14.1% as high. A significant correlation between increased CNV of KIT exons, a high level of angiogenesis (CD34) and increased tumor grade was observed (p< 0.05). A significant correlation between the KIT CNV and the angiogenesis marker was found. Examining KIT expression and CNV has the potential to function as a biomarker for tyrosine kinase inhibitor drugs in breast cancer.

Fasting before or after wound injury accelerates wound healing through the activation of pro-angiogenic SMOC1 and SCG2.

Healing of the chronic diabetic ulceration and large burns remains a clinical challenge. Therapeutic fasting has been shown to improve health. Our study tested whether fasting facilitates diabetic and burn wound healing and explored the underlying mechanism.Methods: The effects of fasting on diabetic and burn wound healing were evaluated by analyzing the rates of wound closure, re-epithelialization, scar formation, collagen deposition, skin cell proliferation and neovascularization using histological analyses and immunostaining. In vitro functional assays were conducted to assess fasting and refeeding on the angiogenic activities of endothelial cells. Transcriptome sequencing was employed to identify the differentially expressed genes in endothelial cells after fasting treatment and the role of the candidate genes in the fasting-induced promotion of angiogenesis was demonstrated.Results: Two times of 24-h fasting in a week after but especially before wound injury efficiently induced faster wound closure, better epidermal and dermal regeneration, less scar formation and higher level of angiogenesis in mice with diabetic or burn wounds. In vitro, fasting alone by serum deprivation did not increase, but rather reduced the abilities of endothelial cell to proliferate, migrate and form vessel-like tubes. However, subsequent refeeding did not merely rescue, but further augmented the angiogenic activities of endothelial cells. Transcriptome sequencing revealed that fasting itself, but not the following refeeding, induced a prominent upregulation of a variety of pro-angiogenic genes, including SMOC1 (SPARC related modular calcium binding 1) and SCG2 (secretogranin II). Immunofluorescent staining confirmed the increase of SMOC1 and SCG2 expression in both diabetic and burn wounds after fasting treatment. When the expression of SMOC1 or SCG2 was down-regulated, the fasting/refeeding-induced pro-angiogenic effects were markedly attenuated.Conclusion: This study suggests that fasting combined with refeeding, but not fasting solely, enhance endothelial angiogenesis through the activation of SMOC1 and SCG2, thus facilitating neovascularization and rapid wound healing.

Angiogenesis and Lymphangiogenesis in Salivary Gland Adenoid Cystic Carcinoma and Mucoepidermoid Carcinoma.

Background and Purpose:This study aimed to investigate the immunohistochemical expression of CD31 and podoplanin in order to examine angiogenesis and lymphangiogenesis, respectively in common malignant tumors of salivary glands. Materials and Methods:Forty formalin-fixed, paraffinated blocks (20 adenoid cystic carcinoma and 20 mucoepidermoid carcinoma blocks) were selected from the medical archives of Amir A’lam Hospital of Tehran, Iran. Sections from the blocks were stained by CD31 and D2-40 markers via immunohistochemistry. Clinical and demographic information was extracted from the patients’ records. Findings:There was a significant difference between tumors in terms of intratumoral microvessel density (MVD) (P< 0.001), total MVD (P< 0.001), and intratumoral lymphatic vessel density (LVD) (P= 0.011). In mucoepidermoid carcinoma, intratumoral MVD and LVD were greater than peritumoral MVD and LVD (P= 0.001 and P< 0.001, respectively). In mucoepidermoid carcinoma, there was no relationship between histological grade with MVD (total, intratumoral or peritumoral) or LVD (total, intratumoral or peritumoral) (P> 0.05). A similar finding was reported with respect to the histopathological grade of adenoid cystic carcinoma (P> 0.05). Conclusion:The higher level of angiogenesis and lymphangiogenesis in mucoepidermoid carcinoma, specifically at the center of tumor, compared to adenoid cystic carcinoma, may be attributed to differences in the clinical behaviors and metastasis of tumors. Moreover, considering the high LVD at the center of tumor in mucoepidermoid carcinoma and infrequency of metastasis to regional lymph nodes in adenoid cystic carcinoma, it can play a significant role in metastasis to regional lymph nodes.

Abstract 2676: Associations of peripheral biomarkers to outcomes to anti-PD-L1 immune checkpoint blockade in metastatic urothelial cancer

Abstract Introduction Immune checkpoint blockade such as inhibition of programmed death-1 (PD-1) or programmed death-ligand 1 (PD-L1) has been shown to be effective in metastatic urothelial cancer (mUC) and many other types of cancers. Despite its robust efficacy, a large proportion of patients still fail to show durable responses; therefore, it is important to understand the mechanisms that drive clinical responses, as well as the pathways associated with lack of response. Circulating peripheral biomarkers are particularly useful as readily accessible and minimally invasive markers to enable monitoring both tumor growth and responses to treatment. Methods Baseline plasma from 337 patients treated with an anti-PD-L1 (atezolizumab) in the mUC Ph2 trial IMvigor210 (NCT02108652) were analyzed using a Myriad-RBM panel. To further characterize the findings, single-cell RNA sequencing (scRNAseq) was performed with PBMC isolated from responders and non-responders (n=5 each) to atezolizumab in the same trial. NanoString gene expression levels of the PBMC isolated from mUC patients in IMvigor210 was used to validate the identified signatures. Results High levels of CXCL9 (HR: 0.692, CI: 0.529-0.903, p=0.006) and leptin (HR: 0.633, CI: 0.485-0.825, p=0.001) were associated with better overall survival (OS), whereas high levels of pro-inflammatory cytokines IL6 (HR: 2.744, CI: 2.084-3.612, p&amp;lt;0.0001) and CXCL8/IL8 (HR: 2.639, CI: 1.997-3.489, p=0.006) were associated with poor OS. In addition, high levels of angiogenesis and cell adhesion proteins such as TIMP1 (HR: 2.317, CI: 1.773-3.029, p&amp;lt;0.0001), VWF (HR: 1.571, CI: 1.207-2.045, p=0.001), and ICAM1 (HR: 1.349, CI: 1.034-1.76, p=0.026) correlated with poor OS. High plasma IL6 levels and high tumor IL6 expression are significantly enriched in non-responders in the T cell excluded phenotype, further suggesting association of IL6 with stroma/angiogenesis. Results from scRNAseq show response (CR/PR) to checkpoint blockade was associated with adaptive immune signature (GZMM, PSMB8, PSMB9) (FDR&amp;lt;0.0001) in lymphoid PBMC. Lack of response (SD/PD) was associated with complement coagulation (PF4, THBS, TIMP1) (FDR&amp;lt;0.0001) and innate inflammation (IL8, IL1B, IL18) (FDR=&amp;lt;0.0001) signatures in the myeloid cells. We confirmed these outcome-associated signatures with OS modeling with gene expression levels in PBMC. Conclusion Peripheral biomarker analysis revealed that adaptive immune signatures to be associated better clinical outcome, whereas innate inflammation and angiogenesis signatures to be correlated with worse clinical outcome to anti-PD-L1 immunotherapy. The link between IL6 and angiogenesis in the tumor microenvironment will be further characterized. Understanding the genes and pathways associated with outcomes will shed light on peripheral biomarker development. Citation Format: Kobe C. Yuen, Vinita Gupta, Congfen Li, Deepali Rishipathak, Edward E. Kadel, Ying-Jiun J. Chen, Leonard D. Goldstein, Zora Modrusan, Sanjeev Mariathasan. Associations of peripheral biomarkers to outcomes to anti-PD-L1 immune checkpoint blockade in metastatic urothelial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2676.

Overexpression of HOXC10 promotes angiogenesis in human glioma via interaction with PRMT5 and upregulation of VEGFA expression.

High levels of angiogenesis are associated with poor prognosis in patients with gliomas. However, the molecular mechanisms underlying tumor angiogenesis remain unclear.Methods: The effect of homeobox C10 (HOXC10) on tube formation, migration, and proliferation of human umbilical vein endothelial cells (HUVECs) and on chicken chorioallantoic membranes (CAMs) was examined. An animal xenograft model was used to examine the effect of HOXC10 on xenograft angiogenesis or the effect of bevacizumab, a monoclonal antibody against vascular endothelial growth factor A (VEGFA), on HOXC10-overexpressing xenografts. A chromatin immunoprecipitation assay was applied to investigate the mechanism in which HOXC10 regulated VEGFA expression.Results: Overexpressing HOXC10 enhanced the capacity of glioma cells to induce tube formation, migration and proliferation of HUVECs, and neovascularization in CAMs, while silencing HOXC10 had the opposite result. We observed that CD31 staining was significantly increased in tumors formed by HOXC10-overexpressing U251MG cells but reduced in HOXC10-silenced tumors. Mechanistically, HOXC10 could transcriptionally upregulate VEGFA expression by binding to its promoter. Strikingly, treatment with bevacizumab, a monoclonal antibody against VEGFA, significantly inhibited the growth of HOXC10-overexpressing tumors and efficiently impaired angiogenesis. Protein arginine methyltransferase 5 (PRMT5) and WD repeat domain 5 (WDR5), both of which regulate histone post-translational modifications, were required for HOXC10-mediated VEGFA upregulation. Importantly, a significant correlation between HOXC10 levels and VEGFA expression was observed in a cohort of human gliomas.Conclusions: This study suggests that HOXC10 induces glioma angiogenesis by transcriptionally upregulating VEGFA expression, and may represent a potential target for antiangiogenic therapy in gliomas.

Gankyrin sustains PI3K/GSK-3β/β-catenin signal activation and promotes colorectal cancer aggressiveness and progression.

High levels of angiogenesis, metastasis and chemoresistance are major clinical features of colorectal cancer (CRC), a lethal disease with a high incidence worldwide. Aberrant activation of Wnt/β-catenin pathway contributes to CRC progression. However, little is known about regulatory mechanisms of the β-catenin activity in cancer progression. Here we report that Gankyrin was markedly upregulated in primary tumor tissues from CRC patients and was associated with poor survival. Moreover, we demonstrated that overexpressing Gankyrin promoted, while knockdown of Gankyrin impaired, the aggressive phenotype of proliferation, angiogenesis, chemoresistance and metastasis of CRC cells both in vitro and in vivo. Importantly, we found a unique molecular mechanism of Gankyrin in CRC cells signaling transduction, that regulated the cross-talk between PI3K/Akt and Wnt/β-catenin signaling pathways, sustaining PI3K/GSK-3β/β-catenin signal activation in CRC. Therefore, these findings not only reveal a mechanism that promotes aggressiveness and progression in CRC, but also provide insight into novel molecular targets for antitumor therapy in CRCs.

Pleiotropic effects of survivin in vascular endothelial cells

ObjectiveTo assess the effects of survivin (SVV)in vascular endothelial cells. MethodsIn this study, we applied a gain-of-function approach and ectopically expressed SVV in rat aortic endothelial cells (RAECs) using a SVV-expressing adenovirus. The resulting SVV expression on the steady-state mRNA and protein level in RAECs was determined by reverse transcription quantitative PCR and Western blot, respectively. Cell viability, apoptosis, and migration were assessed in vitro by CCK-8 assay, flow cytometry, and transwell assay, respectively. The effect of SVV on in vivo angiogenesis was evaluated by immunohistochemistry in nude mice. Non-infected RAECs and those infected with GFP-expressing control adenovirus were used as controls. ResultsCompared to non-infected or control adenovirus-infected RAECs in vitro, SVV-expressing cells had increased viability and migratory capability, but reduced apoptosis. In vivo, SVV-expressing RAECs were associated with a higher level of angiogenesis. ConclusionSVV is a positive regulator of endothelial cell survival and migration, and thus, stabilizes endothelial cells and stimulates angiogenesis.

Influence of Exposure to Chronic Persistent Low-Dose Ionizing Radiation on the Tumor Biology of Clear-Cell Renal-Cell Carcinoma. An Immunohistochemical and Morphometric Study of Angiogenesis and Vascular Related Factors.

Increased angiogenesis is related to boosted growth and malignancy in carcinomas. "Chronic Persistent Low-Dose Ionizing Radiation" (CPLDIR) exposure increases incidence and aggressive behavior of clear-cell renal-cell carcinoma (CCRCC). The aim was to study the biology of angiogenesis, including microvessel density (MVD), in human clear-cell renal-cell carcinomas (CCRCC) originating from a radio-contaminated geographical area (Ukraine) and to compare with similar tumors diagnosed in non-contaminated regions of Europe (Spain, Valencia) and Latin America (Colombia, Barranquilla). MVD was comparatively examined in 124 patients diagnosed with CCRCC from three geographical areas by means of digital micro-imaging and computerized analysis. Additionally, 50 adult normal kidneys were used for controls (autopsy kidneys from Valencia and Barranquilla). Furthermore, an immunohistochemical study of several vascular related growth factors was undertaken using a similar methodology. MVD as well as VEFG are the most discriminating factors associated with an aggressive behavior of CCRCC. Their expression increased in proportion to the level of exposure to chronic low-dose ionizing radiation in Ukrainian patients in the 25 years since the Chernobyl accident substantiated by comparison with the two control groups of renal carcinomas present in non-irradiated areas (Spain and Colombia). No major biological differences relating to angiogenesis appear to exist between the CCRCC diagnosed in two distant geographical areas of the world. HIF-1α expression was similar in all groups, with no statistical significance. Present findings demonstrate the existence of a significant relationship between MVD and VEGF in CCRCC: an increased expression of VEGF is associated with a high level of angiogenesis.

MR-perfusion derived hemodynamic parametric response mapping of bevacizumab efficacy in recurrent glioblastoma

Hemodynamic alterations induced by bevacizumab therapy seem to be of subordinate importance, since the magnitude of parametric response maps (decreased intratumor voxels) is a function of the corresponding pretreatment parameter, and tumors with a high degree of angiogenesis before bevacizumab therapy retain a high level of angiogenesis during therapy, despite a greater antiangiogenic effect of bevacizumab, such that a reversal of the biological behavior and relative prognosis of these tumors does not occur.

URG4/URGCP enhances the angiogenic capacity of human hepatocellular carcinoma cells in vitro via activation of the NF-κB signaling pathway.

BackgroundAngiogenesis is essential for tumor growth. Hepatocellular carcinoma (HCC) is characterized by hypervascularity; high levels of angiogenesis are associated with poor prognosis and a highly invasive phenotype in HCC. Up-regulated gene-4 (URG4), also known as upregulator of cell proliferation (URGCP), is overexpressed in multiple tumor types and has been suggested to act as an oncogene. This study aimed to elucidate the effect of URG4/URGCP on the angiogenic capacity of HCC cells in vitro.MethodsExpression of URG4/URGCP in HCC cell lines and normal liver epithelial cell lines was examined by Western blotting and quantitative real-time PCR. URG4/URGCP was stably overexpressed or transiently knocked down using a shRNA in two HCC cell lines. The human umbilical vein endothelial cell (HUVEC) tubule formation and Transwell migration assays and chicken chorioallantoic membrane (CAM) assay were used to examine the angiogenic capacity of conditioned media from URG4/URGCP-overexpressing and knockdown cells. A luciferase reporter assay was used to examine the transcriptional activity of nuclear factor kappa – light – chain - enhancer of activated B cells (NF-κB). NF-κB was inhibited by overexpressing degradation-resistant mutant inhibitor of κB (IκB)-α. Expression of vascular endothelial growth factor C (VEGFC), tumor necrosis factor-α (TNFα), interleukin (IL)-6, IL-8 and v-myc avian myelocytomatosis viral oncogene homolog (MYC) were examined by quantitative real-time PCR; VEGFC protein expression was analyzed using an ELISA.ResultsURG4/URGCP protein and mRNA expression were significantly upregulated in HCC cell lines. Overexpressing URG4/URGCP enhanced - while silencing URG4/URGCP decreased - the capacity of HCC cell conditioned media to induce HUVEC tubule formation and migration and neovascularization in the CAM assay. Furthermore, overexpressing URG4/URGCP increased - whereas knockdown of URG4/URGCP decreased - VEGFC expression, NF-κB transcriptional activity, the levels of phosphorylated (but not total) IκB kinase (IKK) and IκB-α, and expression of TNFα, IL-6, IL-8 and MYC in HCC cells. Additionally, inhibition of NF-κB activity in HCC cells abrogated URG4/URGCP-induced NF-κB activation and angiogenic capacity.ConclusionsThis study suggests that URG4/URGCP plays an important pro-angiogenic role in HCC via a mechanism linked to activation of the NF-κB pathway; URG4/URGCP may represent a potential target for anti-angiogenic therapy in HCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1378-7) contains supplementary material, which is available to authorized users.

AGK enhances angiogenesis and inhibits apoptosis via activation of the NF-κB signaling pathway in hepatocellular carcinoma.

High levels of angiogenesis and resistance to apoptosis are major clinical features of hepatocellular carcinoma (HCC), a lethal disease with a high incidence worldwide. However, the precise mechanisms underlying these malignant properties remain unclear. Here, we demonstrated that acylglycerol kinase (AGK) is markedly overexpressed in HCC cell lines and clinical tissues. Immunohistochemical analysis of 245 clinical HCC specimens revealed patients with high levels of AGK expression had poorer overall survival compared to patients with low AGK expression. Furthermore, overexpressing AGK significantly enhanced angiogenesis and inhibited apoptosis in vitro and promoted the tumorigenicity of HCC cells in vivo; silencing endogenous AGK had the opposite effects. Importantly, AGK enhanced angiogenesis and inhibited apoptosis in HCC in part via activation of NF-κB signaling. Our findings provide new evidence that AGK plays an important role in promoting angiogenesis and providing resistance to apoptosis, thus AGK may represent a novel therapeutic target for HCC.

Inhibition of TGF-β signaling in mesenchymal stem cells of subchondral bone attenuates osteoarthritis

Osteoarthritis is a highly prevalent and debilitating joint disorder. There is no effective medical therapy for osteoarthritis due to limited understanding of osteoarthritis pathogenesis. We show that TGF–β1 is activated in the subchondral bone in response to altered mechanical loading in an anterior cruciate ligament transection (ACLT) osteoarthritis mouse model. TGF–β1 concentrations also increased in human osteoarthritis subchondral bone. High concentrations of TGF–β1 induced formation of nestin+ mesenchymal stem cell (MSC) clusters leading to aberrant bone formation accompanied by increased angiogenesis. Transgenic expression of active TGF–β1 in osteoblastic cells induced osteoarthritis. Inhibition of TGF–β activity in subchondral bone attenuated degeneration of osteoarthritis articular cartilage. Notably, knockout of the TGF–β type II receptor (TβRII) in nestin+ MSCs reduced development of osteoarthritis in ACLT mice. Thus, high concentrations of active TGF–β1 in the subchondral bone initiated the pathological changes of osteoarthritis, inhibition of which could be a potential therapeutic approach.

The Role of Bcl-2 Family Proteins in Therapy Responses of Malignant Astrocytic Gliomas: Bcl2L12 and Beyond

Glioblastoma (GBM) is a highly aggressive and lethal brain cancer with a median survival of less than two years after diagnosis. Hallmarks of GBM tumors include soaring proliferative indices, high levels of angiogenesis, diffuse invasion into normal brain parenchyma, resistance toward therapy-induced apoptosis, and pseudopallisading necrosis. Despite the recent advances in neurosurgery, radiation therapy, and the development of targeted chemotherapeutic regimes, GBM remains one of the deadliest types of cancer. Particularly, the alkylating agent temozolomide (TMZ) in combination with radiation therapy prolonged patient survival only marginally, and clinical studies assessing efficacies of targeted therapies, foremost ATP mimetics inhibiting the activity of receptor tyrosine kinases (RTKs), revealed only few initial responders; tumor recurrence is nearly universal, and salvage therapies to combat such progression remain ineffective. Consequently, myriad preclinical and clinical studies began to define the molecular mechanisms underlying therapy resistance of GBM tumors, and pointed to the Bcl-2 protein family, in particular the atypical member Bcl2-Like 12 (Bcl2L12), as important regulators of therapy-induced cell death. This review will discuss the multi-faceted modi operandi of Bcl-2 family proteins, describe their roles in therapy resistance of malignant glioma, and outline current and future drug development efforts to therapeutically target Bcl-2 proteins.

Abstract 1601: COX-2 and EP4 receptor as targets to ameliorate tumor growth, lymphangiogenesis, and metastasis in a murine breast cancer model

Abstract We had earlier shown that elevated cyclo-oxygenase (COX)-2 expression by breast cancer cells promotes tumor progression and metastasis by multiple mechanisms: inactivation of host immune cells, stimulation of tumor cell migration and tumor-associated angiogenesis. Furthermore, COX-2 was causally associated with increased VEGF-C expression/secretion in human breast cancer cell lines, corroborated with in situ studies on breast cancer tissue specimens, showing a strong correlation of COX-2 with VEGF-C and lymphangiogenic markers or lymphovascular density, implicated in lymphatic metastasis. VEGF-C upregulation resulted from PGE2-mediated stimulation of EP4, and to a smaller extent, EP1 receptors on breast cancer cells. Since human tumor xeno-transplants in immno-compromised hosts are less than ideal for a therapeutic testing of these findings, in the present study, we developed a syngeneic murine breast cancer model exhibiting lymphangiogenesis and lymphatic metastasis in C3H/HeJ mice, using a highly metastatic, COX-2 expressing C3L5 cell line. This cell line was found to express VEGF-C and VEGF-D mRNA and proteins, and all four EP receptors. Expression of VEGF-C and VEGF-D were suppressed by treatment of cells with COX-2 inhibitors or EP4 antagonist in vitro. C3L5 cells suspended in growth factor-reduced Matrigel were implanted in both inguinal regions in mice that received oral treatments with COX-1/2 inhibitor indomethacin, COX-2 inhibitor celecoxib, EP1 antagonist ONO-8713 or EP4 antagonist ONO-AE3-208, to evaluate tumor growth, metastasis to lymph nodes and the lungs and tumor-associated angiogenesis (CD31 immuno-staining) and lymphangiogenesis (LYVE1 immunostaining). Mice were killed on days 8, 12 and 16. Tumor growth in control vehicle-treated mice was associated with high levels of angiogenesis and lymphangiogenesis at all intervals. VEGF-C protein expression was noted in 20-30% of tumor cells as well as macrophages in situ. Micro-metastasis to the lungs and local lymph nodes was noted as early as day 8 and increased thereafter. Metastasis to distant axillary nodes was seen at day16. Significant reduction in tumor growth, tumor-associated angiogenesis and lymphangiogenesis, and metastasis to the lungs and lymph nodes was achieved by therapies with indomethacin, celecoxib and ONO-AE3-208 but not ONO-8713 as compared to respective vehicle treatments. In conclusion, we have devised a syngeneic mouse breast cancer model mimicking human, showing successful chemo-intervention of tumor growth, tumor-associated angiogenesis/lymphangiogenesis and metastasis to the lungs and lymph nodes with COX inhibitors and EP 4 antagonist. (Supported by grants from the Canadian Breast Cancer Foundation, Ontario chapter and the Ontario Institute of Cancer Research to PKL. EP antagonists were kindly provided by ONO pharmaceuticals, Japan and Celecoxib by Pfizer.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1601.

The temporal correlation of dynamic contrast-enhanced magnetic resonance imaging with tumor angiogenesis in a murine glioblastoma model

Objective: Glioblasomta multiforme (GBM) is a WHO grade IV malignant brain tumor with poor prognosis, despite advances in surgical and adjuvant therapy. GBM is characterized by areas of central necrosis and high levels of angiogenesis, during which increased vascular permeability allows for the extravasation of endothelial progenitor cells to support blood vessel and tumor growth. The purpose of this study was to characterize changes in tumor vascular permeability, vascular density and vessel morphology in vivo during angiogenesis.Methods: An orthotropic murine (GL26) glioblastoma model was used in this study. in vivo serial dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in combination with histologic and molecular genetic analyses was performed to correlate in vivo imaging of vascular development.Results: DCE-MRI revealed a significant change in tumor vessel permeability dependent upon tumor progression and size. Time to max signal intensity displayed a stepwise increase between days 21 and 24 (p<0.05), a critical period before exponential tumor growth during which a significant increase in tumor vascular density and vessel caliber is observed on histology. Furthermore, quantitative real-time PCR revealed a corollary increase in angiogenic signaling molecules before the observed changes on DCE-MRI.Discussion: In vivo changes of orthotopic glioma blood vessel permeability as shown by DCE-MRI correlates with histologic quantification of vascular density and vessel caliber as well as with the molecular expression of angiogenic factors. DCE-MRI is a useful tool for non-invasive in vivo monitoring of angiogenesis in pre-clinical tumor models.