A transcriptome-based signature of pathological angiogenesis predicts breast cancer patient survival.

The specific genes and molecules that drive physiological angiogenesis differ from those involved in pathological angiogenesis, suggesting distinct mechanisms for these seemingly related processes. Unveiling genes and pathways preferentially associated with pathologic angiogenesis is key to understanding its mechanisms, thereby facilitating development of novel approaches to managing angiogenesis-dependent diseases. To better understand these different processes, we elucidated the transcriptome of the mouse retina in the well-accepted oxygen-induced retinopathy (OIR) model of pathological angiogenesis. We identified 153 genes changed between normal and OIR retinas, which represent a molecular signature relevant to other angiogenesis-dependent processes such as cancer. These genes robustly predict the survival of breast cancer patients, which was validated in an independent 1,000-patient test cohort (40% difference in 15-year survival; p = 2.56 x 10-21). These results suggest that the OIR model reveals key genes involved in pathological angiogenesis, and these may find important applications in stratifying tumors for treatment intensification or for angiogenesis-targeted therapies.

Evaluation of the anti-cervical cancer effect of a prodrug ：CBZ-AAN-DOX with hypoxic cell culture and tumor-bearing zebrafish models.

BACKGROUND: Prodrugs are medications or compounds that, after administration, can be converted into pharmacologically active drugs through metabolism. Unlike conventional drugs, prodrugs have reduced adverse or unintended effects, which could become critical limitations in treatments such as chemotherapy. Previously through computer-aided drug design and chemical synthesis, we have obtained and examined a prodrug N-benzyloxycarbonyl-Ala-Asn-Doxorubicin (CBZ-AAN-DOX). CBZ-AAN-DOX is essentially Doxorubicin that is chemically-modified with tripeptides to target Legumain, a highly expressed protein in cancer cells and is involved in tumor metastasis and tumor microvessel formation. The difficulty to test the safety and efficacy of the prodrug (including the pharmacodynamic parameters of CBZ-AAN-DOX on metastasis and invasion of tumors, as well as cardiac and vascular toxicity) primarily comes from the lack of appropriate experimental models. METHODS: Human cervical cancer cell lines CaSki under hypoxic conditions were used to evaluate the cell viability by CCK-8 assay after the prodrug treatment. Western blotting method was performed for Legumain protein determination in the cell culture. Wound healing and transwell invasion assays were performed to determine the effects of the prodrug on tumor metastasis and invasion, respectively. Zebrafish models were constructed for toxicity and angiogenesis visual analysis after in vivo treatment with the prodrug. RESULTS: The CCK-8 results showed that CBZ-AAN-DOX exhibits an IC50 of 28.7 µM in 48 h on CaSki cells that had a lower cell inhibition rate than DOX 80.3 µM for 24 h. Legumain expression was significantly increased in a time-dependent manner in 48 h under hypoxia conditions. The results also showed that 13.9 µM of the prodrug significantly inhibited the migration and invasion of cells and the effects were significantly stronger than that of 41.8 µM of DOX under hypoxia conditions after 48 h. The effects of 160 µM of the prodrug on the survival rate of zebrafish after 72 h and heart-toxicity showed no obvious abnormalities. Cell metastasis and angiogenesis were also inhibited in tumor-bearing zebrafish model. CONCLUSION: The findings in this study demonstrated that CBZ-AAN-DOX is a promising chemotherapy candidate with low toxicity and high efficiency for cervical cancer. Remarkably, the hypoxic culture model together with the zebrafish model serve as a good system for the evaluation of the toxicity, targeting and impact of the prodrug on tumor invasion and metastasis.

Genetic Variants of ANGPT1, CD39, FGF2 and MMP9 Linked to Clinical Outcome of Bevacizumab Plus Chemotherapy for Metastatic Colorectal Cancer.

Angiogenesis pathway genes show substantial genetic variability causing inter-individual differences in responses to anti-angiogenic drugs. We examined 20 single nucleotide polymorphisms (SNPs) in 13 of these genes to predict tumour response and clinical outcome measured as progression free survival (PFS) and overall survival (OS) in 57 patients with metastatic colorectal cancer (mCRC) given bevacizumab plus chemotherapy. SNPs were detected (iPLEX® Assay) in genomic DNA extracted from formalin-fixed paraffin-embedded tumour specimens. The variant allele CD39 rs11188513 was associated with a good tumour response (p = 0.024). Patients homozygous for the wild-type allele FGF2 rs1960669 showed a median PFS of 10.95 months versus 5.44 months for those with at least one variant allele-A (HR 3.30; 95% CI: 1.52-7.14; p = 0.001). Patients homozygous for wild-type MMP9 rs2236416 and rs2274755 showed a median PFS of 9.48 months versus 6 and 6.62 months, respectively, for those with at least one variant allele (p = 0.022, p = 0.043, respectively). OS was also lengthened to 30.92 months (p = 0.034) in carriers of wild-type ANGPT1 rs2445365 versus 22.07 months for those carrying at least one variant allele-A. These gene variants were able to predict clinical outcome and tumour response in mCRC patients given bevacizumab-based therapy.

Expression of the PTEN/FOXO3a/PLZF signalling pathway in pancreatic cancer and its significance in tumourigenesis and progression.

Pancreatic cancer (PC) is one of the most lethal gastrointestinal malignancies. The PTEN/AKT signalling pathway is closely related to the tumourigenesis and progression of PC. The downstream effectors, FOXO3a, PLZF and VEGF, are reported to be involved in angiogenesis, lymph node metastasis and poor survival in PC. By using tissue microarrays and immunohistochemistry, we found, that PTEN, FOXO3a and PLZF expression was significantly decreased in PC specimens compared with that in chronic pancreatitis (CP) specimens, while VEGF expression was significantly increased. Furthermore, the expression of PTEN was positively correlated with that of FOXO3a and PLZF but negatively correlated with that of VEGF. Our results suggest that the PTEN/FOXO3a/PLZF signalling pathway may negatively regulate VEGF expression in PC. Through clinical analysis of 69 PC patients, PTEN, FOXO3a and PLZF expression was found to be significantly decreased in specimens from PC patients with lymph node metastasis and poor prognosis, while VEGF expression was significantly increased. Taken together, these reaults suggest that the PTEN/FOXO3a/PLZF signalling pathway may be capable of inhibiting growth and metastasis in PC by regulating VEGF-mediated angiogenesis, which requires further in vivo and in vitro studies and can potentially be a therapeutic target for PC.

Phase II study of apatinib, a novel tyrosine kinase inhibitor targeting tumor angiogenesis, as second-line treatment for recurrent or advanced cervical cancer patients.

This study was designed to evaluate the efficacy and safety of apatinib, a novel tyrosine kinase inhibitor targeting tumor angiogenesis, as second-line treatment in recurrent or advanced cervical cancer patients. Twenty patients who failed cisplatin/paclitaxel ± bevacizumab treatment received a 4-week cycle of apatinib, with a daily dosage of 500 mg or 250 mg. The follow-up period ranged from 5.9 to 21.3 months (median, 14.0 months). None of the patients achieved a complete response (CR). Nevertheless, a partial response (PR), stable disease (SD) and progressive disease (PD) were observed in three, four and thirteen patients, respectively. The objective response rate (ORR) was 15.0% (95% CI, 2.1%-32.1%), and the disease control rate (DCR) was 35.0% (95% CI, 12.1%-57.9%). Among the 12 patients who were treated with bevacizumab in first-line treatment, two achieved PR and two achieved SD. The ORR and DCR were 16.7% (95% CI, 8.1%-41.4%) and 33.3% (95% CI, 2.0%-64.6%), respectively. The median progression-free survival (PFS) was 5.13 months (95% CI, 2.94-7.32 months), and the median overall survival (OS) was 12.3 months (95% CI, 10.13-14.47 months). The one-year PFS rate was 28.1%, and the one-year OS rate was 44.6%. The most common adverse events were hand-foot syndrome, hypertension, proteinuria, fatigue, nausea, vomiting, anaemia, and neutropenia. Most of the adverse events were of grades 1 and 2. The most frequent grade 3 and 4 adverse events were fatigue, hypertension, and hand-foot syndrome. In summary, apatinib is an effective and well-tolerated second-line treatment for patients with recurrent or advanced cervical cancer.

Polymorphisms in the Angiogenesis-Related Genes EFNB2, MMP2 and JAG1 Are Associated with Survival of Colorectal Cancer Patients.

An individual's inherited genetic variation may contribute to the 'angiogenic switch', which is essential for blood supply and tumor growth of microscopic and macroscopic tumors. Polymorphisms in angiogenesis-related genes potentially predispose to colorectal cancer (CRC) or affect the survival of CRC patients. We investigated the association of 392 single nucleotide polymorphisms (SNPs) in 33 angiogenesis-related genes with CRC risk and survival of CRC patients in 1754 CRC cases and 1781 healthy controls within DACHS (Darmkrebs: Chancen der Verhütung durch Screening), a German population-based case-control study. Odds ratios and 95% confidence intervals (CI) were estimated from unconditional logistic regression to test for genetic associations with CRC risk. The Cox proportional hazard model was used to estimate hazard ratios (HR) and 95% CIs for survival. Multiple testing was adjusted for by a false discovery rate. No variant was associated with CRC risk. Variants in EFNB2, MMP2 and JAG1 were significantly associated with overall survival. The association of the EFNB2 tagging SNP rs9520090 (p < 0.0001) was confirmed in two validation datasets (p-values: 0.01 and 0.05). The associations of the tagging SNPs rs6040062 in JAG1 (p-value 0.0003) and rs2241145 in MMP2 (p-value 0.0005) showed the same direction of association with overall survival in the first and second validation sets, respectively, although they did not reach significance (p-values: 0.09 and 0.25, respectively). EFNB2, MMP2 and JAG1 are known for their functional role in angiogenesis and the present study points to novel evidence for the impact of angiogenesis-related genetic variants on the CRC outcome.

Myeloid-derived suppressor cells endow stem-like qualities to multiple myeloma cells by inducing piRNA-823 expression and DNMT3B activation.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) and cancer stem cells (CSCs) are two important cellular components in the tumor microenvironment, which may modify the cancer phenotype and affect patient survival. However, the crosstalk between MDSCs and multiple myeloma stem cells (MMSCs) are relatively poorly understood. METHODS: The frequencies of granulocytic-MDSCs (G-MDSCs) in MM patients were detected by flow cytometry and their association with the disease stage and patient survival were analyzed. RT-PCR, flow cytometry, western blot and sphere formation assays were performed to investigate the effects of G-MDSCs, piRNA-823 and DNA methylation on the maintenance of stemness in MM. Then a subcutaneous tumor mouse model was constructed to analyze tumor growth and angiogenesis after G-MDSCs induction and/or piRNA-823 knockdown in MM cells. RESULTS: Our clinical dataset validated the association between high G-MDSCs levels and poor overall survival in MM patients. In addition, for the first time we showed that G-MDSCs enhanced the side population, sphere formation and expression of CSCs core genes in MM cells. Moreover, the mechanism study showed that G-MDSCs triggered piRNA-823 expression, which then promoted DNA methylation and increased the tumorigenic potential of MM cells. Furthermore, silencing of piRNA-823 in MM cells reduced the stemness of MMSCs maintained by G-MDSCs, resulting in decreased tumor burden and angiogenesis in vivo. CONCLUSION: Altogether, these data established a cellular, molecular, and clinical network among G-MDSCs, piRNA-823, DNA methylation and CSCs core genes, suggesting a new anti-cancer strategy targeting both G-MDSCs and CSCs in MM microenvironment.

Exosome-mediated secretion of LOXL4 promotes hepatocellular carcinoma cell invasion and metastasis.

BACKGROUND: Lysyl oxidase-like 4 (LOXL4) has been found to be dysregulated in several human malignancies, including hepatocellular carcinoma (HCC). However, the role of LOXL4 in HCC progression remains largely unclear. In this study, we investigated the clinical significance and biological involvement of LOXL4 in the progression of HCC. METHODS: LOXL4 expression was measured in HCC tissues and cell lines. Overexpression, shRNA-mediated knockdown, recombinant human LOXL4 (rhLOXL4), and deletion mutants were applied to study the function of LOXL4 in HCC. Exosomes derived from HCC cell lines were assessed for the ability to promote cancer progression in standard assays. The effects of LOXL4 on the FAK/Src pathway were examined by western blotting. RESULTS: LOXL4 was commonly upregulated in HCC tissues and predicted a poor prognosis. Elevated LOXL4 was associated with tumor differentiation, vascular invasion, and tumor-node-metastasis (TNM) stage. Overexpression of LOXL4 promoted, whereas knockdown of LOXL4 inhibited cell migration and invasion of HCC in vitro, and overexpressed LOXL4 promoted intrahepatic and pulmonary metastases of HCC in vivo. Most interestingly, we found that HCC-derived exosomes transferred LOXL4 between HCC cells, and intracellular but not extracellular LOXL4 promoted cell migration by activating the FAK/Src pathway dependent on its amine oxidase activity through a hydrogen peroxide-mediated mechanism. In addition, HCC-derived exosomes transferred LOXL4 to human umbilical vein endothelial cells (HUVECs) though a paracrine mechanism to promote angiogenesis. CONCLUSIONS: Taken together, our data demonstrate a novel function of LOXL4 in tumor metastasis mediated by exosomes through regulation of the FAK/Src pathway and angiogenesis in HCC.

Chemerin and Cancer.

Chemerin is a multifunctional adipokine with established roles in inflammation, adipogenesis and glucose homeostasis. Increasing evidence suggest an important function of chemerin in cancer. Chemerin's main cellular receptors, chemokine-like receptor 1 (CMKLR1), G-protein coupled receptor 1 (GPR1) and C-C chemokine receptor-like 2 (CCRL2) are expressed in most normal and tumor tissues. Chemerin's role in cancer is considered controversial, since it is able to exert both anti-tumoral and tumor-promoting effects, which are mediated by different mechanisms like recruiting innate immune defenses or activation of endothelial angiogenesis. For this review article, original research articles on the role of chemerin and its receptors in cancer were considered, which are listed in the PubMed database. Additionally, we included meta-analyses of publicly accessible DNA microarray data to elucidate the association of expression of chemerin and its receptors in tumor tissues with patients' survival.

New Insights about the Wnt/ß-Catenin Signaling Pathway in Primary Bone Tumors and Their Microenvironment: A Promising Target to Develop Therapeutic Strategies?

Osteosarcoma and Ewing sarcoma are the most common malignant primary bone tumors mainly occurring in children, adolescents and young adults. Current standard therapy includes multidrug chemotherapy and/or radiation specifically for Ewing sarcoma, associated with tumor resection. However, patient survival has not evolved for the past decade and remains closely related to the response of tumor cells to chemotherapy, reaching around 75% at 5 years for patients with localized forms of osteosarcoma or Ewing sarcoma but less than 30% in metastatic diseases and patients resistant to initial chemotherapy. Despite Ewing sarcoma being characterized by specific EWSR1-ETS gene fusions resulting in oncogenic transcription factors, currently, no targeted therapy could be implemented. It seems even more difficult to develop a targeted therapeutic strategy in osteosarcoma which is characterized by high complexity and heterogeneity in genomic alterations. Nevertheless, the common point between these different bone tumors is their ability to deregulate bone homeostasis and remodeling and divert them to their benefit. Therefore, targeting different actors of the bone tumor microenvironment has been hypothesized to develop new therapeutic strategies. In this context, it is well known that the Wnt/ß-catenin signaling pathway plays a key role in cancer development, including osteosarcoma and Ewing sarcoma as well as in bone remodeling. Moreover, recent studies highlight the implication of the Wnt/ß-catenin pathway in angiogenesis and immuno-surveillance, two key mechanisms involved in metastatic dissemination. This review focuses on the role played by this signaling pathway in the development of primary bone tumors and the modulation of their specific microenvironment.

Long noncoding RNA n339260 promotes vasculogenic mimicry and cancer stem cell development in hepatocellular carcinoma.

Vasculogenic mimicry (VM) refers to the unique capability of aggressive tumor cells to mimic the pattern of embryonic vasculogenic networks. Cancer stem cells (CSC) represent a subpopulation of tumor cells endowed with the capacity for self-renewal and multilineage differentiation. Previous studies have indicated that CSC may participate in the formation of VM. With the advance of high-resolution microarrays and massively parallel sequencing technology, long noncoding RNAs (lncRNAs) are suggested to play a critical role in tumorigenesis and, in particular, the development of human hepatocellular carcinoma (HCC). Currently, no definitive relationship between lncRNA and VM formation has been described. In the current study, we demonstrated that expression of the lncRNA, n339260, is associated with CSC phenotype in HCC, and n339260 level correlated with VM, metastasis, and shorter survival time in an animal model. Overexpression of n339260 in HepG2 cells was associated with a significant increase in CSC. Additionally, the appearance of VM and vascular endothelial (VE)-cadherin, a molecular marker of VM, was also induced by n339260 overexpression. Using a short hairpin RNA approach, n339260 was silenced in tumor cells, and knockdown of n339260 was associated with reduced VM and CSC. The results of this study indicate that n339260 promotes VM, possibly by the development of CSC. The related molecular pathways may be used as novel therapeutic targets for the inhibition of HCC angiogenesis and metastasis.

LIFR promotes tumor angiogenesis by up-regulating IL-8 levels in colorectal cancer.

Leukemia inhibitory factor receptor (LIFR) has been documented as a cancer promoter and to be present at high levels in various types of tumor tissues. In our search for molecules prognostic of colorectal cancer (CRC), we found high levels of LIFR in CRC tissue samples. Further analyses revealed that LIFR was indeed prognostic of CRC patient survival, and was associated with tumor size, lymphatic metastasis and stages. LIFR was found to promote tumor growth, metastasis and angiogenesis both in vitro and in vivo. High levels of LIFR in CRC facilitated proliferation and migration of endothelial cells, resulting in an increase in angiogenic activity. Moreover, interleukin 8 (IL-8) was found to play a role in the LIFR induced angiogenesis. IL-8 levels were correlated with LIFR levels in CRC tissues, whereas depletion of IL-8 led to a reduced angiogenic activity of LIFR in CRC cells. In addition, LIFR increased phosphorylation level of Erk, which regulates il-8 transcription. We conclude that LIFR is possibly a valuable prognostic marker for CRC. Our results also implicate a mechanism by which LIFR regulates tumor angiogenesis through Erk/IL-8 pathway, and that LIFR could be a potential therapeutic target for CRC.

Anti-angiogenic therapy for high-grade glioma.

BACKGROUND: This is an updated version of the original Cochrane Review published in September 2014. The most common primary brain tumours in adults are gliomas. Gliomas span a spectrum from low to high grade and are graded pathologically on a scale of one to four according to the World Health Organization (WHO) classification. High-grade glioma (HGG) carries a poor prognosis. Grade IV glioma is known as glioblastoma and carries a median survival in treated patients of about 15 months. Glioblastomas are rich in blood vessels (i.e. highly vascular) and also rich in a protein known as vascular endothelial growth factor (VEGF) that promotes new blood vessel formation (the process of angiogenesis). Anti-angiogenic agents inhibit the process of new blood vessel formation and promote regression of existing vessels. Several anti-angiogenic agents have been investigated in clinical trials, both in newly diagnosed and recurrent HGG, showing preliminary promising results. This review was undertaken to report on the benefits and harms associated with the use of anti-angiogenic agents in the treatment of HGGs. OBJECTIVES: To evaluate the efficacy and toxicity of anti-angiogenic therapy in people with high-grade glioma (HGG). The intervention can be used in two broad groups: at first diagnosis as part of 'adjuvant' therapy, or in the setting of recurrent disease. SEARCH METHODS: We conducted updated searches to identify published and unpublished randomised controlled trials (RCTs), including the Cochrane Central Register of Controlled Trials (CENTRAL; 2018, Issue 9), MEDLINE and Embase to October 2018. We handsearched proceedings of relevant oncology conferences up to 2018. We also searched trial registries for ongoing studies. SELECTION CRITERIA: RCTs evaluating the use of anti-angiogenic therapy to treat HGG versus the same therapy without anti-angiogenic therapy. DATA COLLECTION AND ANALYSIS: Review authors screened the search results and reviewed the abstracts of potentially relevant articles before retrieving the full text of eligible articles. MAIN RESULTS: After a comprehensive literature search, we identified 11 eligible RCTs (3743 participants), of which 7 were included in the original review (2987 participants). There was significant design heterogeneity in the included studies, especially in the response assessment criteria used. All eligible studies were restricted to glioblastomas and there were no eligible studies evaluating other HGGs. Ten studies were available as fully published peer-reviewed manuscripts, and one study was available in abstract form. The overall risk of bias in included studies was low. This risk was based upon low rates of selection bias, detection bias, attrition bias and reporting bias. The 11 studies included in this review did not show an improvement in overall survival with the addition of anti-angiogenic therapy (pooled hazard ratio (HR) of 0.95, 95% confidence interval (CI) 0.88 to 1.02; P = 0.16; 11 studies, 3743 participants; high-certainty evidence). However, pooled analysis from 10 studies (3595 participants) showed improvement in progression-free survival with the addition of anti-angiogenic therapy (HR 0.73, 95% CI 0.68 to 0.79; P < 0.00001; high-certainty evidence).We carried out additional analyses of overall survival and progression-free survival according to treatment setting and for anti-angiogenic therapy combined with chemotherapy compared to chemotherapy alone. Pooled analysis of overall survival in either the adjuvant or recurrent setting did not show an improvement (HR 0.93, 95% CI 0.86 to 1.02; P = 0.12; 8 studies, 2833 participants; high-certainty evidence and HR 0.99, 95% CI 0.85 to 1.16; P = 0.90; 3 studies, 910 participants; moderate-certainty evidence, respectively). Pooled analysis of overall survival for anti-angiogenic therapy combined with chemotherapy compared to chemotherapy also did not clearly show an improvement (HR 0.92, 95% CI 0.85 to 1.00; P = 0.05; 11 studies, 3506 participants; low-certainty evidence). The progression-free survival in the subgroups all showed findings that demonstrated improvements in progression-free survival with the addition of anti-angiogenic therapy. Pooled analysis of progression-free survival in both the adjuvant and recurrent setting showed an improvement (HR 0.75, 95% CI 0.69 to 0.82; P < 0.00001; 8 studies, 2833 participants; high-certainty evidence and HR 0.64, 95% CI 0.54 to 0.76; P < 0.00001; 2 studies, 762 participants; moderate-certainty evidence, respectively). Pooled analysis of progression-free survival for anti-angiogenic therapy combined with chemotherapy compared to chemotherapy alone showed an improvement (HR 0.72, 95% CI 0.66 to 0.77; P < 0.00001; 10 studies, 3464 participants). Similar to trials of anti-angiogenic therapies in other solid tumours, adverse events related to this class of therapy included hypertension and proteinuria, poor wound healing, and the potential for thromboembolic events, although generally, the rate of grade 3 and 4 adverse events was low (< 14.1%) and in keeping with the literature. The impact of anti-angiogenic therapy on quality of life varied between studies. AUTHORS' CONCLUSIONS: The use of anti-angiogenic therapy does not significantly improve overall survival in newly diagnosed people with glioblastoma. Thus, there is insufficient evidence to support the use of anti-angiogenic therapy for people with newly diagnosed glioblastoma at this time. Overall there is a lack of evidence of a survival advantage for anti-angiogenic therapy over chemotherapy in recurrent glioblastoma. When considering the combination anti-angiogenic therapy with chemotherapy compared with the same chemotherapy alone, there may possibly be a small improvement in overall survival. While there is strong evidence that bevacizumab (an anti-angiogenic drug) prolongs progression-free survival in newly diagnosed and recurrent glioblastoma, the impact of this on quality of life and net clinical benefit for patients remains unclear. Not addressed here is whether subsets of people with glioblastoma may benefit from anti-angiogenic therapies, nor their utility in other HGG histologies.

Rictor regulates the vasculogenic mimicry of melanoma via the AKT-MMP-2/9 pathway.

Vasculogenic mimicry (VM)-positive melanomas are usually associated with poor prognosis. Rictor, the key component of the rapamycin-insensitive complex of mTOR (mTORC2), is up-regulated in several cancers, especially in melanomas with poor prognosis. The aim of this study was to investigate the role of Rictor in the regulation of VM and the mechanism underlying this possible regulation. VM channels were found in 35 of 81 tested melanoma samples and high Rictor expression correlated with VM structures. Moreover, Kaplan-Meier survival curves indicated that VM structures and high Rictor expression correlated with shorter survival in patients with melanoma. In vitro, Rictor knockdown by short hairpin RNA (shRNA) significantly inhibited the ability of A375 and MUM-2B melanoma cells to form VM structures, as evidenced by most tubes remaining open. Cell cycle analysis revealed that Rictor knockdown blocked cell growth and resulted in the accumulation of cells in G2/M phase, and cell migration and invasion were greatly affected after Rictor down-regulation. Western blotting assays indicated that down-regulating Rictor significantly inhibited the phosphorylation of AKT at Ser473 and Thr308 , which subsequently inhibited the expression and activity of downstream MMP-2/9, as confirmed by real-time PCR and gelatin Zymography. MK-2206, a small-molecule inhibitor of AKT, similarly inhibited the activity of AKT and secretion of MMP-2/9, further supporting that Rictor down-regulation inhibits the phosphorylation of AKT and activity of downstream MMP-2/9 to affect VM formation. In conclusion, Rictor plays an important role in melanoma VM via the Rictor-AKT-MMP-2/9 signalling pathway.

Increased vasohibin-1 expression is associated with metastasis and poor prognosis of renal cell carcinoma patients.

The microvascular density detected by markers of endothelial cells (ECs), such as CD31 and CD34, is considered to be a biomarker for angiogenesis, and it is generally associated with the malignant potential of solid tumors. However, there is a conflicting relationship between the microvascular density and prognosis in clear-cell renal cell carcinoma (ccRCC) patients. It may be explained by the suggestion that the microvascular density cannot fully reflect the angiogenic activity in ccRCC, as the markers of ECs are expressed by both quiescent and activated ECs. To investigate the real angiogenic activity, we examined vasohibin-1 (VASH1), a recently identified regulator of angiogenesis, which was demonstrated to be specifically expressed by ECs of newly formed blood vessels. Expression of VASH1 and CD34 were immunohistochemically examined in 116 primary untreated ccRCCs, 10 metastatic untreated ccRCCs, and 9 metastatic ccRCCs treated with sunitinib. ECs in the tumor microvessels were sporadically immunostained for VASH1, although no VASH1 staining was observed in the non-neoplastic renal tissues. CD34 was ubiquitously expressed by all ECs in both ccRCC and non-neoplastic renal tissues. Multivariate Cox analysis indicated that an elevated VASH1 density, but not microvascular density, was a significant and independent predictor of overall survival (odds ratio, 7.71; P=0.003). The microvascular density was significantly decreased in the sunitinib-treated metastases compared with untreated tumors (P=0.001). On the other hand, the VASH1 density was significantly higher in the metastatic ccRCCs treated with sunitinib compared with non-treated ones (P=0.010), indicating that VASH1 may be associated with the resistance of ECs to sunitinib treatment. Thus, VASH1 expression may reflect the actual activity of angiogenesis, and VASH1 can serve as a new prognostic and predictive biomarker in patients with ccRCC.

Anti-angiogenesis for cancer revisited: Is there a role for combinations with immunotherapy?

Angiogenesis is defined as the formation of new blood vessels from preexisting vessels and has been characterized as an essential process for tumor cell proliferation and viability. This has led to the development of pharmacological agents for anti-angiogenesis to disrupt the vascular supply and starve tumor of nutrients and oxygen, primarily through blockade of VEGF/VEGFR signaling. This effort has resulted in 11 anti-VEGF drugs approved for certain advanced cancers, alone or in combination with chemotherapy or other targeted therapies. But this success had only limited impact on overall survival of cancer patients and rarely resulted in durable responses. Given the recent success of immunotherapies, combinations of anti-angiogenics with immune checkpoint blockers have become an attractive strategy. However, implementing such combinations will require a better mechanistic understanding of their interaction. Due to overexpression of pro-angiogenic factors in tumors, their vasculature is often tortuous and disorganized, with excessively branched leaky vessels. This enhances vascular permeability, which in turn is associated with high interstitial fluid pressure, and a reduction in blood perfusion and oxygenation. Judicious dosing of anti-angiogenic treatment can transiently normalize the tumor vasculature by decreasing vascular permeability and improving tumor perfusion and blood flow, and synergize with immunotherapy in this time window. However, anti-angiogenics may also excessively prune tumor vessels in a dose and time-dependent manner, which induces hypoxia and immunosuppression, including increased expression of the immune checkpoint programmed death receptor ligand (PD-L1). This review focuses on revisiting the concept of anti-angiogenesis in combination with immunotherapy as a strategy for cancer treatment.

Vascular Invasion and Metastasis is Predictive of Outcome in Barcelona Clinic Liver Cancer Stage C Hepatocellular Carcinoma.

Background: Patients with Barcelona Clinic Liver Cancer (BCLC) stage C hepatocellular carcinoma (HCC) have variable long-term outcomes. Better delineation of prognosis is important for clinical trial enrollment and clinical practice in an era of precision medicine. We hypothesized that stratification of patients with BCLC stage C HCC by presence of vascular invasion and/or metastasis improves prognostic discrimination. Methods: Using a prospectively maintained database, we identified 234 patients diagnosed with BCLC stage C HCC between 2005 and 2015. Patients were stratified into 3 groups based on tumor characteristics: (1) vascular invasion alone, (2) metastasis alone, and (3) vascular invasion and metastasis. Overall survival (OS) was compared using a Cox model. A subgroup analysis was performed based on extent of vascular invasion and site of metastasis. Results: The cohort comprised 123 patients (53%) with vascular invasion alone, 34 (15%) with metastasis alone, and 77 (33%) with both vascular invasion and metastasis. Median survival was 5.7, 3.9, and 3.0 months, respectively (P<.01). Patients with vascular invasion or metastasis alone had significantly better survival compared with those with vascular invasion and metastasis (adjusted hazard ratio [HR],0.68; 95% CI, 0.49-0.94, and HR, 0.61; 95% CI, 0.39-0.96, respectively). Compared with tumoral invasion of branch portal veins, involvement of the main portal vein was associated with worse survival (HR, 2.13; 95% CI, 1.29-3.49). OS did not differ by site of metastasis. Conclusions: Stratification of patients within the BCLC stage C staging subgroup by vascular invasion and presence of metastasis further discriminates patient prognosis. This substratification may have implications for therapy and more accurate prognostic features.

Perfusion Parameters on Breast Dynamic Contrast-Enhanced MRI Are Associated With Disease-Specific Survival in Patients With Triple-Negative Breast Cancer.

OBJECTIVE: The aim of this study was to investigate the association between perfusion parameters on MRI performed before treatment and survival outcome (disease-free survival [DFS], disease-specific survival [DSS]) in patients with triple-negative breast cancer (TNBC). MATERIALS AND METHODS: Sixty-one patients (median age, 50 years; age range, 27-77 years) with TNBC (tumor size on MRI: median, 25.5 mm; range, 11.0-142.0 mm) were included. We analyzed clinical and pathologic variables and MRI parameters. Cox proportional hazards models were used to determine associations with survival outcome. RESULTS: The median follow-up time was 46.1 months (range, 13.9-58.4 months). Eleven of 61 (18.0%) patients had events (i.e., local, regional, or distant recurrence or contralateral breast cancer) and seven (11.5%) died of breast cancer. Among the pretreatment variables, a larger tumor size on MR images (hazard ratio [HR] = 1.024, p = 0.003) was associated with worse DFS at univariate analysis. In multivariate pretreatment models for DSS, a higher fractional volume of extravascular extracellular space per unit volume of tissue (ve) value (HR = 1.658, p = 0.038), higher peak enhancement (HR = 1.843, p = 0.018), and a larger tumor size on MR images (HR = 1.060, p = 0.001) were associated with worse DSS. In multivariate posttreatment models, a larger pathologic tumor size (HR for DFS, 1.074 [p = 0.005]; HR for DSS, 1.050 [p = 0.042]) and metastasis in surgically resected axillary lymph nodes (HR for DFS, 5.789 [p = 0.017]; HR for DSS, 23.717 [p = 0.005]) were associated with worse survival outcome. CONCLUSION: A higher ve value, higher peak enhancement, and larger tumor size of the primary tumor on pretreatment MRI were independent predictors of worse DSS in patients with TNBC.

Combined Analysis of Vascular Endothelial Growth Factor Expression with Cyclooxygenase-2 and Mast Cell Density in Oral Squamous Cell Carcinoma.

BACKGROUND: Overexpression of vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2) and increasing mast cell density (MCD) in premalignant and malignant oral lesions have been documented. However, their correlation with clinicopathologic parameters and survival rate in oral squamous cell carcinoma (OSCC) is not completely clear. This study aimed to assess these subjects. METHODS: VEGF, COX-2, and mast cell tryptase expression were examined immunohistochemically in 57 cases of OSCC. The relationships between the markers' expression and clinicopathologic data were assessed using bivariate and multivariate analysis. RESULTS: Spearman's rank correlation coefficient showed a significant correlation between VEGF and COX-2 expression (r = 0.462, p < 0.001), as well as between VEGF expression and MCD (r = 0.306, p < 0.001). Multivariate analysis showed no significant correlation between the markers' immunoexpression and overall survival (OS), but a significant correlation between mode of invasion and OS [hazard ratio 0.362 (95% CI: 0.138- 0.974); p = 0.038] was observed. An association between MCD and gender (p = 0.042) was also found, as MCD was higher in males. CONCLUSION: The significant correlation of VEGF expression with COX-2 expression and MCD may represent the roles of COX-2 and MCD in tumor angiogenesis by modulating VEGF production. However, VEGF, COX-2, and MCD are not useful indicators to predict prognosis in OSCC. Nevertheless, the mode of invasion can be considered as an independent prognostic factor in OSCC patients.

Angiogenesis Inhibitors in NSCLC.

Angiogenesis is a complex biological process that plays a relevant role in sustaining the microenvironment, growth, and metastatic potential of several tumors, including non-small cell lung cancer (NSCLC). Bevacizumab was the first angiogenesis inhibitor approved for the treatment of patients with advanced NSCLC in combination with chemotherapy; however, it was limited to patients with non-squamous histology and first-line setting. Approval was based on the results of two phase III trials (ECOG4599 and AVAIL) that demonstrated an improvement of about two months in progression-free survival (PFS) in both trials, and in the ECOG4599 trial, an improvement in overall survival (OS) also. Afterwards, other antiangiogenic agents, including sunitinib, sorafenib, and vandetanib have been unsuccessfully tested in first and successive lines. Recently, two new antiangiogenic agents (ramucirumab and nintedanib) produced a significant survival benefit in second-line setting. In the REVEL study, ramucirumab plus docetaxel prolonged the median OS of patients with any histology NSCLC when compared with docetaxel alone (10.4 versus 9.1 months, hazard ratio (HR) 0.857, p = 0.0235). In the LUME-Lung 1 study, nintedanib plus docetaxel prolonged the median PFS of patients with any tumor histology (p = 0.0019), and improved OS (12.6 versus 10.3 months) in patients with adenocarcinoma. As a result, it became a new option for the second-line treatment of patients with advanced NSCLC and adenocarcinoma histology. Identifying predictive biomarkers to optimize the benefit of antiangiogenic drugs remains an ongoing challenge.