Interleukin-8 and its receptor CXCR2 in the tumour microenvironment promote colon cancer growth, progression and metastasis

BACKGROUND: Colorectal cancer (CRC) is a leading cause of death in the United States. Increased level of interleukin-8 (IL-8) and CXCR2 on tumours and in the tumour microenvironment has been associated with CRC growth, progression and recurrence in patients. Here, we aimed to evaluate the effects of tissue microenvironment-encoded IL-8 and CXCR2 on colon cancer progression and metastasis.

METHODS: A novel immunodeficient, skin-specific IL-8-expressing transgenic model was generated to evaluate colon cancer growth and metastasis. Syngeneic mouse colon cancer cells were grafted in CXCR2 knockout (KO) mice to study the contribution of CXCR2 in the microenvironment to cancer growth.

RESULTS: Elevated levels of IL-8 in the serum and tumour microenvironment profoundly enhanced the growth of human and mouse colon cancer cells with increased peri-tumoural angiogenesis, and also promoted the extravasation of the cancer cells into the lung and liver. The tumour growth was inhibited in CXCR2 KO mice with significantly reduced tumour angiogenesis and increased tumour necrosis.

CONCLUSION: Increased expression of IL-8 in the tumour microenvironment enhanced colon cancer growth and metastasis. Moreover, the absence of its receptor CXCR2 in the tumour microenvironment prevented colon cancer cell growth. Together, our study demonstrates the critical roles of the tumour microenvironment-encoded IL-8/CXCR2 in colon cancer pathogenesis, validating the pathway as an important therapeutic target.

Assessing the in vivo efficacy of biologic antiangiogenic therapies

PURPOSE: To review key clinical issues underlying the assessment of in vivo efficacy when using antiangiogenic therapies for cancer treatment.

METHODS: Literature relevant to use of antiangiogenic therapies in cancer was reviewed, with particular emphasis on the assessment of in vivo efficacy of these agents, as well as additional angiogenic factors that could play a role in escape from angiogenesis inhibition.

RESULTS: In order to grow and metastasize, tumors need to continually acquire new blood supplies; therefore, therapeutic inhibition of angiogenesis has become a component of anticancer treatment for many tumor types. Bevacizumab, a humanized monoclonal antibody directed at vascular endothelial growth factor A (VEGF-A), has shown activity in combination with chemotherapy in metastatic colorectal cancer. Nevertheless, the use of antiangiogenic therapies remains suboptimal; specifically, optimal dose, duration of therapy, and combination of agents remain unknown. Also, at present, it is not possible to determine which patients are most likely to respond to a given form of antiangiogenic therapy. There has been increased recognition of alternative pathways possibly associated with disease progression in patients undergoing antiangiogenic therapy targeted at VEGF-A. Multiligand-targeted antiangiogenic therapies, such as ziv-aflibercept (formerly known as aflibercept, VEGF Trap), are currently undergoing clinical evaluation. Ziv-aflibercept forms monomeric complexes with VEGF-A, VEGF-B, and PlGF, which have a long half-life, allowing optimization of ziv-aflibercept doses and angiogenic blockage.

CONCLUSIONS: Although antiangiogenic therapies have increased treatment options for cancer patients, their use is limited by a lack of established and standardized methodology to evaluate their efficacy in vivo. Circulating endothelial cells, hypertension, and several molecular and imaging-based markers have potential for use as biomarkers in these patients and may better define appropriate patient populations.

DNA microarray profiling of genes differentially regulated by the histone deacetylase inhibitors vorinostat and LBH589 in colon cancer cell lines

BACKGROUND: Despite the significant progress made in colon cancer chemotherapy, advanced disease remains largely incurable and novel efficacious chemotherapies are urgently needed. Histone deacetylase inhibitors (HDACi) represent a novel class of agents which have demonstrated promising preclinical activity and are undergoing clinical evaluation in colon cancer. The goal of this study was to identify genes in colon cancer cells that are differentially regulated by two clinically advanced hydroxamic acid HDACi, vorinostat and LBH589 to provide rationale for novel drug combination partners and identify a core set of HDACi-regulated genes.

METHODS: HCT116 and HT29 colon cancer cells were treated with LBH589 or vorinostat and growth inhibition, acetylation status and apoptosis were analyzed in response to treatment using MTS, Western blotting and flow cytometric analyses. In addition, gene expression was analyzed using the Illumina Human-6 V2 BeadChip array and Ingenuity Pathway Analysis.

RESULTS: Treatment with either vorinostat or LBH589 rapidly induced histone acetylation, cell cycle arrest and inhibited the growth of both HCT116 and HT29 cells. Bioinformatic analysis of the microarray profiling revealed significant similarity in the genes altered in expression following treatment with the two HDACi tested within each cell line. However, analysis of genes that were altered in expression in the HCT116 and HT29 cells revealed cell-line-specific responses to HDACi treatment. In addition a core cassette of 11 genes modulated by both vorinostat and LBH589 were identified in both colon cancer cell lines analyzed.

CONCLUSION: This study identified HDACi-induced alterations in critical genes involved in nucleotide metabolism, angiogenesis, mitosis and cell survival which may represent potential intervention points for novel therapeutic combinations in colon cancer. This information will assist in the identification of novel pathways and targets that are modulated by HDACi, providing much-needed information on HDACi mechanism of action and providing rationale for novel drug combination partners. We identified a core signature of 11 genes which were modulated by both vorinostat and LBH589 in a similar manner in both cell lines. These core genes will assist in the development and validation of a common gene set which may represent a molecular signature of HDAC inhibition in colon cancer.

EGF61 polymorphism predicts complete pathologic response to cetuximab-based chemoradiation independent of KRAS status in locally advanced rectal cancer patients

BACKGROUND: Cetuximab has shown significant clinical activity in metastatic colon cancer. However, cetuximab-containing neoadjuvant chemoradiation has not been shown to improve tumor response in locally advanced rectal cancer patients in recent phase I/II trials. We evaluated functional germline polymorphisms of genes involved in epidermal growth factor receptor pathway, angiogenesis, antibody-dependent cell-mediated cytotoxicity, DNA repair, and drug metabolism, for their potential role as molecular predictors for clinical outcome in locally advanced rectal cancer patients treated with preoperative cetuximab-based chemoradiation.

METHODS: 130 patients (74 men and 56 women) with locally advanced rectal cancer (4 with stage II, 109 with stage III, and 15 with stage IV, 2 unknown) who were enrolled in phase I/II clinical trials treated with cetuximab-based chemoradiation in European cancer centers were included. Genomic DNA was extracted from formalin-fixed paraffin-embedded tumor samples and genotyping was done by using PCR-RFLP assays. Fisher's exact test was used to examine associations between polymorphisms and complete pathologic response (pCR) that was determined by a modified Dworak classification system (grade III vs. grade IV: complete response).

RESULTS: Patients with the epidermal growth factor (EGF) 61 G/G genotype had pCR of 45% (5/11), compared with 21% (11/53) in patients heterozygous, and 2% (1/54) in patients homozygous for the A/A allele (P < 0.001). In addition, this association between EGF 61 G allele and pCR remained significant (P = 0.019) in the 59 patients with wild-type KRAS.

CONCLUSION: This study suggested EGF A+61G polymorphism to be a predictive marker for pCR, independent of KRAS mutation status, to cetuximab-based neoadjuvant chemoradiation of patients with locally advanced rectal cancer.

Molecular predictors of response to antiangiogenesis therapies

Angiogenesis is a crucial component of tumor growth and metastasis. Targeting the vascular endothelial growth factor pathway represents therapeutic potentials for treating cancer. To date, 3 Food and Drug Administration-approved agents targeting angiogenesis have been developed, bevacizumab, sunitinib, and sorafenib. However, no validated biomarkers are available to identify those patients who are likely to benefit from antiangiogenesis therapy. Molecular biomarker research in antiangiogenesis inhibition is an actively growing field. Although current data are extremely promising, it is still uncertain which biomarker(s) can reliably predict their efficacy. With increasing numbers of inhibitors being developed, the need for biomarkers is more critical than ever. This review will focus on translational research that strives to identify molecular biomarkers (tissue, circulating and genomic) for approved antiangiogenesis therapies that can indicate benefit, resistance, and toxicity.

Prognostic significance of combining VEGFA, FLT1 and KDR mRNA expression in lung cancer

Angiogenesis is important in cancer progression. Promising results in clinical trials have indicated that targeting vascular epidermal growth factor (VEGF) signaling may prolong lung cancer patient survival. In particular, various studies have implicated VEGFA as a potential prognostic marker in lung cancer, although prognostication using the expression of VEGF receptors (VEGFRs), such as fms-related tyrosine kinase 1 (FLT1; also known as VEGFR1) and kinase insert domain receptor (KDR; also known as VEGFR2), has produced varied results in different lung cancer studies. The present study aimed to investigate the prognostic significance of these three factors, alone or in combination. mRNA expression data were extracted from four independent lung cancer cohorts totaling 583 patients, and the association between mRNA expression and survival was investigated by performing statistical analyses. When VEGFA, FLT1 and KDR expression were considered alone, only VEGFA demonstrated a significant association with patient survival consistently across all four datasets (P<0.05). Patients with a high expression of VEGFA and one of the two receptors were associated with significantly worse survival than patients expressing low levels of VEGFA and the particular receptor (P<0.05). Notably, patients with a high level expression of all three genes in their tumor specimens were associated with a significantly shorter survival time compared with patients exhibiting a low level expression of one, two or all three genes (P<0.05). The results indicate that a high level of VEGFA expression and its receptors may be required for cancer progression. Therefore, these three factors should be considered together as a prognostic indicator for lung cancer patients.

Pulpal Neuropeptide Y levels in relation to pain experience

Neuropeptide Y is a 36 amino acid peptide that belongs to the pancreatic polypeptide family. It co-localises with adrenaline in sympathetic nerves and is released upon sympathetic activation resulting in vasoconstriction. In addition to its vascular effects NPY is also thought to have a role in pain modulation, angiogenesis and immunomodulation. Objectives: The aim of this study was to quantify the levels of NPY in human dental pulp tissue from intact and grossly carious teeth and to relate these results to pain experience. Methods: A total of 48 permanent teeth [mean age 32.1(+/- 11.2 years)] were included in the study, of these 22 were intact and 26 were grossly carious. In the grossly carious group, 17 teeth were reported painful prior to extraction and the remainder were reported non-painful. NPY was measured using a sensitive and specific radioimmunoassay which has been previously described. Pain was scored as either present or absent in all the teeth studied. Results: Of particular interest in this study was the finding that NPY levels were significantly higher in dental pulp tissue from non-painful grossly carious teeth (p= 0.006) compared with painful grossly carious teeth. Conclusions: The increased levels of NPY reported in non-painful grossly carious teeth may suggest a role for NPY in pain modulation in human dental pulp.

The prognostic and therapeutic value of EpHA2 in early colorectal cancer (CRC).

Background: EpHA2 is a 130 kD transmembrane glycoprotein belonging to ephrin receptor subfamily and involved in angiogenesis/tumour neovascularisation. High EpHA2 mRNA level has recently been implicated in cetuximab resistance. Previously, we found high EpHA2 levels in a panel of invasive colorectal cancer (CRC) cells, which was associated with high levels of stem-cell marker CD44. Our aim was to investigate the prognostic value of EpHA2 and subsequently correlate expression levels to known clinico-pathological variables in early stage CRC. Methods: Tissue samples from 509 CRC patients were analysed. EpHA2 expression was measured using IHC. Kaplan-Meier graphs were used. Univariate and multivariate analyses employed Cox Proportional Hazards Ratio (HR) method. A backward selection method (Akaike’s information criterion) was used to determine a refined multivariate model. Results: EpHA2 was highly expressed in CRC adenocarcinoma compared to matched normal colon tissue. In support of our preclinical invasive models, strong correlation was found between EpHA2 expression and CD44 and Lgr5 staining (p<0.001). In addition, high EpHA2 expression significantly correlated with vascular invasion (p=0.03).HR for OS for stage II/III patients with high EpHA2 expression was 1.69 (95%CI: 1.164-2.439; p=0.003). When stage II/III was broken down into individual stages, there was significant correlation between high EpHA2 expression and poor 5-years OS in stage II patients (HR: 2.18; 95%CI: 1.28-3.71; p=0.005).HR in the stage III group showed a trend to statistical significance (HR: 1.48; 95%CI=0.87-2.51; p=0.05). In both univariate and multivariate analyses of stage II patients, high EpHA2 expression was the only significant factor and was retained in the final multivariate model. Higher levels of EpHA2 were noted in our RAS and BRAF mutant CRC cells, and silencing EpHA2 resulted in significant decreases in migration/invasion in parental and invasive CRC sublines. Correlation between KRAS/NRAS/BRAFmutational status and EpHA2 expression in clinical samples is ongoing. Conclusions: Taken together, our study is the first to indicate that EpHA2 expression is a predictor of poor clinical outcome and a potential novel target in early stage CRC.

Neuropeptides regulate expression of angiogenic growth factors in pulpal fibroblasts

In the dental pulp angiogenesis is crucial for tooth development and a prerequisite for successful repair following injury and inflammation. The role of neuropeptides in pulpal inflammation has been well documented but their role in the regulation of angiogenesis in the dental pulp has not been elucidated. Objectives: The aim was to profile the expression of angiogenic growth factors produced by pulp fibroblasts and to study the effects of neuropeptides on their expression. Methods: Human pulp fibroblasts derived from healthy molar teeth were stimulated with neuropeptides previously identified in dental pulp, namely, Substance P (SP), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP) and calcitonin related gene peptide (CGRP) for 24 and 48 hrs. Simultaneous expression of ten growth factors was quantified using a novel human angiogenesis array (Ray Biotech, USA). Results: Pulp fibroblasts expressed human angiogenic growth factors, VEGF, bFGF, PDGF-BB, HGF, ANG2, HB-EGF, PIGF, angiogenin and leptin. Among the growth factors expressed VEGF, angiogenin and HGF were abundantly expressed compared to others. Neuropeptides induced variable effects on the expression of the angiogenic factors: CGRP potently up-regulated VEGF, bFGF, HGF and PIGF after 24 hr, while NPY tended to down regulate growth factors after 24 hr in culture but markedly up regulated ANG2, bFGF and leptin after 48 hr. SP down regulated expression of all angiogenic growth factors except for leptin, while VIP induced a small increase in expression of each growth factor, irrespective of time. Conclusion: Pulp fibroblasts express a range of angiogenic growth factors including angiogenin and leptin. Neuropeptides regulate the expression of these factors, suggesting an additional role for neuropeptides in the regulation of inflammation and healing in the dental pulp.
This work is supported by TC White Research Fund

INFLUENCE OF NOX4 NADPH OXIDASE ON GENERATION AND ANGIOGENIC FUNCTION OF INDUCED PLURIPOTENT STEM CELL-DERIVED ENDOTHELIAL CELLS

Human induced pluripotent stem (iPS) cell-derived endothelial cells (ECs) hold clear potential for therapeutic angiogenesis as a novel strategy for ischaemic disease. Recently, we have developed a novel method for direct reprogramming of partial iPS (PiPS) cells, which unlike iPS cells, are generated before pluripotency so do not form tumours, and may be differentiated into ECs with characteristic morphology and pro-angiogenic actions. Our previous work showed that PiPS-derived ECs are capable of forming vascular-like tubes both in vitro and in vivo and promoting re-endothelialisation of ischemic tissue, with greater effectiveness versus mature ECs.

Interestingly, our preliminary data demonstrate that Nox NADPH oxidases, which are reported to influence stem cell function, are progressively induced during PiPs/PiPS-EC differentiation and in response to hypoxia, with Nox4 demonstrating highest expression. As this isoform is an established regulator of angiogenesis, we hypothesize that Nox4 plays a key role in modulating PiPS-EC generation and angiogenic function.

The aim of this project is therefore to investigate: (1) the specific role of Nox4 in direct reprogramming of PiPS cells and differentiation to PiPS-ECs; (2) whether genetic manipulation of Nox4 influences in vitro function of PiPs-ECs and their ability to promote in vivo angiogenesis. This will be achieved by employing established in vitro functional assays and an experimental model of hindlimb ischaemia with assessment of relevant end-points. Identification of a key role for Nox4 in regulating PiPS-EC generation/function may inform selective targeting of this isoform to enhance the efficiency of PiPS-EC differentiation and their capacity to treat ischemic disease.

STAT3 activation in circulating monocytes contributes to neovascular age-related macular degeneration

Infiltrating macrophages are critically involved in pathogenic angiogenesis such as neovascular age-related macular degeneration (nAMD). Macrophages originate from circulating monocytes and three subtypes of monocyte exist in humans: classical (CD14+CD16-), non-classical (CD14-CD16+) and intermediate (CD14+CD16+) monocytes. The aim of this study was to investigate the role of circulating monocyte in neovascular age-related macular degeneration (nAMD). Flow cytometry analysis showed that the intermediate monocytes from nAMD patients expressed higher levels of CX3CR1 and HLA-DR compared to those from controls. Monocytes from nAMD patients expressed higher levels of phosphorylated Signal Transducer and Activator of Transcription 3 (pSTAT3), and produced higher amount of VEGF. In the mouse model of choroidal neovascularization (CNV), pSTAT3 expression was increased in the retina and RPE/choroid, and 49.24% of infiltrating macrophages express pSTAT3. Genetic deletion of the Suppressor of Cytokine Signalling 3 (SOCS3) in myeloid cells in the LysM-Cre+/-:SOCS3fl/fl mice resulted in spontaneous STAT3 activation and accelerated CNV formation. Inhibition of STAT3 activation using a small peptide LLL12 suppressed laser-induced CNV. Our results suggest that monocytes, in particular the intermediate subset of monocytes are activated in nAMD patients. STAT3 activation in circulating monocytes may contribute to the development of choroidal neovascularisation in AMD.

Beneficial Effects of Berberine on Oxidized LDL-induced Cytotoxicity in Human Retinal Müller Cells

PURPOSE. Limited mechanistic understanding of diabetic retinopathy (DR) has hindered therapeutic advances. Berberine, an isoquinolone alkaloid, has shown favorable effects on glucose and lipid metabolism in animal and human studies, but effects on DR are unknown. We previously demonstrated intraretinal extravasation and modification of LDL in human diabetes, and toxicity of modified LDL to human retinal M¨uller cells. We now explore pathogenic effects of modified LDL on M¨uller cells, and the efficacy of berberine in mitigating this cytotoxicity. METHODS. Confluent human M¨uller cells were exposed to in vitro–modified ‘highly oxidized, glycated (HOG-) LDL versus native-LDL (N-LDL; 200 mg protein/L) for 6 or 24 hours, with/ without pretreatment with berberine (5 lM, 1 hour) and/or the adenosine monophosphate (AMP)-activated protein kinase (AMPK) inhibitor, Compound C (5 lM, 1 hour). Using techniques including Western blots, reactive oxygen species (ROS) detection assay, and quantitative real-time PCR, the following outcomes were assessed: cell viability (CCK-8 assay), autophagy (LC3, Beclin-1, ATG-5), apoptosis (cleaved caspase 3, cleaved poly-ADP ribose polymerase), oxidative stress (ROS, nuclear factor erythroid 2-related factor 2, glutathione peroxidase 1, NADPH oxidase 4), angiogenesis (VEGF, pigment epithelium-derived factor), inflammation (inducible nitric oxide synthase, intercellular adhesion molecule 1, IL-6, IL-8, TNF-a), and glial cell activation (glial fibrillary acidic protein). RESULTS. Native-LDL had no effect on cultured human M¨uller cells, but HOG-LDL exhibited marked toxicity, significantly decreasing viability and inducing autophagy, apoptosis, oxidative stress, expression of angiogenic factors, inflammation, and glial cell activation. Berberine attenuated all the effects of HOG-LDL (all P < 0.05), and its effects were mitigated by AMPK inhibition (P < 0.05). CONCLUSIONS. Berberine inhibits modified LDL-induced M¨uller cell injury by activating the AMPK pathway, and merits further study as an agent for preventing and/or treating DR.