Sarah P. Gunningham

The angiogenic switch for vascular endothelial growth factor (VEGF)-A, VEGF-B, VEGF-C, and VEGF-D in the adenoma–carcinoma sequence during colorectal cancer progression

Angiogenesis is essential for tumour growth and metastasis. It is controlled by angiogenic factors, one of the most important being vascular endothelial growth factor (VEGF)‐A. Although its role has been demonstrated in many tumour types including colorectal carcinoma (CRC), the importance of the newer family members in adenoma, invasive tumour growth, and progression to a metastatic phenotype has been poorly characterized in CRC. The aim of this study was to determine the role and timing of the VEGF angiogenic switch during CRC progression. We measured the gene expression of VEGF ligands (VEGF‐A, VEGF‐B, VEGF‐C, and VEGF‐D) and their receptors (VEGFR‐1, VEGFR‐2, and VEGFR‐3), in normal colorectal tissues (n = 20), adenomas (n = 10), and in CRC (n = 71) representing different Dukes stages using ribonuclease protection assay, semi‐quantitative relative reverse transcriptase polymerase chain reaction, together with the pattern of their expression by immunohistochemistry. VEGF‐A mRNA was the most abundant in colorectal tissue, followed by VEGF‐B, VEGF‐C, and VEGF‐D. VEGF‐A and VEGF‐B mRNAs were significantly more abundant in adenomas (p = 0.0003 and p = 0.04 respectively) compared with normal tissues, while VEGF‐A and VEGF‐C were significantly increased in carcinomas compared with normal tissues (p = 0.0006 and p = 0.0009 respectively). A significantly greater amount of VEGF‐C mRNA was present in carcinomas compared with adenomas (p = 0.03), whereas there was a significant reduction of VEGF‐B in carcinomas compared with adenomas (p = 0.0002). VEGF‐D mRNA was significantly more abundant in normal tissues than in adenomas (p = 0.0001) and carcinomas (p < 0.0001). In normal tissues distant from the primary tumour, there was a significantly greater amount of VEGF‐A and VEGF‐D mRNA in patients with Dukes B and Dukes C respectively, compared with Dukes A stage tumours (p = 0.04 and p = 0.01 respectively). Immunohistochemistry showed low basal levels of all ligands in histologically normal tissues and their expression in the epithelium of tumours reflected the levels of mRNA expression identified. VEGF‐A and VEGF‐C mRNA levels correlated significantly with tumour grade (p = 0.01 and p = 0.01 respectively) and tumour size (p = 0.001 and p = 0.01 respectively), but not with patient age, sex, presence of infiltrative margin, lymphocytic response, vascular invasion, Dukes stage, or lymph node involvement (p > 0.05). VEGF‐B mRNA correlated with an infiltrative margin (p = 0.04) but no other clinicopathological variable, and expression of VEGF‐D demonstrated no association with any parameter examined. VEGFR‐1 was significantly correlated with tumour grade (p = 0.02), Dukes stage (p < 0.001), and lymph node involvement (p = 0.004), VEGFR‐2 with lymph node involvement (p = 0.02), and VEGFR‐3 did not correlate with any of the clinicopathological variables tested. These results suggest that VEGF‐A and VEGF‐B play a role early in tumour development at the stage of adenoma formation and that VEGF‐C plays a role in advanced disease when there is more likelihood of metastatic spread. The finding of increased levels of VEGF‐A and VEGF‐D expression in normal tissues collected from a site distant from the primary tumour indicates changes in the surrounding tumour environment that may enhance the subsequent spread of tumour cells. Copyright

Expression of the angiopoietins and their receptor Tie2 in human renal clear cell carcinomas; regulation by the von Hippel-Lindau gene and hypoxia

Angiogenesis is essential for tumour growth and metastasis, and is co‐ordinated by several classes of angiogenic factors. To determine the significance and regulation of the angiopoietin (Ang) pathway in highly vascular human renal cell carcinomas (RCCs), this study has investigated the expression of the Ang‐1, Ang‐2, Ang‐4, and Tie2 genes in a series of normal (n = 26) and neoplastic (n = 45; clear cell n = 35, papillary n = 10) human kidney tissues, examined the pattern of Ang‐2 and Tie2 protein expression, and correlated expression with clinicopathological variables. The effect of the von Hippel‐Lindau (VHL) gene and hypoxia in the renal cell lines RCC786‐0 and RCC4 has also been investigated. Ang‐1, Ang‐2 and Tie2, but not Ang‐4 mRNA, were detected in normal and tumour samples. A significant increase in Ang‐2 (p < 0.001) and a decrease in Tie2 receptor mRNA (p = 0.001) were observed, but no significant difference was observed in Ang‐1 mRNA abundance between normal kidney and RCC (p = 0.37). Immunohistochemistry for Ang‐2 showed strong expression in vascular endothelium and weak expression in tumour cells, whereas Tie2 was expressed exclusively on endothelium. Tie2 gene expression was positively correlated with Ang‐2 expression in cancers (p = 0.001) and showed a borderline significant association with Ang‐1 (p = 0.06), but there was no significant relationship between Ang‐1 and Ang‐2 (p = 0.69). No significant relationships were observed in clear cell carcinomas between Ang‐1, Ang‐2 and Tie2 mRNA abundance and patient sex, patient age, or tumour size (p > 0.05). However, there was significantly greater Ang‐1 (p = 0.02), Ang‐2 (p = 0.03), and Tie2 (p = 0.04) mRNA abundance in clear cell than in chromophil RCCs. Ang‐2 gene expression was down‐regulated by hypoxia in VHL wild‐type RCC786‐0 and RCC4 transfectants (p = 0.0002 and p = 0.04, respectively), mirroring the low expression in human tumour cells. These data suggest that it is endothelial induction of Ang‐2 in tumours that regulates vessel stability and supports targeting Tie2 as an effective novel anti‐angiogenic therapy in clear cell RCCs. Copyright

Expression of vascular endothelial growth factor D is associated with hypoxia inducible factor (HIF-1α) and the HIF-1α target gene DEC1, but not lymph node metastasis in primary human breast carcinomas

Background: Vascular endothelial growth factor D (VEGF-D) induces angiogenesis and lymphangiogenesis. Nodal metastasis is recognised as a powerful prognostic marker in breast carcinoma, but the molecular mechanisms underlying this process are unknown. Although it has been suggested that VEGF-D may regulate nodal metastasis, this is based largely on animal models, its role in human disease being unclear. Aims: To measure the pattern and degree of VEGF-D protein expression in normal and neoplastic human breast tissues. Methods: The pattern and degree of VEGF-D expression was measured in normal tissue and invasive carcinomas, and expression was correlated with clinicopathological parameters, hypoxia markers, and survival. Because other VEGF family members are affected by oestrogen, whether VEGF-D is regulated by oestrogen in breast cancer cell lines was also assessed. Results: VEGF-D was significantly positively associated with hypoxia inducible factor (HIF-1α) (p = 0.03) and the HIF-1α regulated gene DEC1 (p = 0.001), but not lymph node status, the number of involved lymph nodes, patient age, tumour size, tumour grade, lymphovascular invasion, oestrogen receptor, progesterone receptor, c-erb-B2, or tumour histology (all p>0.05). There was no significant relation between tumour VEGF-D expression and relapse free (p = 0.78) or overall (p = 0.94) survival. VEGF-D expression was enhanced by oestrogen in MCF-7 and T47D breast cancer cells, and was blocked by hydroxytamoxifen. Conclusion: These findings support a role for hypoxia and oestrogen induced VEGF-D in human breast cancer and also suggest that tamoxifen and related oestrogen antagonists may exert some of their antitumour effects through the abrogation of VEGF-D induced function.

VEGF-B expression in human primary breast cancers is associated with lymph node metastasis but not angiogenesis.

Angiogenesis is essential for tumour growth and metastasis. It is regulated by numerous angiogenic factors, one of the most important being vascular endothelial growth factor (VEGF). Recently VEGF‐B, a new VEGF family member that binds to the tyrosine kinase receptor flt‐1, has been identified. Although the importance of VEGF has been shown in many human tumour types, the contribution of VEGF‐B to tumour neovascularization is unknown in any tumour type. This study therefore measured the mRNA level of VEGF‐B and its receptor flt‐1 by ribonuclease protection assay and the pattern of VEGF‐B expression by immunohistochemistry in 13 normal breast samples and 68 invasive breast cancers. Flt‐1 expression was significantly higher in tumours than in normal breast (p=0.02) but no significant difference was seen in VEGF‐B between normal and neoplastic breast (p=0.3). There was a significant association between VEGF‐B and node status (p=0.02) and the number of involved nodes (p=0.01), but not with age (p=0.7), size (p=0.6), oestrogen receptor (ER) (p=0.2), grade (p=0.5) or vascular invasion (p=0.16). No significant relationship was present between VEGF‐B and flt‐1 (p=0.2) or tumour vascularity (p=0.4). VEGF‐B was expressed mostly in the cytoplasm of tumour cells, although occasional stromal components including fibroblasts and endothelial cells were also positive. No difference in VEGF‐B expression was observed adjacent to regions of necrosis, in keeping with this VEGF family member not being hypoxically regulated. These findings suggest that VEGF‐B may contribute to tumour progression by a non‐angiogenic mechanism, possibly by increasing plasminogen activators and hence metastasis, as has been described in vitro. Measurement of VEGF‐B together with other angiogenic factors may identify a poor prognostic patient group, which may benefit from anti‐VEGF receptor therapy targeted to flt‐1 (VEGFR1) as well as kdr (VEGFR2). Copyright

Immunohistochemical analysis of cancer stem cell markers in invasive breast carcinoma and associated ductal carcinoma in situ: relationships with markers of tumor hypoxia and microvascularity ☆,☆☆

We performed immunohistochemical analysis of 3 cancer stem cell-related markers (CD44(+)/CD24(-/low), aldehyde dehydrogenase [ALDH]-1, CD133) in 94 invasive ductal carcinomas and assessed relationships with markers of hypoxia (carbonic anhydrase IX [CAIX]), tumor microvessel density (CD31), and clinicopathologic variables. Overall, 10% of tumors were CD44(+)/CD24(-/low), 13% were ALDH-1(+), 25% were CD133(+), 35% were immunonegative, and 1 tumor was immunopositive for all 3 markers. Associated ductal carcinoma in situ (DCIS) was present in 48% of tumors. Marker immunopositivity was detected in DCIS in 13% (CD44(+)/CD24(-/low)), 7% (ALDH-1(+)), and 32% (CD133(+)) of these tumors and was more likely present in DCIS when also detected in the invasive compartment (P = .03, P = .001, and P = .009, respectively). CD44(+)/CD24(-/low) cells were more common in progesterone receptor-negative tumors (P < .01), and ALDH-1(+) cells were more common in estrogen receptor-negative tumors (P < .01). CD133(+) cells were more common in patients younger than 50 years (P < .05) and in high grade (P < .01), localized (P < .05), and estrogen receptor-negative (P < .001), progesterone receptor-negative (P = .02), and triple-negative breast cancers (P < .001). CD44(+)/CD24(-/low) (P = .06) and CD133(+) (P = .02) tumor cells were more common in CAIX(+) versus CAIX(-) tumors, whereas ALDH-1(+) tumors had a higher mean microvessel density than did ALDH-1(-) tumors (P = .002). No significant relationships were observed between the markers studied and survival for 5 years. Our study demonstrated the presence of cancer stem cell marker-positive tumor cells in DCIS as well as invasive breast cancer and showed that CD44(+)/CD24(-/low) and CD133(+) cells were more frequently observed in hypoxic regions of tumor, whereas ALDH-1(+) cells more commonly colocalized to tumors with high microvessel density.

Anti-vascular agent Combretastatin A-4-P modulates Hypoxia Inducible Factor-1 and gene expression

BackgroundA functional vascular network is essential for the survival, growth and spread of solid tumours, making blood vessels a key target for therapeutic strategies. Combretastatin A-4 phosphate (CA-4-P) is a tubulin-depolymerising agent in Phase II clinical trials as a vascular disrupting agent. Not much is known of the molecular effect of CA-4-P under tumour conditions. The tumour microenvironment differs markedly from that in normal tissue, specifically with respect to oxygenation (hypoxia). Gene regulation under tumour conditions is governed by hypoxia inducible factor 1 (HIF-1), controlling angiogenic and metastatic pathways.MethodsWe investigated the effect of CA-4-P on factors of the upstream and downstream signalling pathway of HIF-1 in vitro.ResultsCA-4-P treatment under hypoxia tended to reduce HIF-1 accumulation in a concentration-dependent manner, an effect which was more prominent in endothelial cells than in cancer cell lines. Conversely, CA-4-P increased HIF-1 accumulation under aerobic conditions in vitro. At these concentrations of CA-4-P under aerobic conditions, nuclear factor κB was activated via the small GTPase RhoA, and expression of the HIF-1 downstream angiogenic effector gene, vascular endothelial growth factor (VEGF-A), was increased.ConclusionOur findings advance the understanding of signal transduction pathways involved in the actions of the anti-vascular agent CA-4-P.

Quantification of in vitro and in vivo angiogenesis stimulated by ovine forestomach matrix biomaterial.

Ovine forestomach matrix (OFM) biomaterial acts as a biomimetic of native extracellular matrix (ECM) by providing structural and functional cues to orchestrate cell activity during tissue regeneration. The ordered collagen matrix of the biomaterial is supplemented with secondary ECM-associated macromolecules that function in cell adhesion, migration and communication. As angiogenesis and vasculogenesis are critical processes during tissue regeneration we sought to quantify the angiogenic properties of the OFM biomaterial. In vitro studies demonstrated that soluble OFM components stimulated human umbilical vein endothelial cell (HUVEC) migration and increased vascular sprouting from an aorta. Blood vessel density and branch points increased in response to OFM in an ex ovo chicken chorioallantoic membrane (CAM) assay. The OFM biomaterial was shown to undergo remodeling in a porcine full-thickness excisional model and gave rise to significantly more blood vessels than wounds treated with small intestinal submucosa decellularized ECM or untreated wounds.

The alternative lengthening of telomeres pathway may operate in non-neoplastic human cells†

The alternative lengthening of telomeres (ALT) mechanism represents an alternative to the enzyme telomerase in the maintenance of mammalian telomeres in 25–60% of sarcomas and a minority of carcinomas (about 5–15%). ALT‐positive cells are distinguished by long and heterogeneous telomere length distributions by terminal restriction fragment (TRF) Southern blotting. Another diagnostic marker of ALT is discrete nuclear co‐localized signals of telomeric DNA and the promyelocytic leukaemia protein (PML), referred to as ALT‐associated PML bodies (APBs). Recently, we detected smaller sized co‐localized PML and telomere DNA (APB‐like) bodies in endothelial cells adjacent to astrocytoma tumour cells in situ. In this study, we examined a wide variety of non‐neoplastic tissues, and report that co‐localized signals of PML and telomere DNA are present in endothelial, stromal, and some epithelial cells. Co‐localized signals of PML and telomere DNA showed an increased frequency in non‐neoplastic cells with DNA damage. These results suggest that a mechanism similar to that in ALT‐positive tumours also operates in non‐neoplastic cells, which may be activated by DNA damage. Copyright

Professional killer cell deficiencies and decreased survival in pulmonary arterial hypertension

Increasing evidence implicates lymphocytes in pulmonary arterial hypertension (PAH) pathogenesis. Rats deficient in T‐lymphocytes show increased propensity to develop PAH but when injected with endothelial progenitor cells are protected from PAH (a mechanism dependent on natural killer (NK) cells). A decreased quantity of circulating cytotoxic CD8+ T‐lymphocytes and NK cells are now reported in PAH patients; however, the effect of lymphocyte depletion on disease outcome is unknown.

The G-Protein–Coupled Receptor CLR Is Upregulated in an Autocrine Loop with Adrenomedullin in Clear Cell Renal Cell Carcinoma and Associated with Poor Prognosis

Purpose: The G-protein–coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) and its ligand peptide adrenomedullin (encoded by ADM gene) are implicated in tumor angiogenesis in mouse models but poorly defined in human cancers. We therefore investigated the diagnostic/prognostic use for CLR in human tumor types that may rely on adrenomedullin signaling and in clear cell renal cell carcinoma (RCC), a highly vascular tumor, in particular. Experimental Design: In silico gene expression mRNA profiling microarray study (n = 168 tumors) and cancer profiling cDNA array hybridization (n = 241 pairs of patient-matched tumor/normal tissue samples) were carried out to analyze ADM mRNA expression in 13 tumor types. Immunohistochemistry on tissue microarrays containing patient-matched renal tumor/normal tissues (n = 87 pairs) was conducted to study CLR expression and its association with clinicopathologic parameters and disease outcome. Results: ADM expression was significantly upregulated only in RCC and endometrial adenocarcinoma compared with normal tissue counterparts (P < 0.01). CLR was localized in tumor cells and vessels in RCC and upregulated as compared with patient-matched normal control kidney (P < 0.001). Higher CLR expression was found in advanced stages (P < 0.05), correlated with high tumor grade (P < 0.01) and conferred shorter overall survival (P < 0.01). Conclusions: In human tissues ADM expression is upregulated in cancer type–specific manner, implicating potential role for adrenomedullin signaling in particular in RCC, where CLR localization suggests autocrine/paracrine mode for adrenomedullin action within the tumor microenvironment. Our findings reveal previously unrecognized CLR upregulation in an autocrine loop with adrenomedullin in RCC with potential application for this GPCR as a target for future functional studies and drug development. Clin Cancer Res; 19(20); 5740–8. ©2013 AACR.