Margaret J. Currie

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

Low Ascorbate Levels Are Associated with Increased Hypoxia-Inducible Factor-1 Activity and an Aggressive Tumor Phenotype in Endometrial Cancer

Activation of the transcription factor hypoxia-inducible factor (HIF)-1 allows solid tumors to thrive under conditions of metabolic stress. Because HIF-1 is switched off by hydroxylation reactions that require ascorbate, inadequate intracellular ascorbate levels could contribute to HIF-1 overactivation. In this study, we investigated whether the ascorbate content of human endometrial tumors [known to be driven by HIF-1 and vascular endothelial growth factor (VEGF)] influenced HIF-1 activity and tumor pathology. We measured protein levels of HIF-1alpha and three downstream gene products [glucose transporter 1 (GLUT-1), Bcl-2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3), and VEGF], as well as the ascorbate content of tumor and patient-matched normal endometrial tissue samples. HIF-1alpha and its downstream gene products were upregulated in tumor tissue, with the highest levels being present in high-grade tumors. High-grade tumors also had reduced capacity to accumulate ascorbate compared with normal tissue; however, all grades contained tumors with low ascorbate content. Tumors with the highest HIF-1alpha protein content were ascorbate deficient. Low ascorbate levels were also associated with elevated VEGF, GLUT-1, and BNIP3 protein levels and with increased tumor size, and there was a significant association between low tissue ascorbate levels and increased activation of the HIF-1 pathway (P = 0.007). In contrast, tumors with high ascorbate levels had lesser levels of HIF-1 activation. This study shows for the first time a likely in vivo relationship between ascorbate and HIF-1, with low tumor tissue ascorbate levels being associated with high HIF-1 activation and tumor growth.

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

Cancer disparities in indigenous Polynesian populations: Māori, Native Hawaiians, and Pacific people

Polynesia consists of several islands that are scattered across a vast triangle in the Pacific, and include New Zealand, Hawaii, and the Pacific islands. There are reported differences in the types of cancer and epidemiologies seen among communities in these islands, the reasons for which are diverse and complex. In this Review, we describe patterns of cancer incidence, mortality, and survival in indigenous populations compared with populations of European origin in Polynesia, and highlight the limited available data for Pacific populations. Additionally, we document the current knowledge of the underlying biology of cancers in these populations, and report risk factors that differ between ethnicities, including smoking, viral infections, and obesity. Disparities in measures of health are highlighted, as are evident differences in knowledge of tumour biology and cancer management between majority and minority populations.

Association of angiopoietin-2, C-reactive protein and markers of obesity and insulin resistance with survival outcome in colorectal cancer.

Background:This study investigated the relationship of obesity, insulin resistance, inflammation and angiogenesis with cancer progression and survival in a colorectal cancer cohort.Methods:Clinical and pathological data, along with anthropometric and follow-up data, were collected from 344 consecutive colorectal cancer patients. Serum samples at diagnosis were analysed by immunoassay for adiponectin, C-reactive protein (CRP), vascular endothelial growth factor-A (VEGF-A), angiopoietin-2 (Ang-2), insulin-like growth factor-1 (IGF-1), insulin and C-peptide.Results:Serum Ang-2 and VEGF-A levels increased with tumour T stage (P=0.007 and P=0.025, respectively) and N stage (P=0.02 and P=0.03, respectively), and correlated with CRP levels (r=0.43, P<0.001 and r=0.23, P<0.001, respectively). Angiopoietin-2 correlated with C-peptide (r=0.14, P=0.007) and VEGF-A with IGF-1 in males (r=0.25, P=0.001). Kaplan–Meier analysis showed that patients with high serum levels of CRP and Ang-2 had significantly reduced survival (both P⩽0.001). After adjusting for tumour stage and age, Ang-2 remained a significant predictor of survival. The CRP levels were inversely associated with survival in American Joint Committee on Cancer stage II patients (P=0.038), suggesting that CRP could be used to support treatment decisions in this subgroup. Serum markers and anthropometric measures of obesity correlated with each other, but not with survival.Conclusion:Our study supports the concept that obesity-related inflammation, rather than obesity itself, is associated with colorectal cancer progression and survival. The study confirms serum Ang-2 as a predictive marker for outcome of colorectal cancer.

First and subsequent nonmelanoma skin cancers: incidence and predictors in a population of New Zealand renal transplant recipients

BACKGROUND Renal transplant recipients (RTRs) have an increased risk of developing nonmelanoma skin cancers (NMSCs). The aims of this study were to determine the incidence and subsequent history of NMSCs in RTRs, together with risk factors. METHODS All patients transplanted between July 1972 and March 2007, and followed up at Christchurch Hospital, New Zealand, were studied. Immunosuppression regimens were mostly prednisone, azathioprine, cyclosporine and prednisone, mycophenolate mofetil, cyclosporine since 1998. RESULTS Of 384 RTRs, 96 developed at least one NMSC. The median time to first NMSC was 18.3 years (95% CI 14.2, 22.9) from transplant, as estimated by survival analysis. Individual predictors of first NMSC in RTRs were older age at first transplant (P < 0.0001), male sex (P = 0.006) and initial immunosuppression regimen (P = 0.001); only age (P < 0.0001) and male gender (P = 0.003) were significant predictors in a joint model. The mean rate of subsequent NMSCs was 1.67 per year (95% CI = 1.32, 2.11). Older age at first renal transplant (P = 0.009) or at discovery of the first NMSC (P = 0.01) was associated with a higher annual rate of new NMSC following the discovery of the first NMSC. The median survival time to a second NMSC was 2.2 years (CI 1.4, 3.0). Fourteen patients died of metastatic squamous cell carcinoma (15% case fatality). CONCLUSIONS NMSCs are a major health issue for RTRs, especially in older males. Once RTRs have developed their first NMSC, ongoing surveillance and prompt treatment are essential.

Intracellular ascorbate enhances hypoxia-inducible factor (HIF)-hydroxylase activity and preferentially suppresses the HIF-1 transcriptional response

Hypoxia-inducible factor (HIF)-1 drives the transcription of hundreds of genes to support cell survival under conditions of microenvironmental and metabolic stress. HIF-1 is downregulated by iron-containing 2-oxoglutarate-dependent enzymes that require ascorbate as a cofactor. The HIF hydroxylases control both protein stability and the formation of an active transcription complex and, consequently, ascorbate could affect HIF-1α stabilization and/or gene expression, but the relative effect of ascorbate on these separate processes has not been well characterized. In this study we examined the effects of known intracellular ascorbate concentrations on both processes in response to various means of hydroxylase inhibition, including CoCl2, NiCl2, desferrioxamine, dimethyloxalylglycine, and hypoxia. Ascorbate inhibited HIF-1 activity most dramatically with all mechanisms of iron competition. In addition, HIF-1-dependent gene expression was effectively prevented by ascorbate and was inhibited even under conditions that allowed HIF-1α protein stabilization. This suggests that (1) ascorbate acts primarily to stabilize and reduce the iron atom in the hydroxylase active site and (2) the asparagine hydroxylase controlling HIF-1 transcriptional activity is particularly susceptible to fluctuations in intracellular ascorbate. These findings suggest that ascorbate plays a significant role in supporting HIF-hydroxylase function and that it could thereby modulate the cell survival response.

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.