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

Abstract

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-1alpha) (p = 0.03) and the HIF-1alpha 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.

Figure 1

(A) Vascular endothelial growth factor D (VEGF-D) staining in luminal ductal cells with occasional staining in myoepithelial cells of normal breast. (B) Expression in areas of cystic disease, with enhanced VEGF-D expression in duct epithelial cells (inset: nerve and stromal cells that are also immunopositive). (C) Heterogeneous staining of VEGF-D in malignant epithelium in a poorly differentiated tumour and (D) a well differentiated tumour with granular staining at the apical pole of glands. (E) Enhanced VEGF-D immunostaining at the periphery of a tumour (arrows) without upregulation in an adjacent area of necrosis (asterisk). (F) Strong staining for VEGF-D in a lymphatic tumour embolus.

Figure 2

Representative reverse transcriptase (RT) polymerase chain reaction showing ethidium bromide stained vascular endothelial growth factor D (VEGF-D) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression in a panel of oestrogen receptor positive (MCF-7 and T47D) and negative (BT20, MDA-MD-231, MDA-MD-435, MDA-MD-453, MDA-MD-468, and SKBR3) breast carcinoma cell lines with human umbilical vein endothelial cell RT+ and RT− controls (2 μg total RNA used for each sample).

Figure 3

Vascular endothelial growth factor D (VEGF-D) mRNA expression in MCF-7 and T47D breast carcinoma cell lines incubated in hormone free RPMI 1640 medium or hormone free RPMI 1640 medium supplemented with 17β estradiol (E; 10⁻⁹M) for two and 18 hours. Gene expression was measured by relative reverse transcriptase polymerase chain reaction, standardised to 18S, and expressed as mean (SEM).

Figure 4

Vascular endothelial growth factor D (VEGF-D) in T47D breast carcinoma cell lines incubated in hormone free RPMI 1640 medium or hormone free RPMI 1640 medium supplemented with 17β estradiol (E; 10⁻⁹M) and/or 4-hydroxytamoxifen (HT; 10⁻⁶M) for 18 hours. Gene expression was measured by relative reverse transcriptase polymerase chain reaction, standardised to 18S, and expressed as mean (SEM).