Na + /H + exchanger regulatory factor 1 (NHERF1) and angiogenesis in familial breast cancer

1. FIRENZE 7- 9 Settembre 2009 Na+/H+ exchanger regulatory factor 1 (NHERF1) and angiogenesis in familial breast cancer A Mangia*, A Malfettone*, C Salvatore**, B Stea*, G Simone** and A Paradiso* *Clinical Experimental Oncology Laboratory, National Cancer Institute-Bari ** Histopathology Unit National Cancer Institute-Bari

2. BACKGROUND • Na+/H+ exchanger regulatory factor 1 (NHERF1) is a scaffolding protein that recruits membrane and cytoplasmic proteins into functional complexes. Our recent evidences demonstrated that in breast cancer NHERF1 overexpression is associated with increased tumor hypoxia and poor prognosis. • Hypoxia is implicated in tumour proliferation and angiogenesis that interests neoplastic regions. In fact, the hypoxia-inducible factor-1 (HIF-1), mediating transcriptional activation of vascular endothelial growth factor (VEGF) gene, is considered the central initiator of angiogenesis activity in tumours.

3. AIM of the study Aim of this study is to determine NHERF1 expression on a series of familial and sporadic breast cancer patients and to examine the relationship with other tumour progression markers (HIF-1, VEGFR 1, HER2/neu).

4. MATERIALS AND METHODS Tissue specimens NHERF1, VEGFR1, HIF1α proteins expression were analysed by immunohistochemistry on a tissue microarray (TMA), including 94 familial and 93 sporadic breast tumors. Cytoplasmic, membrane and nuclear NHERF1 reactivity was analysed.

5. Immunohistochemistry Immunohistochemistry was performed on breast tumours distributed across the 4 TMAs utilizing standard procedure for sampling, fixation and paraffin embedded. Histological sections (4m) were incubated with anti-NHERF1 (1:150, EBP50 PA1-090), HIF-1 (1:100, H-206), VEGFR1 (1:100, Flt-1 C-17) and HER2/neu (1:100, NCL-L CB11) overnight at 4°C. The bound antibody was visualized using a biotinylated secondary antibody, peroxidase-labelled streptavidin, and AEC substrate-chromogen (LSAB2 System-HRP;Dako). The slides were counterstained with H&E. For negative controls, the primary antibody was omitted and replaced by PBS.

6. Legend: a) Six of 94 familial patients were bilateral tumours b) Nx, no axillary lymph node dissection c) evaluation on whole section Clinicopathological characteristics of 94 familial and 93 sporadic breast cancer patients based on the family history of disease

7. Familial breast cancer patients * byχ2 In familial patients, high levels of nuclear NHERF1 were associated with positive HIF-1α tumours (p=0.003), while cytoplasmic NHERF1 expression was associated with negative HIF-1α (p=0.002). Moreover, cytoplasmic NHERF1 overexpression was associated with VEGFR1 positivity (p=0.009).

8. Immunohistochemical study on TMAs of familial tumours A B Cytoplasmic and nuclear NHERF1 protein expression Nuclear HIF-1a expression Cytoplasmic VEGFR1 expression (A= X100 magnification; B= X200)

9. Sporadic breast cancer patients * byχ2 In sporadic patients, nuclear NHERF1 protein is significantly correlated with positive HIF-1α expression (p=0.019), but any significant association between cytoplasmic NHERF1 and both HIF-1α and VEGFR1 was found. This is in agreement with a pivotal role of NHERF1 in more aggressive tumours such as familiar breast cancers. Interestingly, the tumours overexpressing HER2/neu showed a significant association with high cytoplasmic NHERF1levels (p= 0.007).

10. NHERF1 and HER2/neu expressionin sporadic tumours A B Membranous NHERF1 protein expression Cytoplasmic and nuclear NHERF1 protein expression HER2/neuexpression (A= X100 magnification; B= X200)

11. CONCLUSIONS • In familial breast cancers with respect to sporadic tumours, NHERF1 protein resulted strongly related with HIF-1α expression and more importantly with VEGFR1 reactivity. • In this context, we suggest an emerging role of NHERF1 in tumour angiogenesis.