1. 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 FIRENZE 7- 9 Settembre 2009

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 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). AIM of the study

4. MATERIALS AND METHODS 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. Tissue specimens

5. 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. Immunohistochemistry

6. Clinicopathological characteristics of 94 familial and 93 sporadic breast cancer patients based on the family history of disease a Familial cases (n=94) Sporadic cases (n=93) p value Age ≤ 45 > 45 < 0.001 49 15 45 78 Tumour size T1 T2-4 Tis 0.960 38 37 59 53 3 3 Lymph node status Negative Positive b Nx 0.165 35 39 57 45 8 9 Histological grade G1-G2 G3 Not recorded 0.128 55 56 41 28 4 9 PgR ≤10% positive cells >10% positive cells 0.214 40 43 60 49 c ER ≤10% positive cells >10% positive cells 0.025 35 19 64 70 MIB1 ≤20% positive cells >20% positive cells 0.011 35 47 65 45 a) Six of 94 familial patients were bilateral tumours b) Nx, no axillary lymph node dissection c) evaluation on whole section Legend :

7. 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). Markers of Angiogenesis cytoplasmic NHERF1 (median value = 40%) nuclear NHERF1 (median value = 0%) %p value*% HIF-1α Cells Negative (median 0%) 430.0021/ Cells Positive (median >0%) 30/70.003 VEGFR1 Cells Negative (median 0%) 33/// Cells Positive (median >0%) 440.009// * by χ2 Familial breast cancer patients

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

9. 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 NHERF1 levels (p= 0.007). Markers of tumour progression cytoplasmic NHERF1 (median value = 35%) nuclear NHERF1 (median value = 0%) % p value* % HIF-1α Cells Negative (median <8.50%) //3 Cells Positive (median >8.50%) //120.019 VEGFR1 Cells Negative (median 0%) // / / Cells Positive (median >0%) /// / HER2/neu Negative (0; 1+) 28 / / Positive (3+) 410.007 // Sporadic breast cancer patients * by χ2

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

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.