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

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.

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

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

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

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 < Tumour size T1 T2-4 Tis Lymph node status Negative Positive b Nx Histological grade G1-G2 G3 Not recorded PgR ≤10% positive cells >10% positive cells c ER ≤10% positive cells >10% positive cells MIB1 ≤20% positive cells >20% positive cells a) Six of 94 familial patients were bilateral tumours b) Nx, no axillary lymph node dissection c) evaluation on whole section Legend :

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%) / Cells Positive (median >0%) 30/ VEGFR1 Cells Negative (median 0%) 33/// Cells Positive (median >0%) // * by χ2 Familial breast cancer patients

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)

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%) // VEGFR1 Cells Negative (median 0%) // / / Cells Positive (median >0%) /// / HER2/neu Negative (0; 1+) 28 / / Positive (3+) // Sporadic breast cancer patients * by χ2

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

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.