NHERF1 PROTEIN EXPRESSION IN BREAST CANCER A Mangia*, I Suriano*, A Malfettone*, A Bellizzi*, O Popescu**, R Daprile**, B Stea*, G Simone** and A Paradiso* *Clinical Experimental Oncology Laboratory, National Cancer Institute-Bari ** Histopathology Unit National Cancer Institute-Bari

BACKGROUND  NHERF1 (Na + /H + exchanger regulator factor) is a PDZ domain containing protein that recruits membrane receptors/transporters and cytoplasmic signalling proteins into functional complexes. NHERF1 expression is altered in breast cancer but its effective role in mammary carcinogenesis remains undefined.  We further clarify the role of NHERF1 breast progression exploring the tissue distribution and cellular localization of NHERF1 protein in human normal tissue (NT), atypical ductal hyperplasia (ADH), carcinoma in situ (CIS), invasive carcinoma (IBC), synchronous metastatic lymph node and distant metastasis of a series of breast cancer.

MATERIALS AND METHODS According to pathological classification, the study was performed on: a) 35 ADH; b) 14 CIS and the surrounding non-cancerous tissues; c) 22 IBC from node negative patients (N0M0) and the surrounding non-cancerous tissues; d) 19 IBC, the surrounding non-cancerous tissues and synchronous metastatic lymph nodes from node positive patients (N1M0); e) 10 IBC, the surrounding non-cancerous tissues, synchronous metastatic lymph nodes and distant metastasis from patients with metastatic disease (N1M1).

Immunohistochemistry was performed on breast tissue specimens by utilizing standard procedure for sampling, fixation, paraffin inclusion and slides preparation. Histological sections of 4  m were incubated with rabbit polyclonal antibody EBP50, 1:150 dilution (PA1-090 Affinity Bioreagents, Golden, CO) overnight at 4°C. The bound antibody was visualized using a biotinylated secondary antibody, peroxidase-labelled streptavidin, and AEC substrate- chromogen (LSAB2 System-HRP;DakoCytomation). The slides were counterstained with H&E and mounted with permanent mounting media. Paraffin-embedded cells pellets from MCF-7 cell lines, expressing high levels of NHERF1, were used as positive controls. For negative controls, the primary antibody was omitted and replaced by PBS. Immunohistochemistry  Immunohistochemistry

Immunohistofluorescence studies were performed on 24 formalin-fixed tumor tissue sections selected among those with overexpressed HER2/neu (moAb, clone CB11 - Novocastra Laboratories Ltd, UK), scored 2+ and 3+. For antigen retrieval pretreatment, serial sections of 3  m were immersed in a 10 mM citrated buffer, pH 6.0, at 95°C, 40 min. Then, slides were washed in dH 2 O, treated 10 min with 0.2% BSA in PBS to block non specific protein binding, and incubated with NHERF1, 1:150 dilution (PA1-090 Affinity Bioreagents, Golden, CO) and HER2/neu antibody 1:40 dilution, overnight at 4°C in a humidified chamber. For negative controls, slide sections that were positive for staining were treated with 0.2% BSA instead of the primary antibody. Images were obtained on a BX40 microscope (Olympus) with a SenSys 1401E-Photometrics CCD camera. Immunohistofluorescence  Immunohistofluorescence

A NHERF1 immunopositivity was present mostly as apical membranous immunoreactivity, especially at the luminal aspects of morphologically normal cells (A) or as both cytoplasmic and membranous localizaton in ADH and CIS (B,C). RESULTS NHERF1 localization and expression in different compartment of breast tissue RESULTS NHERF1 localization and expression in different compartment of breast tissue C B

D E F NHERF1 positivity was present mostly as cytoplasmic staining in IBC (D) and methachronus distant metastases (E). In synchronous metastatic lymph node tissues (F) NHERF1 immunoreactivity was present exclusively as cytoplasmic localization. NHERF1 localization and expression in different compartments of breast tissue

Cellular distribution of NHERF1 in breast tissues  Significantly higher cytoplasmic-levels of the protein resulted in both synchronous metastatic lymph nodes and distant metastases with respect to the corresponding normal tissues (p= , p= respectively) and CIS (p=0.002, p=0.03 respectively).  In NT, ADH and CIS tissues, NHERF1 was present mainly in apical membrane with expression levels significantly higher than the cytoplasmic amount.  Notably, the cytoplasmic amount of NHERF1 detected in IBC tissues was significantly increased with respect to the corresponding cytoplasmic levels of the protein observed in NT and CIS tissues ( p=0.0001, p=0.007 respectively ).

Cytoplasmic accumulations of NHERF1 in paired samples of NT and CIS tissue Cytoplasmic accumulations of NHERF1 in paired samples of NT and IBC tissue The cytoplasmic NHERF1 expression levels in paired CIS and NT from 12 patients (fig A) and in paired IBC and NT from 44 patients (fig B), showed a heterogeneous behaviour with some cases increasing but other lowering NHERF1 expression levels in the different sites. AB

NHERF1 characteristics in tumour progression No significant difference in mean-levels of NHERF1 cytoplasmic expression was found when both synchronous metastatic lymph node either distant metastases were compared with the IBC tissues.

Intense NHERF1 staining was found at the apical surface of morphologically normal epithelial cells, while HER2/neu was found as basolateral and in intraepithelial junctions (A). This localization is present also in CIS (B). : Green signal : HER2/neu : Red signal: NHERF1 A B NHERF1 and HER2/neu co-localization

In IBC (A) and in synchronous metastatic lymph node (B), NHERF1 lost its exclusively apical localization in the tumor epithelial cells, becoming mostly cytosolic in no longer polarized cells of the tumor nests. A B : Green signal : HER2/neu : Red signal: NHERF1

In tumor cells from metachronous distant metastases NHERF1, still localized in the sub-membrane region, was found to be co- localized with HER2/neu, suggesting an important role of these two proteins in driving invasion and metastasis to distant tissue. In these metastatic samples large areas of NHERF1 nuclear localization were present, suggesting a role of NHERF1 also in trascription pathways necessary for this process. : Green signal : HER2/neu : Red signal: NHERF1

CONCLUSIONS  We suppose that breast cancerogenesis is characterized by an increased cytoplasmic expression of NHERF1 as the tumor progresses suggesting a role in this process. The switch from apical membrane to cytoplasmic expression is compatible with a dual role of NHERF1 as a tumor suppressor or tumor promoter dependent on its subcellular localization.  In invasive tumor and distant metastasis, the sub-membrane NHERF1, still present, is able to interact with HER2/neu, probably regulating its trafficking.  Their interaction could be of some relevance in the metastatization processes.

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