High expression of focal adhesion kinase (p125FAK) in node-negative breast cancer is related to overexpression of HER-2/neu and activated Akt kinase but does not predict outcome

Abstract

Introduction: Focal adhesion kinase (FAK) regulates multiple cellular processes including growth, differentiation, adhesion, motility and apoptosis. In breast carcinoma, FAK overexpression has been linked to cancer progression but the prognostic relevance remains unknown. In particular, with regard to lymph node-negative breast cancer it is important to identify high-risk patients who would benefit from further adjuvant therapy.

Methods: We analyzed 162 node-negative breast cancer cases to determine the prognostic relevance of FAK expression, and we investigated the relationship of FAK with major associated signaling pathways (HER2, Src, Akt and extracellular regulated kinases) by immunohistochemistry and western blot analysis.

Results: Elevated FAK expression did not predict patient outcome, in contrast to tumor grading (P = 0.005), Akt activation (P = 0.0383) and estrogen receptor status (P = 0.0033). Significant positive correlations were observed between elevated FAK expression and HER2 overexpression (P = 0.001), as well as phospho-Src Tyr-215 (P = 0.021) and phospho-Akt (P < 0.001), but not with phospho-ERK1/2 (P = 0.108). Western blot analysis showed a significant correlation of FAK Tyr-861 activation and HER2 overexpression (P = 0.01).

Conclusions: Immunohistochemical detection of FAK expression is of no prognostic significance in node-negative breast cancer but provides evidence that HER2 is involved in tumor malignancy and metastatic ability of breast cancer through a novel signaling pathway participating FAK and Src.

Figure 1

Immunohistochemistry for focal adhesion kinase (FAK) in ductal carcinoma in situ, invasive breast carcinonoma and normal breast tissue. (a) Strongest FAK expression was frequently seen in ductal carcinoma in situ (arrowhead). Notice the missing staining in normal breast tissue (arrow). (b) Invasive breast carcinoma cases (arrowhead) exhibited an equal or less intense FAK immunostaining then adjacent ductal carcinoma in situ (arrow) in all but one case. Original magnification, × 400.

Figure 2

Representative immunohistochemical staining of a Her-2/neu and focal adhesion kinase (FAK)-overexpressing tumor for phospho-Src Tyr-215 and phospho-Src Tyr-416. Light micrograph displaying (a) strong phospho-Src Tyr-215 and (b) missing phospho-Src Tyr-416 staining in serial sections of a Her-2/neu-overexpressing and FAK-overexpressing invasive breast cancer sample as analyzed by immunohistochemistry. Inset: immunohistochemical staining showing FAK overexpression of the same sample. Original magnification, × 400.

Figure 3

Representative immunohistochemical staining of serial sections of three tumors for phospho-Akt, focal adhesion kinase (FAK) and HER2. (A1)–(A3) HER2: tumors with strong HER2 staining intensity (DAKO score 3+) (A1), weak HER2 staining (DAKO score 1+) (A2) and missing HER2 staining (DAKO score 0) (A3). (B1)–(B3) Tumors showing strong HER2 immunostaining (A1) more frequently exhibited strong FAK immunostaining (B1), whereas tumors expressing weak or no HER2 immunostaining were more often associated with weak or no FAK immunostaining (B2, B3). (C1)–(C3) Phospho-Akt: the percentage of phospho-Akt positively stained tumor cells ranged from more than 80% (C1) to 45% (C2) or was lower than 45% (C3). Higher HER2 (A1) and FAK (B1) staining intensites are correlated with higher percentage of phospho-Akt positively stained tumor cells (C1, C2). Original magnification, × 200.

Figure 4

Upregulation of tyrosine phosphorylation of focal adhesion kinase (FAK) at tyrosine 861 in HER2-overexpressing breast tumors. Western blot analysis: lysates of seven breast cancer samples were analyzed by immunoblotting using antibodies that recognize total FAK and distinct tyrosine sites on Src and FAK kinases. Tumor samples were put in order according to the HER2 expression status (HerceptTest™) and the blot was probed with anti-HER2 antibody to analyze the exact amount of HER2 protein.

Figure 5

Scheme depicting signalling transduction in breast cancer involving HER2, Src, focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3K), Akt and Erk.