Increased Expression and Activation of FAK in Small-Cell Lung Cancer Compared to Non-Small-Cell Lung Cancer

Abstract

Introduction: Focal adhesion kinase (FAK) plays a crucial role in cancer development and progression. FAK is overexpressed and/or activated and associated with poor prognosis in various malignancies. However, in lung cancer, activated FAK expression and its prognostic value are unknown.

Methods: FAK and activated FAK (phospho-FAK Y397) expressions were analyzed by multiplex immunofluorescence staining in formalin-fixed paraffin-embedded tissues from 95 non-small-cell lung cancer (NSCLC) and 105 small-cell lung cancer (SCLC) patients, and 37 healthy donors. The FAK staining score was defined as the percentage (%) of FAK-stained tumor area multiplied by (×) FAK mean intensity and phospho-FAK staining score as the (% of phospho-FAK-stained area of low intensity × 1) + (% of phospho-FAK-stained area of medium intensity × 2) + (% of the phospho-FAK-stained area of high intensity × 3). FAK and phospho-FAK staining scores were compared between normal, NSCLC, and SCLC tissues. They were also tested for correlations with patient characteristics and clinical outcomes.

Results: The median follow-up time after the first treatment was 42.5 months and 6.4 months for NSCLC and SCLC patients, respectively. FAK and phospho-FAK staining scores were significantly higher in lung cancer than in normal lung and significantly higher in SCLC compared to NSCLC tissues (p < 0.01). Moreover, the ratio between phospho-FAK and FAK staining scores was significantly higher in SCLC than in NSCLC tissues (p < 0.01). However, FAK and activated FAK expression in lung cancer did not correlate with recurrence-free and overall survival in NSCLC and SCLC patients.

Conclusions: Total FAK and activated FAK expressions are significantly higher in lung cancer than in normal lung, and significantly higher in SCLC compared to NSCLC, but are not prognostic biomarkers in this study.

Keywords: FAK; expression; lung cancer; multiplex immunofluorescence staining; non-small-cell lung cancer; phospho-FAK; prognosis; small-cell lung cancer; targeted therapy.

Figure 1

Illustration of focal adhesion kinase (FAK) and phospho-FAK staining quantification on a tissue microarray section of non-small-cell lung cancer (NSCLC) stained by multiplex immunofluorescence (IF) immunohistochemistry (IHC). (A) Tissue microarray (TMA) sections were sequentially stained by mIF with an antibody against phospho-FAK (red signal) and FAK (orange signal), followed by the Hoechst nuclear marker (blue signal). After whole slide fluorescence image acquisitions, IHC was performed with a tumor marker using an antibody against pan-cytokeratin CKAE1-AE3 (CK, brown signal) on the same slide and digitalized with a slide scanner. (B) Each TMA plug was then automatically delineated via the image analysis tool Oncotopix version 2017.2 (Visiopharm). (C) CK-positive tumor regions were semi-automatically delineated from CK-negative stroma. (D) These tumor regions, detected on the brightfield scan, were transposed to the aligned fluorescent scan with the Visiopharm Tissue Align module. (E) FAK and phospho-FAK stained areas were finally detected and quantified as illustrated for phospho-FAK in Figure D.2., with staining detection according to three thresholds of intensity (low, yellow; medium, orange; high, red), while Figure D.1. shows phospho-FAK staining without the mask. Original magnification: A, B, C: 1×; D: 2×; E: 20×.

Figure 2

Illustrations of FAK and phospho-FAK (Y397) expression evaluated by multiplex immunofluorescence (IF) immunohistochemistry (IHC) in lung cancer and normal lung tissues. (A) Lung adenocarcinoma with the absence of phospho-FAK expression but homogenous cytoplasmic FAK staining (orange) in the tumor core, adjacent non-tumoral bronchi, and some stromal cells (including vessels and lymphoid structures). (B) Lung adenocarcinoma with nuclear phospho-FAK staining (red) and homogenous cytoplasmic FAK staining (orange). (C) Lung squamous carcinoma with the absence of phospho-FAK expression but weak cytoplasmic FAK staining. (D) Lung squamous carcinoma with nuclear phospho-FAK staining (red) and homogenous cytoplasmic FAK staining (orange). (E) Small-cell lung cancer with nuclear phospho-FAK staining (red) and cytoplasmic FAK staining (orange). (F) Normal lung with cytoplasmic FAK staining in bronchi and some stromal cells (including vessels and lymphoid structures). (G) Lung squamous carcinoma used as a negative control, showing the absence of phospho-FAK and FAK staining. Original magnification: 20×; scale bar: 50 µm.

Figure 3

Quantification of FAK and phospho-FAK (Y397) expression evaluated by multiplex immunofluorescence immunohistochemistry in 37 normal lungs, 95 non-small-cell lung cancer (NSCLC), and 105 small-cell lung cancer (SCLC) tissues: (A) FAK staining score: Percentage (%) of FAK-stained tumor area multiplied by (x) FAK mean intensity, (B) phospho-FAK (Y397) staining score: (% of phospho-FAK-stained tumor area of low intensity × 1) + (% of phospho-FAK-stained tumor area of medium intensity × 2) + (% of phospho-FAK-stained tumor area of high intensity × 3), and (C) ratio between phospho-FAK and FAK staining scores. Each dot represents one sample. Data presented as the mean ± S.D. p-values were obtained using linear models and adjusted for multiple testing using the Bonferroni method.

Figure 4

Quantification of nuclear FAK and nuclear phospho-FAK (Y397) expression evaluated by multiplex immunofluorescence immunohistochemistry in 37 normal lung, 95 non-small-cell lung cancer (NSCLC), and 105 small-cell lung cancer (SCLC) tissues: (A) Nuclear FAK staining score: Percentage (%) of FAK-stained nucleus area multiplied by (×) nuclear FAK mean intensity, (B) nuclear phospho-FAK (Y397) staining score: (% of phospho-FAK-stained nucleus area of low intensity × 1) + (% of phospho-FAK-stained nucleus area of medium intensity × 2) + (% of phospho-FAK-stained nucleus area of high intensity × 3). Each dot represents one sample. Data presented as the mean ± S.D. p-values were obtained using linear models and adjusted for multiple testing using the Bonferroni method.

Figure 5

Quantification of (A) FAK and (B) phospho-FAK expression evaluated by Western blot (WB), with normalization to glyceraldehyde 3-phosphate dehydrogenase (GAPDH ) expression, in nine normal lungs, 30 non-small-cell lung cancer (NSCLC), and 10 small-cell lung cancer (SCLC) tissue lysates. Each dot represents one sample. Data presented as the mean ± S.D. Significance determined by the Kruskal-Wallis test. (C) Illustration of a representative WB of FAK and phospho-FAK (Y397) expression in normal lung, NSCLC, and SCLC tissue lysates. All the WB are represented in Supplementary Figure S1.