Immunohistochemical evaluation of ROCK activation in invasive breast cancer

Patients with primary breast carcinoma were retrieved from the surgical pathology file of the hospital from 1990 to 1999. The clinicopathological data including age, histologic type, grade, nodal status, stage at diagnosis, date of surgery, follow up data, and ER/PR/HER2 data were collected from the pathology and medical records. Overall survival was defined as the time from operation to death related to breast cancer. The study protocol was approved by the Institutional Review Board of Taipei Veterans General Hospital, Taiwan. In this retrospective study, the sample collection followed the ethical standards of the World Association’s Declaration of Helsinki and the need for informed consent was waived by the Institutional Review Board of the Taipei Veterans General Hospital, Taiwan.

All specimens were fixed in 10 % neutral buffered formalin. After reviewing the original histopathology slides for confirmation of the presence of the required tissues, two hundred and seventy-five tumor samples from patients with breast cancer were used in this study. The tumor samples according to their original diagnoses were classified as carcinomas in situ (CIS, n = 56), invasive carcinomas (IC, n = 116) and invasive carcinomas with metastasis (ICM, n = 103). Tumor samples larger than 1 cm, which were available and adequate for building tissue arrays with 2 mm tissue cylinders from 2 to 3 appropriate areas, were selected from each case. Two cores from representative areas of the tumors, or three cores from the tumors with heterogeneous features or those with available metastatic tumors were selected to construct tissue microarrays (TMAs). All patient identifiers were delinked from the tissues in the TMAs.

Tissue sections were immunostained using anti-pS1333 ROCKI and anti-pS1366 ROCKII antibodies on a Bond-max immunostainer (Leica Microsystems, Newcastle, UK). The production and validation of anti-pS1333-ROCKI and anti-pS1366-ROCKII antibodies have been described previously [24, 25]. Tissue sections were deparaffinized in xylene, rehydrated through serial dilutions of alcohol, and washed in phosphate-buffered saline (pH 7.2). On-board heat-induced antigen retrieval in pH 9.0 ethylenediamine tetraacetic acid (EDTA) for 30 min was performed. Sections were incubated with the primary antibodies (1: 750 for pS1333 ROCKI; 1:3200 for pS1366 ROCKII) for 60 min at room temperature. Visualization was performed using a VBS Refine polymer detection system (Leica Microsystems). ROCKII S1366 phosphopeptide or nonphosphopeptide (0.3 μg/ml) was added for the peptide competition experiment. All sections were counterstained with hematoxylin. Both nuclear staining and cytoplasmic staining of ROCKI and ROCKII phosphorylation were evaluated. The percentage of tumor cells with perceptible ROCK phosphorylation signal of in the nucleus was recorded for nuclear staining. Cytoplasmic staining was graded as negative/weak (no staining or <10 % faint staining), moderate (10–50 % area with intermediate staining), and strong (>50 % area with intense staining). Stains for ER (clone 6 F11, Leica Biosystems, Newcastle, UK, 1:100), PR (clone 16, Leica Biosystems, 1:150), HER2 (polyclone A0485, Dako, Glostrup, Denmark, 1:900) and Ki-67 (clone MIB-1, Dako, Glostrup, Denmark, 1:75) were performed. The evaluations of ER, PR, and HER2 followed previously reported instructions [26, 27]. One percent or more tumor cells exhibiting nuclear staining were regarded as positive for ER and PR [26]. HER2 positivity was defined by complete intense membrane staining in more than 10 % tumor cells [27]. The percentage of Ki67 positive tumor cells derived from four high-power fields (400×) was averaged for the Ki67 labeling index.

HEK293T cells were maintained in Dulbecco's modified Eagle’s medium (DMEM) supplemented with 10 % (v/v) fetal bovine serum (FBS) in a humidified atmosphere of 5 % CO₂/95 % air at 37 °C. For siRNA transfection experiments, 5 × 10⁵ of cells were transfected with or without of siRNA targeting human ROCKII (Dharmacon smartpool) by Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA). After 2 days, cells were than transfected with pEGFP-RhoAV14 by TurboFect reagent (Thermo Fisher Scientific) for 16 h. Cell were trypsinzied and collected by centrifugation at 900 rpm for 3 min. The cell pellets were fixed in 10 % neutral buffered formalin for 48 h, centrifuged and processed to paraffin cell block in the automatic tissue processor. A parallel set of cell lysate was prepared for the examination the protein expression levels of ROCKII and GFP-RhoAV14 by Western blot analysis with anti-ROCKII and anti-RhoA antibodies.

Chi-square test for trend was used to compare the distributions of categorical variables. Differences between continuous variables were compared using the Mann–Whitney U test. Univariate Cox regression was performed for survival analyses. The survival curve was plot using Kaplan-Meier method. Their differences were compared by log-rank test. Multivariate Cox regression model was used to adjust the influence of significant prognostic factors. The statistical difference was considered significant when the P value was less than 0.05.

The clinical and pathological characteristics including age, grade, ER/PR/HER2 status, and follow up of study cohort underlying the tumor type category are shown in Table 1.

The IHC results of ROCKI S1333 phosphorylation and ROCKII S1366 phosphorylation stratified by tumor classification are listed in Table 2. The status of ROCKI and ROCKII activation was determined by IHC staining with anti-pS1333 ROCKI and anti-pS1366 ROCKII antibody, respectively. Both ROCKI and ROCKII activation signals were observed in the cytoplasm and nucleus of tumor cells. The nuclear ROCKII signals were observed more frequently in the ICM 51/103 (50 %) cases than in the IC 35/116 (30 %) and CIS 11/56 (20 %) cases (P < 0.001). In addition, the percentage of tumor cells with perceptible ROCKII phosphorylation signal was evaluated, and the mean percentage of cells with nuclear signals was found to be significantly higher in ICM cases (20.8 %) than in IC (8.7 %) and CIS (6.9 %) cases (P = 0.003). ROCKI activation signal in nucleus was observed with no significant differences among CIS, IC or ICM cases (Table 2). Both ROCKI and ROCKII activation signals were observed in the cytoplasm with no significant differences among CIS, IC or ICM cases (Table 2), either. Overall, these data suggest that nuclear ROCKII activation signal is associated with tumor metastasis in invasive breast cancer.

The representative IHC staining of ROCKII S1366 phosphorylation is showed in Figure 1. Three different breast carcinoma samples were shown and the ROCKII S1366 phosphorylation was detected negative or positive clearly at different proportional of nucleus and/or cytoplasm of tumor cells. To confirm the binding specificity of the anti-pS1366 ROCKII antibody, the ROCKII S1366 phosphorylated and non-phosphorylated peptides were used for antibody neutralization. The staining signal of a tumor sample revealing ROCII S1366 phosphorylation positive signal both in cytosol and nuclei could be abolished by competition with phosphorylated S1366 peptide but not by non-phosphorylated peptide, indicating the specificity of detection (Fig. 2).

To further validate the specificity, HEK293T cells were used to prepare cell blocks for IHC staining of ROCKII S1366 phosphorylation. We found that the ROCKII S1366 phosphorylation signal was significant increased by cells expression of constitutively active RhoAV14. Depletion of the endogenous ROCKII by siRNA transfection diminished the staining signal, confirming that the signal derived from ROCKII (Fig. 3a). Of note, the ROCKII S1366 phosphorylation level determined in cells without GFP-RhoAV14 transfection was so low that the effect of ROCKII depletion was inconspicuous. Enhancement of ROCKII activation by GFP-RhoAV14 expression highlights the decrease of ROCKII S1366 phosphorylation signal in knockdown cells. The ROCKII S1366 phosphorylation and protein expression of ROCKII and GFP-RhoAV14 were also confirmed by Western blot analysis (Fig. 3b).

The positive staining of ROCKII activation signal in the invasive breast cancer (IC and ICM) respect to clinicopathologic features is listed in Table 3. The nuclear ROCKII S1366 phosphorylation signal was significantly stronger in tumors at advanced stage (P = 0.003), and correlated with ER negative (P = 0.002), PR negative (P = 0.017), HER2 positive status (P = 0.017) and high Ki67 labeling index (P = 0.044). There was no significant correlation between nuclear ROCKII activation signal and patient age (< 50 years vs. ≥ 50 years) or histological grade (Table 3). The IHC results of nuclear ROCKII S1366 phosphorylation stratified by molecular classification in invasive breast cancer are listed in Table 4. Our results show that nuclear ROCKII S1366 phosphorylation signal was significantly lower in the luminal types group compared to HER2-enriched group (P < 0.001) and to triple negative group (P = 0.044) (Table 4).

Nuclear ROCKII activation signal was a significant prognostic factor, as revealed by univariate Cox regression analyses (Hazard ratio = 1.013, P = 0.004). The best cut-off value for the proportion of nuclear ROCKII activation to predict survival was 30 %; there was significant difference in the survival between cases with nuclear ROCKII activation signal ≥ 30 % (median survival 142 months) and cases with nuclear ROCKII activation signal < 30 % (median survival 257 months) (P < 0.001, Fig. 4). The relevance of nuclear ROCKII activation to other prognostic variables was then studied by multivariate analyses. Tumor stage was the most significant one among the prognostic variables studied such as patient age, histologic grade, tumor stage, ER, PR and HER2 status, and nuclear ROCKII activation (Table 5, model 1). Tumor stage is a complex function including tumor size, lymph node status and distant metastasis. Since nuclear ROCKII activation signal was significantly higher in ICM cases (Table 2) and associated with late tumor stage (Table 3), being highly dependent on tumor stage might confound the prognostic value of nuclear ROCKII activation. We then removed tumor stage form the multivariate analysis and found that the prognostic significance of nuclear ROCKII activation was revalidated (Hazard ratio = 2.116, P = 0.016; Table 5, model 2). This data further support the correlation of nuclear ROCKII activation with late tumor stage as well as metastasis.