Up-regulation of LIMK1 expression in prostate cancer is correlated with poor pathological features, lymph node metastases and biochemical recurrence.

Abstract This study aimed to explore the association between LIM domain kinase 1 (LIMK1) expression in prostate cancer (PCa) tissues with advanced pathological features, lymph node metastases and biochemical recurrence. A total of 279 PCa specimens from patients who underwent radical prostatectomy and 50 benign prostatic hyperplasia (BPH) specimens were collected to construct tissue microarray, which were subjected to immunohistochemical staining for LIMK1 expression subsequently. Logistic and Cox regression analysis were used to evaluate the relationship between LIMK1 expression and clinicopathological features of patients with PCa. Immunohistochemical staining assay demonstrated that LIMK1 expression was significantly higher in PCa than BPH specimens (77.1% vs 26.0%; P < .001). LIMK1 expression was significantly higher in positive lymph node specimens than corresponding PCa specimens (P = .002; P < .001). Up‐regulation of LIMK1 was associated with prostate volume, prostate‐specific antigen, prostate‐specific antigen density, Gleason score, T stage, lymph node metastases, extracapsular extension and seminal vesicle invasion, and positive surgical margin. Multivariate logistic regression analysis demonstrated that LIMK1 was an independent risk factor for PCa lymph node metastasis (P < .05). Multivariate Cox regression analysis revealed that the up‐regulation of LIMK1 was an independent risk factor for biochemical recurrence. Kaplan‐Meier analysis indicated that up‐regulation LIMK1 was associated with shortened biochemical‐free survival (BFS) after radical prostatectomy (P < .001). In conclusion, LIMK1 was significantly up‐regulated in PCa and positive lymph node specimens and correlated with lymph node metastasis and shortened BFS of PCa. The underlying molecular mechanism of LIMK1 in PCa should be further evaluated.


| INTRODUC TI ON
Prostate cancer (PCa) is one of the most commonly diagnosed male malignancies and the second leading cause of cancer-related death in men. 1,2 Unfortunately, most androgen-dependent PCa (ADPC) inevitably progresses to castration-resistant prostate cancer (CRPC) after androgen ablation therapy. 3,4 Metastasis is a complicated and multi-step process. Invasion and distant metastasis are significantly associated with the prognosis of PCa. The prognosis was poor in patients with metastatic PCa because no curative treatment is currently available.
Co-ordinated reorganization of the actin cytoskeleton is essential to tumour invasion and metastasis. 5-7 LIM kinase 1 (LIMK1) is one of the members of the LIM kinase protein family. 6,8 Previous studies 6,[8][9][10][11] demonstrated that LIMK played an essential role in regulating the polymerization of actin through phosphorylation and inactivation of cofilin, which acted as the only downstream effector of LIMK1.
Cofilin can be inactivated by LIMK1 when its Ser3 site was phosphorylated. 12 Inactivated cofilin lost the ability of binding to actin filaments which improved the stability of F-actin, resulting in the change of actin cytoskeleton. 13 Several studies have now confirmed that the expression of LIMK1 is consistently elevated in the many kinds of tumours including breast cancer, 6,14 ovarian cancer, 15,16 colon cancer 17,18 and gastric cancer. 19,20 However, researchers have paid little attention to the role of LIMK1 in prostate cancer. Several studies have reported that LIMK can promote the invasive and metastatic ability of tumours. 8,17 What is more, it also participated in many kinds of biological behaviours including angiogenesis, proliferation, cell cycle and migration. 14,[21][22][23] Thus, LIMK1 has great potential to be a therapeutic target to prevent the invasion and metastasis of PCa.
This study hypothesized that LIMK1 was high expression in PCa and was involved in the invasion and metastasis of PCa. The expression of LIMK1 in PCa was determined by immunohistochemistry, and the relationship between the expression of LIMK1 and the invasion, metastasis, and prognosis of PCa was analysed.

| Ethics
This study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Fujian Medical University. Written informed consents were obtained from all patients.

| Tissue specimen and data collection
A total of 279 specimens of PCa tissue were collected from the patients who underwent radical prostatectomy from January 2012 to September 2015. All specimens were pathologically confirmed as primary prostate adenocarcinoma. Patients without any preoperative endocrine therapy, chemotherapy, radiotherapy, and immunotherapy were included.
The age of patients ranged from 48 to 78 years, with an average of 68.45 ± 6.92 years. The stage of PCa was classified based on the Union for International Cancer Control (UICC)-TNM classification. Fifty benign prostatic hyperplasia specimens were treated as control.

| Follow-up
Patients with total prostate-specific antigen (PSA) level less than 0.01 ng/mL were followed up for 1 month after radical prostatectomy. The follow-up started from the date of the operation until the occurrence of the biochemical relapse. The follow-up time ranged from 6 to 36 months. The exclusion criteria were as follows: patients who received adjuvant radiotherapy and/or endocrine therapy during follow-up, with positive lymph node metastasis and with insufficient follow-up data. Finally, a total of 163 patients were included in the analysis of biochemical recurrence. The follow-up protocol 24 was as follows: the first month after surgery; then every 3 months after surgery for 2 years; and afterwards every 6 months since the third year.
The follow-up was ended when biochemical recurrence occurred before September 2015. The total follow-up time was 9 to 95 months, and the median follow-up time was 55 months; the total biochemical recurrence rate was 17.18% (28/163), and the median biochemical recurrence time was 25 months (ranged from 10 to 67 months).

| Construction of tissue microarray
The paraffin-embedded benign prostatic hyperplasia and PCa specimens were obtained from the department of pathology of the First Affiliated Hospital of Fujian Medical University. The paraffin-embedded specimens were sliced and underwent H&E staining. The representative areas of the H&E staining sections were evaluated and confirmed by a senior pathologist in order to construct tissue microarray. A tissue microarray maker was designed to generate tissue microarrays by using 2 × 2 mm tissue cores in each case. Finally, tissue microarrays contain 5 × 10 tissue cores for both PCa and benign prostatic hyperplasia specimens in each were obtained and then be sliced continuously into 4-μm-thick sections.

| Immunohistochemistry
In the present study, staining of LIMK1 was performed by immu- Ltd.) by a high-pressure cooker 3 minutes for antigen retrieval.
The sections were then examined by light microscopy (Olympus) by two blinded pathologists. Any discrepancies were resolved by re-reviewing the sections. Two semi-quantitative methods and the total LIMK1 immunostaining score methods including staining intensity and the proportion of positive cells were described as follows. [25][26][27][28] The immunohistochemistry score of LIMK1 consists of two parts, including staining intensity and the proportion of positive cells. We classified the stating intensity as 0, absent; 1, weak; 2, moderate; and 3, strong.

| The different expression of LIMK1 between benign prostatic hyperplasia and PCa tissues
A total of 215 cases of LIMK1-positive expression were seen in 279 cases of PCa tissues, while a total 13 cases of LIMK1-positive expression were observed in 50 cases of benign prostatic hyperplasia tissues.
LIMK1 was mainly expressed in the cytoplasm of positive cells. The results demonstrated that the positive expression rate of LIMK1 in PCa tissues was significantly higher than that of benign prostatic hyperplasia tissues (77.1% vs 26.0%, respectively; P < .001; Table 1; Figure 1).

| The different expression of LIMK1 between PCa tissues and corresponding lymph node metastases specimens
A total of 40 cases of positive lymph node metastases specimens and corresponding PCa tissues were collected for

| The relationship between LIMK1 expression and clinicopathological features of PCa patients
The expression of LIMK1 was significantly associated with the prostate volume, PSA level, PSA density, Gleason score, T stage, lymph node metastases, extracapsular extension and seminal vesicle invasion, and positive surgical margin (P < .05, Table 3). However, the expression of LIMK1 was not associated with the age of patients.
Moreover, the strong staining of LIMK1 was seen in the low differentiation, high stage and lymph node metastasis specimens ( Figure 3).
Subsequently, subgroup analysis stratified by the lymph node metastasis was analysed. The results demonstrated that lymph node metastases were significantly associated with the preoperative PSA level, postoperative Gleason score, extracapsular extension, seminal vesicle invasion, positive surgical margin and the LIMK1 expression (P < .05, Table 4). However, lymph node metastases were not associated with the age, body mass index, prostate volume, the proportion of positive biopsy cores and PSA density (P > .05, Table 4).
The multivariate logistic regression analysis demonstrated that LIMK1 was independent risk factor for PCa lymph node metastasis (P < .001, Table 5).

| The relationship between LIMK1 expression and biochemical recurrence
Univariate Cox regression analysis demonstrated that higher proportion of positive biopsy cores, T stage, Gleason score, extracapsular extension, positive surgical margin and LIMK1 expression was associated with biochemical recurrence (P < .05, Table 6). Multivariate Cox regression analysis revealed that upregulation of LIMK1 was independent risk factor for biochemical recurrence (P < .05, Table 6).

| D ISCUSS I ON
The LIMK family consists of LIMK1 and LIMK2, which belongs to the serine protein kinase, and associated with actin polymerization and microtubule depolymerization. 29 The expression of LIMK1 was mainly seen in the cytoplasm and can freely shuttle between normal nucleus and cytoplasm. 9 The expression of LIMK was elevated in many kinds of tumours, especially highly invasive malignancies. LIMK 1 plays an important role in the invasion and metastases of tumours by regulating the actin cytoskeleton molecules. 14,15,17 Recently, the significance of LIMK1 in tumorigenesis has aroused extensive concern. 6,19 There are many mechanisms regulating the activation of LIMKI. The activated LIMK1 is responsible for the stability of the cytoskeleton and the bond of external stimulation of the cells. 9 The LIMK1 was in the cytoplasm and rapidly migrates back and forth between the nucleus and the cytoplasm. 11,29 When the cytoskeleton is assembled, LIMK1 deactivates the cofilin by phosphorylation of 3 serine residues, which reverses the process of actin depolymerization. 8 It is reported that LIMK1 played an important role in regulating the transportation process of lysosome and endosome. 6  In general, however, there is still a lack of an effective way to predict the risk of lymph node metastasis preoperatively and evaluate the benefit obtained from the lymph node dissection. In this study,  biochemical recurrence of PCa remains low. 41 There is still no ideal and reliable marker for predicting the tumour growth, invasion and metastasis. 42 Sen et al 43 reported that the serum LIMK1 level in patients with hepatocellular carcinoma was significantly higher than those in patients with liver cirrhosis and normal individuals. The diagnostic accuracy of LIMK1 in the diagnosis of hepatocellular carcinoma was higher than that of AFP. In the study of cytotoxic chemotherapy of triple-negative breast cancer, the expression of LIMK1 was associated with the prognosis of the cytotoxic chemotherapy. 44  There are some limitations in this study. Firstly, this study was retrospectively designed. Secondly, the prognostic power of the number of positive lymph nodes with that of lymph node ratio was not analysed in this study.
In conclusion, LIMK1 was significantly up-regulated in PCa and positive lymph node specimens and correlated with lymph node metastasis and shortened BFS of PCa. The underlying molecular mechanism of LIMK1 in PCa should be further evaluated.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

AUTH O R CO NTR I B UTI O N S
NX, XYX and JBH conceived and designed the experiments; YPW and YZL performed the experiments; SHC, XLS and XDL analysed the data; NX, XYX, YW and QSZ contributed reagents/materials/ analysis tools; and YPW, JBH, YZL and HC wrote the paper. All authors read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this article.