LIM kinase 1 interacts with myosin-9 and alpha-actinin-4 and promotes colorectal cancer progression

Abstract

Background: LIM kinase 1 (LIMK1) is a key regulator of the cytoskeletal organisation involved in cell proliferation and migration. Even though LIMK1 is frequently dysregulated in epithelial cancers, the role and mechanisms of LIMK1 in colorectal cancer (CRC) remains unclear.

Methods: Immunohistochemical analysis was performed to examine the expression and clinical significance of LIMK1 in CRC samples. Loss- and gain-of-function assay was performed to investigate the effects of aberrant expression on cellular biological behaviour of CRC cells in vitro and in vivo. Immunoblotting and immunoprecipitation was used to screen LIMK1-related signalling pathways and downstream factors.

Results: In this study, our results showed that LIMK1 was upregulated in CRC tissues and localised in both the cytoplasm and the nucleus of CRC cells. Overexpression of LIMK1 in cytoplasmic and nuclear subcellular compartments was closely related to tumour metastasis and poor prognosis of CRC patients. Enhanced expression of cytoplasmic and nuclear LIMK1 significantly increased cell proliferation and migration by driving epithelial-mesenchymal transition and activating the PI3K/Akt signal pathway in vitro as well as promoting growth and metastasis of CRC xenografts, whereas opposite effects were achieved in LIMK1-silenced cells. Furthermore, we identified two tumour metastasis-associated proteins, MYH9 and ACTN4, as direct targets of LIMK1, which were required for a LIMK1-mediated aggressive phenotype.

Conclusions: These findings indicate that LIMK1 plays a critical role in promoting CRC progression at subcellular level. Our findings provide new insights into the metastasis of CRC and advocate for the development of clinical intervention strategies against advanced CRC.

Figure 1

Both cytoplasmic and nuclear LIMK1 are associated with a poor prognostic phenotype of CRCs. (A) Western blot analysis of LIMK1 in CRC tissues (T) and adjacent non-tumour tissues (N). The immunosignal was quantified using densitometric scanning software, and the relative protein abundance was determined by normalisation with β-actin. (B) IF analysis of LIMK1 protein expression in normal mucosa tissues and CRC tissues (original magnification × 2400). (C) IHC analysis of LIMK1 protein expression in CRC tissues and adjacent non-tumour tissues. Representative photographs of IHC staining in CRC tissues and adjacent non-tumour tissues are shown. (D) Kaplan–Meier survival curves and univariate analyses (log-rank) for CRC patients with distinct expression levels and subcellular localisation of LIMK1. (E) IF analysis for subcellular localisation of LIMK1 in CRC cell lines (original magnification × 2400). (F) Western blot analysis of cytoplasmic and nuclear fractions from SW620 and LOVO. Nuclear segregation is assayed by total H3K4. Cytoplasmic segregation is assayed by GAPDH. LIMK1 is assayed with anti-LIMK1 antibody. (G) Western blot analysis for the expression of LIMK1 in CRC cell lines. IHC, immunohistochemistry. A full colour version of this figure is available at the British Journal of Cancer journal online.

Figure 2

Exogenous LIMK1 localised to the cytoplasm and nucleus contributes to aggressive phenotypes in vitro. (A) HA-alone and three HA-tagged LIMK1 proteins are depicted graphically and colour coded. HA was fused to the N-terminus of LIMK1 cDNA. Exogenous NLS and NES tags were fused to the N-terminus of HA. (B) SW480 and HCT116 cells were transiently transfected with HA, HA-NES-LIMK1, HA-NLS-LIMK1, and HA-LIMK1 vectors. Western blot analysis was performed to detect the exogenous and total expression of LIMK1 using anti-HA and anti-LIMK1. (C) IF assay was used to visualise subcellular localisation of LIMK1 in treated SW480 cells (original magnification × 2400). Exogenous NLS and NES sequences target HA-NLS-LIMK1 and HA-NES-LIMK1 proteins to the nuclear and cytoplasmic subcellular compartments. (D) The effect of LIMK1 on cell proliferation was evaluated by CCK-8 assay. (E) The invading cells of the transwell assay were counted under a microscope in five randomly selected fields. Bars represent the number of invaded cells. (F) Wound healing assay was performed to evaluate the motility. Bars represent migration index of treated or control cells. The distance migrated by treated cells was relative to that migrated by control cells. Representative figures are shown. The asterisk (*) indicates P<0.05. The double asterisk (**) indicates P<0.01. A full colour version of this figure is available at the British Journal of Cancer journal online.

Figure 3

Silencing of LIMK1 suppresses cell proliferation and migration in vitro. (A) SW600 cells were transiently transfected with LIMK1 siRNAs. Western blotting analysis of LIMK1 was performed to evaluate interfering efficiency. (B) The effect of LIMK1 siRNA on cell proliferation was evaluated by CCK-8 assay. (C) The invading cells of the transwell assay were counted under a microscope in five randomly selected fields. Bars represent the number of invaded cells. (D) Wound healing assay was performed to evaluate the motility ability. Bars represent migration index of treated or control cells. The distance migrated by treated cells was relative to that migrated by control cells. Representative figures were shown. The double asterisk (**) indicates P<0.01. The hash (#) indicates P>0.05. A full colour version of this figure is available at the British Journal of Cancer journal online.

Figure 4

Subcellular localisation of endogenous LIMK1 overexpression promotes CRC growth and progression in vivo. (A) Western blot analysis was used to detect the endogenous overexpression of LIMK1 in stable subcellular transfectants. (B) IF assay was used to visualise subcellular localisation of LIMK1 in stable transfectants (original magnification × 2400). (C) Tumour cells were injected subcutaneously into the back of nude mice to evaluate tumour growth. Representative figure of tumours formed is shown. (D) Tumour weight and volume in the back of nude mice injected with indicated cells was measured. The data of all primary tumours are expressed as mean±s.d. Scatter plots of tumour weight derived from indicated cells at 30 d after subcutaneous implantation. (E) The representative photographs of H&E and LIMK1 staining of subcutaneous tumour are shown. Proliferative ability was measured by the Ki-67 proliferative labelling index. (F) Tumour cells were injected into nude mice through the tail vein to evaluate the lung homing potential of cells. The number of metastatic lung nodules in individual mice was counted under the microscope. The magnification areas indicated metastatic nodes in the lung. The asterisk (*) indicates P<0.05. The double asterisk (**) indicates P<0.01. A full colour version of this figure is available at the British Journal of Cancer journal online.

Figure 5

LIMK1 localised to the cytoplasm and nucleus induces EMT and activates the PI3K/Akt signalling pathway. (A) Western blot analysis of EMT markers (E-cadherin, β-catenin, vimentin, N-cadherin, snail) in indicated cells treated with LIMK1 vector. (B) Western blot analysis of phosphorylated PTEN and AKT at Ser473 and Thr308 in indicated cells treated with LIMK1 vector. The immunosignal was quantified using densitometric scanning software, and relative protein abundance was determined by normalisation with β-actin. Each bar represents the mean±s.d. The results were reproduced in three independent experiments. The asterisk (*) indicates P<0.05. A full colour version of this figure is available at the British Journal of Cancer journal online.

Figure 6

MYH9 and ACTN4 are downstream factors of LIMK1. (A) Immunoprecipitation of the whole proteins from SW480/HA-LIMK1, SW480/HA-NES-LIMK1 and SW480/HA-NLS-LIMK1 cells with anti-HA antibody, respectively. (B) Validation of endogenous interaction between LIMK1 and MYH9 or ACTN4 in indicated cells. The input sample contained 2% of the total proteins used for the immunoprecipitation. (C) The subcellular localisation of MYH9 or ACTN4 and its co-localisation with LIMK1 in indicated cells was assessed by confocal microscopy (original magnification × 2400). A full colour version of this figure is available at the British Journal of Cancer journal online.