Molecular Biology Program, University of Colorado Denver, 12801 East 17th Ave, Aurora, CO 80045, USA.
Mol Cancer. 2011 Jun 18;10:75. doi: 10.1186/1476-4598-10-75.
LIM kinase 1 (LIMK1) is expressed in both cytoplasmic and nuclear compartments, and is a key regulator of cytoskeletal organization involved in cell migration and proliferation. LIMK1 levels are increased in several human cancers, with LIMK1 over-expression in prostate and breast cancer cells leading to tumor progression. While it has been presumed that the mechanism by which LIMK1 promotes cancer progression is via its cytoplasmic effects, the role of nuclear vs cytoplasmic LIMK1 in the tumorigenic process has not been examined.
To determine if cytoplasmic or nuclear LIMK1 expression correlated with breast cancer, we performed immunohistochemical (IHC) analysis of breast tissue microarrays (TMAs), The IHC analysis of breast TMAs revealed that 76% of malignant breast tissue samples strongly expressed LIMK1 in the cytoplasm, with 52% of these specimens also expressing nuclear LIMK1. Only 48% of benign breast samples displayed strong cytoplasmic LIMK1 expression and 27% of these expressed nuclear LIMK1. To investigate the respective roles of cytoplasmic and nuclear LIMK1 in breast cancer progression, we targeted GFP-LIMK1 to cytoplasmic and nuclear subcellular compartments by fusing nuclear export signals (NESs) or nuclear localization sequences (NLS), respectively, to the amino-terminus of GFP-LIMK1. Stable pools of MDA-MB-231 cells were generated by retroviral transduction, and fluorescence microscopy revealed that GFP alone (control) and GFP-LIMK1 were each expressed in both the cytoplasm and nucleus of MDA-MB-231 cells, whereas NLS-GFP-LIMK1 was expressed in the nucleus and NES-GFP-LIMK1 was expressed in the cytoplasm. Western blot analyses revealed equal expression of GFP-LIMK1 and NES-GFP-LIMK1, with NLS-GFP-LIMK1 expression being less but equal to endogenous LIMK1. Also, Western blotting revealed increased levels of phospho-cofilin, phospho-FAK, phospho-paxillin, phospho-Src, phospho-AKT, and phospho-Erk1/2 in cells expressing all GFP-LIMK1 fusions, compared to GFP alone. Invasion assays revealed that all GFP-LIMK1 fusions increased MDA-MB-231 cell invasion ~1.5-fold, compared to GFP-only control cells. Tumor xenograft studies in nude mice revealed that MDA-MB-231 cells stably expressing GFP-LIMK, NLS-GFP-LIMK1 and NES-GFP-LIMK1 enhanced tumor growth 2.5-, 1.6- and 4.7-fold, respectively, compared to GFP-alone.
Taken together, these data demonstrate that LIMK1 activity in both the cytoplasmic and nuclear compartments promotes breast cancer progression, underscoring that nuclear LIMK1 contributes to the transforming function of LIMK1.
LIM 激酶 1（LIMK1）表达于细胞质和核区室，是细胞迁移和增殖中细胞骨架组织的关键调节因子。在几种人类癌症中 LIMK1 水平增加，前列腺癌和乳腺癌细胞中的 LIMK1 过表达导致肿瘤进展。虽然人们推测 LIMK1 促进癌症进展的机制是通过其细胞质效应，但核区室与细胞质区室中 LIMK1 在肿瘤发生过程中的作用尚未被研究。
为了确定细胞质或核区室 LIMK1 的表达是否与乳腺癌相关，我们对乳腺癌组织微阵列（TMA）进行了免疫组织化学（IHC）分析。对乳腺癌 TMA 的 IHC 分析表明，76%的恶性乳腺癌组织样本在细胞质中强烈表达 LIMK1，其中 52%的标本还表达核区室 LIMK1。只有 48%的良性乳腺样本显示强烈的细胞质 LIMK1 表达，其中 27%的标本表达核区室 LIMK1。为了研究细胞质和核区室 LIMK1 在乳腺癌进展中的各自作用，我们通过将核输出信号（NES）或核定位序列（NLS）分别融合到 GFP-LIMK1 的氨基末端，将 GFP-LIMK1 靶向到细胞质和核区室亚细胞区室。通过逆转录病毒转导生成 MDA-MB-231 细胞的稳定池，荧光显微镜显示 GFP 本身（对照）和 GFP-LIMK1 均在 MDA-MB-231 细胞的细胞质和核区室中表达，而 NLS-GFP-LIMK1 则在核区室中表达，NES-GFP-LIMK1 则在细胞质中表达。Western blot 分析显示 GFP-LIMK1 和 NES-GFP-LIMK1 的表达相等，NLS-GFP-LIMK1 的表达较少但与内源性 LIMK1 相等。此外，Western blot 分析显示，与 GFP 对照细胞相比，表达所有 GFP-LIMK1 融合蛋白的细胞中磷酸化 cofilin、磷酸化 FAK、磷酸化 paxillin、磷酸化 Src、磷酸化 AKT 和磷酸化 Erk1/2 的水平增加。侵袭实验显示，与 GFP 对照细胞相比，所有 GFP-LIMK1 融合蛋白均使 MDA-MB-231 细胞侵袭增加约 1.5 倍。裸鼠肿瘤异种移植研究显示，稳定表达 GFP-LIMK、NLS-GFP-LIMK1 和 NES-GFP-LIMK1 的 MDA-MB-231 细胞的肿瘤生长分别增强 2.5 倍、1.6 倍和 4.7 倍，与 GFP 对照相比。
综上所述，这些数据表明，细胞质和核区室中的 LIMK1 活性均促进乳腺癌进展，突出表明核区室 LIMK1 有助于 LIMK1 的转化功能。
