Nuclear and cytoplasmic LIMK1 enhances human breast cancer progression.
Nuclear and cytoplasmic LIMK1 enhances human breast cancer progression.
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DOI：
10.1186/1476-4598-10-75
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发表时间：
2011-06-18
期刊：
影响因子：
37.3
通讯作者：
Gutierrez-Hartmann A
中科院分区：
文献类型：
--
作者：
McConnell BV;Koto K;Gutierrez-Hartmann A
LIM激酶1（LIMK1）在细胞质和细胞核区室中表达，是参与细胞迁移和增殖的细胞骨架组织的关键调节因子。LIMK1水平在几种人类癌症中增加，其中LIMK1在前列腺癌和乳腺癌细胞中过表达导致肿瘤进展。虽然已经推测LIMK1促进癌症进展的机制是通过其细胞质效应，但尚未研究细胞核与细胞质LIMK1在肿瘤发生过程中的作用。为了确定细胞质或细胞核LIMK1表达是否与乳腺癌相关，我们对乳腺组织微阵列（TMAs）进行免疫组织化学（IHC）分析。乳腺TMAs的IHC分析显示，76%的恶性乳腺组织样品在细胞质中强烈表达LIMK1，其中52%的这些标本还表达细胞核LIMK1。只有48%的良性乳腺样本显示出较强的细胞质LIMK1表达，其中27%表达细胞核LIMK1。为了研究细胞质和核LIMK1在乳腺癌进展中各自的作用，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靶向细胞质和核亚细胞区室。通过逆转录病毒转导产生MDA-MB-231细胞的稳定池，荧光显微镜显示单独的GFP（对照）和GFP-LIMK 1各自在MDA-MB-231细胞的细胞质和细胞核中表达，而NLS-GFP-LIMK 1在细胞核中表达，NES-GFP-LIMK 1在细胞质中表达。Western印迹分析显示GFP-LIMK1和NES-GFP-LIMK1的相等表达，NLS-GFP-LIMK1的表达低于但等于内源性LIMK1。此外，蛋白质印迹显示，与单独的GFP相比，在表达所有GFP-LIMK1融合体的细胞中，磷酸-cofilin、磷酸-FAK、磷酸-桩蛋白、磷酸-Src、磷酸-AKT和磷酸-Erk1/2的水平增加。侵袭测定显示，与仅GFP的对照细胞相比，所有GFP-LIMK1融合物使MDA-MB-231细胞侵袭增加约1.5倍。在裸鼠中的肿瘤异种移植研究显示，与单独的GFP相比，稳定表达GFP-LIMK、NLS-GFP-LIMK 1和NES-GFP-LIMK 1的MDA-MB-231细胞分别增强肿瘤生长2.5倍、1.6倍和4.7倍。总之，这些数据表明，LIMK1在细胞质和细胞核区室中的活性促进乳腺癌的进展，强调细胞核LIMK1有助于LIMK1的转化功能。
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 cytoplamsic 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.
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