Triple-negative breast cancer (TNBC) is a highly metastatic breast cancer subtype and due to the lack of hormone receptors and HER2 expression, TNBC has limited therapeutic options with chemotherapy being the primary choice for systemic therapy. LIM Domain Kinase 2 (LIMK2) is a serine/threonine kinase that plays an important role in the regulation of actin filament dynamics. Here, we show that LIM domain kinase 2 (LIMK2) is overexpressed in TNBC, and short-hairpin RNA (shRNA)-mediated LIMK2 knockdown or its pharmacological inhibition blocks metastatic attributes of TNBC cells. To determine the mechanism by which LIMK2 promotes TNBC metastatic progression, we performed stable isotope labeling by amino acids in cell culture (SILAC) based unbiased large-scale phosphoproteomics analysis. This analysis identified 258 proteins whose phosphorylation was significantly reduced due to LIMK2 inhibition. Among these proteins, we identified SRSF protein kinase 1 (SRPK1), which encodes for a serine/arginine protein kinase specific for the SR (serine/arginine-rich domain) family of splicing factors. We show that LIMK2 inhibition blocked SRPK1 phosphorylation and consequentially its activity. Furthermore, similar to LIMK2, genetic inhibition of SRPK1 by shRNAs or its pharmacological inhibition blocked the metastatic attributes of TNBC cells. Moreover, the pharmacological inhibition of LIMK2 blocked metastatic progression in mice without affecting primary tumor growth. In sum, these results identified LIMK2 as a facilitator of distal TNBC metastasis and a potential target for preventing TNBC metastatic progression.

三阴性乳腺癌 (TNBC) 是一种高度转移性乳腺癌亚型，由于缺乏激素受体和 HER2 表达，三阴性乳腺癌的治疗选择有限，化疗是全身治疗的主要选择。LIM 结构域激酶 2 (LIMK2) 是一种丝氨酸/苏氨酸激酶，在调节肌动蛋白丝动力学中起重要作用。在这里，我们显示 LIM 结构域激酶 2 (LIMK2) 在 TNBC 中过度表达，而短发夹 RNA (shRNA) 介导的 LIMK2 敲低或其药理抑制作用可阻断 TNBC 细胞的转移属性。为了确定 LIMK2 促进 TNBC 转移进展的机制，我们通过基于无偏倚的大规模磷酸蛋白质组学分析的细胞培养 (SILAC) 中的氨基酸进行了稳定同位素标记。该分析确定了 258 种蛋白质，其磷酸化因 LIMK2 抑制而显着降低。在这些蛋白质中，我们鉴定了 SRSF 蛋白激酶 1 (SRPK1)，它编码特定于 SR（富含丝氨酸/精氨酸结构域）家族剪接因子的丝氨酸/精氨酸蛋白激酶。我们显示 LIMK2 抑制阻止了 SRPK1 磷酸化并因此阻止其活性。此外，与 LIMK2 类似，shRNA 对 SRPK1 的遗传抑制或其药理学抑制阻断了 TNBC 细胞的转移特性。此外，LIMK2 的药理抑制作用可阻止小鼠的转移进展，而不影响原发性肿瘤的生长。总之，这些结果将 LIMK2 确定为远端 TNBC 转移的促进剂和预防 TNBC 转移进展的潜在目标。