Lung cancer is the second most commonly occurring cancer. The ability to metastasize and spread to distant locations renders the tumor more aggressive. Members of the Rho subfamily of small GTP-binding proteins (GTPases) play a central role in the regulation of the actin cytoskeleton and in cancer cell migration and metastasis. In this study we investigated the role of the RhoA/Cdc42 GAP, StarD13, a previously described tumor suppressor, in malignancy, migration and invasion of the lung cancer cells A549. We knocked down StarD13 expression in A549 lung cancer cells and tested the effect on cell migration and invadopodia formation using time lapse imaging and invasion assays. We also performed rescue experiments to determine the signaling pathways downstream of StarD13 and transfected the cells with FRET biosensors for RhoGTPases to identify the proteins involved in invadopodia formation. We observed a decrease in the level of expression of StarD13 in lung tumor tissues compared to normal lung tissues through immunohistochemistry. StarD13 also showed a lower expression in the lung adenocarcinoma cell line A549 compared to normal lung cells, WI38. In addition, the depletion of StarD13 increased cell proliferation and viability in WI38 and A549 cells, suggesting that StarD13 might potentially be a tumor suppressor in lung cancer. The depletion of StarD13, however, inhibited cell motility, conversely demonstrating a positive regulatory role in cell migration. This was potentially due to the constitutive activation of RhoA detected by pull down and FRET assays. Surprisingly, StarD13 suppressed cell invasion by inhibiting Cdc42-mediated invadopodia formation. Indeed, TKS4 staining and invadopodia assay revealed that StarD13 depletion increased Cdc42 activation as well as invadopodia formation and matrix degradation. Normal lung cells depleted of StarD13 also produced invadopodia, otherwise a unique hallmark of invasive cancer cells. Cdc42 knock down mimicked the effects of StarD13, while overexpression of a constitutively active Cdc42 mimicked the effects of its depletion. Finally, immunostaining and FRET analysis revealed the absence of StarD13 in invadopodia as compared to Cdc42, which was activated in invadopodia at the sites of matrix degradation. In conclusion, StarD13 plays distinct roles in lung cancer cell migration and invasion through its differential regulation of Rho GTPases.

中文翻译：

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在肺腺癌迁移和侵袭过程中，rho GTPases 的差异调节揭示了肿瘤抑制因子 StarD13 在侵袭伪足调节中的新作用。

肺癌是第二常见的癌症。转移和扩散到远处位置的能力使肿瘤更具侵袭性。小 GTP 结合蛋白 (GTPase) 的 Rho 亚家族成员在调节肌动蛋白细胞骨架和癌细胞迁移和转移中发挥核心作用。在这项研究中，我们调查了 RhoA/Cdc42 GAP、StarD13（一种先前描述的肿瘤抑制因子）在肺癌细胞 A549 的恶性、迁移和侵袭中的作用。我们敲低了 A549 肺癌细胞中的 StarD13 表达，并使用延时成像和侵袭测定测试了对细胞迁移和侵袭伪足形成的影响。我们还进行了救援实验以确定 StarD13 下游的信号通路，并用 RhoGTPases 的 FRET 生物传感器转染细胞，以确定参与入侵伪足形成的蛋白质。我们通过免疫组织化学观察到与正常肺组织相比，肺肿瘤组织中 StarD13 的表达水平降低。与正常肺细胞 WI38 相比，StarD13 在肺腺癌细胞系 A549 中的表达也较低。此外，StarD13 的消耗增加了 WI38 和 A549 细胞的细胞增殖和活力，表明 StarD13 可能是肺癌的肿瘤抑制因子。然而，StarD13 的消耗抑制了细胞运动，相反地证明了在细胞迁移中的积极调节作用。这可能是由于下拉和 FRET 分析检测到的 RhoA 的组成型激活。令人惊讶的是，StarD13 通过抑制 Cdc42 介导的侵袭伪足形成来抑制细胞侵袭。事实上，TKS4 染色和侵袭伪足分析表明 StarD13 消耗增加了 Cdc42 活化以及侵袭伪足的形成和基质降解。耗尽 StarD13 的正常肺细胞也会产生侵袭伪足，否则是侵袭性癌细胞的独特标志。Cdc42 击倒模拟了 StarD13 的影响，而组成型活性 Cdc42 的过度表达模拟了其消耗的影响。最后，免疫染色和 FRET 分析显示，与 Cdc42 相比，侵袭伪足中不存在 StarD13，后者在基质降解位点的侵袭伪足中被激活。综上所述，