Department of Natural Sciences, School of Arts and Sciences, Lebanese American University, P.O. Box: 13-5053. Chouran, Beirut, 1102 2801, Lebanon.
Department of Pediatrics HemeOnc division, Weill Cornell Medicine, Joan & Sanford I. Weill Medical College of Cornell University, New York, USA.
Cell Commun Signal. 2020 Sep 8;18(1):144. doi: 10.1186/s12964-020-00635-5.
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. Video abstract.
肺癌是第二大常见癌症。其转移和扩散到远处的能力使其更具侵袭性。小分子 GTP 结合蛋白（GTPases）Rho 亚家族成员在调节肌动蛋白细胞骨架和癌细胞迁移及转移中发挥核心作用。在这项研究中，我们研究了 RhoA/Cdc42 GAP 蛋白 StarD13 的作用，StarD13 是一种先前描述的肿瘤抑制因子，在肺癌细胞 A549 的恶性程度、迁移和侵袭中发挥作用。
我们敲低了肺癌细胞 A549 中的 StarD13 表达，并通过延时成像和侵袭实验检测其对细胞迁移和侵袭小窝形成的影响。我们还进行了挽救实验，以确定 StarD13 的下游信号通路，并转染 RhoGTPases 的 FRET 生物传感器，以鉴定参与侵袭小窝形成的蛋白。
通过免疫组织化学，我们观察到与正常肺组织相比，肺癌组织中 StarD13 的表达水平降低。StarD13 在肺腺癌细胞系 A549 中的表达也低于正常肺细胞 WI38。此外，StarD13 的耗竭增加了 WI38 和 A549 细胞的增殖和活力，这表明 StarD13 可能是肺癌的潜在肿瘤抑制因子。然而，StarD13 的耗竭抑制了细胞的运动，这表明其在细胞迁移中具有正向调节作用。这可能是由于通过下拉和 FRET 实验检测到 RhoA 的组成性激活。令人惊讶的是，StarD13 通过抑制 Cdc42 介导的侵袭小窝形成来抑制细胞侵袭。事实上，TKS4 染色和侵袭小窝实验显示，StarD13 的耗竭增加了 Cdc42 的激活以及侵袭小窝的形成和基质降解。耗竭 StarD13 的正常肺细胞也产生了侵袭小窝，否则这是侵袭性癌细胞的独特特征。Cdc42 的敲低模拟了 StarD13 的作用，而过表达组成性激活的 Cdc42 则模拟了其耗竭的作用。最后，免疫染色和 FRET 分析显示，与在基质降解部位激活的 Cdc42 相比，StarD13 在侵袭小窝中不存在。
总之，StarD13 通过对 Rho GTPases 的差异调节，在肺癌细胞迁移和侵袭中发挥不同的作用。视频摘要。
