背景介紹: 許多的證據顯示癌症幹細胞 (cancer stem cells) 具有自我再生、藥物抗性和細胞遷移的能力，且Wnt訊號傳遞途徑的過度活化，容易促使癌症幹細胞的產生，導致不同癌症的藥物治療抗性及遠端轉移，但SFRPs (secreted frizzled related proteins) 胞外蛋白能拮抗Wnt訊號傳遞途徑。先前本實驗室的研究已證實新穎的Rab37 small GTPase透過胞釋作用 (excytosis) 參與調控貨物蛋白 (cargo) 的分泌，進而抑制肺癌細胞的轉移。我們利用蛋白質體學分析結果得知SFRP1可能為Rab37的囊泡運輸 (vesicle trafficking) 貨物蛋白；然而，Rab37蛋白在調控癌症幹性所扮演之角色以及是否介導SFRP1分泌目前仍未知。
目的: 本研究以肺癌為模式探討Rab37是否介導SFRP1蛋白分泌而抑制癌症幹性的相關分子機制。
實驗結果: 我們在肺癌細胞中以shRNA方式將Rab37蛋白表達抑制或於細胞的培養液 (conditioned medium) 加入SFRP1抑制劑後，透過癌細胞球團 (tumor sphere) 分析及定量聚合酶連鎖反應的結果顯示肺癌細胞球團的生成、癌幹特性的轉錄因子及Wnt下游相關基因的表現量皆有增加的趨勢；在癌細胞內穩定表達Rab37蛋白或經過添加SFRP1重組蛋白的處理後，則會抑制肺癌細胞幹性。在免疫缺陷的裸鼠體內植入50顆Rab37蛋白低表達的肺癌細胞即可產生腫瘤，顯示Rab37蛋白低表達的肺癌細胞具癌幹特性；特別的是，這種裸鼠癌幹特性可以因為加入SFRP1重組蛋白而抑制腫瘤生長。我們透過收集Rab37野生 (wild-type)、活化態 (GTP active mutant)、非活化態 (GDP inactive mutant) 穩定表達細胞的培養液及離析胞內囊泡，以及免疫螢光染色與全反射倒立螢光顯像，進一步確認Rab37以GTPase活性模式調控SFRP1蛋白的胞外運輸。最後在我們的臨床肺癌病人檢體中發現Rab37低表達伴隨著SFRP1蛋白的減少及癌幹指標Oct4高表現，這類病人的存活率及無病變存活期有明顯降低的現象。
實驗結論: 本研究提供肺癌細胞、動物及病人臨床的結果證實Rab37透過胞吐作用將SFRP1蛋白釋放至胞外，進而抑制Wnt訊號傳遞途徑及癌幹特性。未來可以針對此 Rab37-SFRP1-Wnt 訊息路徑找尋有潛力的肺癌治療策略。

Background: Emerging evidence suggests that cancer stem cells (CSCs) are responsible for tumor initiation, tumorigenicity, and therapeutic resistance. In addition, activated Wnt signaling pathway occurs frequently in the CSCs of many cancer types. We previously characterized that Rab37 small GTPase mediates exocytosis of tissue inhibitor of metalloproteinase 1 leading to suppression of tumor metastasis. Notably, secreted frizzled-related protein 1 (SFRP1) was identified as a putative cargo by our Rab37-mediated secretomic analysis. SFRP1 is known to be an extracellular antagonist of the Wnt signaling pathway, which is critical for cancer stemness.
Purpose: We investigated whether Rab37-mediated SFRP1 secretion suppressed lung CSC properties and whether activation of Rab37-SFRP axis exerted therapeutic value.
Results: We showed that knockdown of Rab37 at the cell-based assays or addition of SFRP1 inhibitor in conditioned medium (CM) increased self-renewal capacity and up-regulated the expression of stemness-related genes and downstream target genes of Wnt signal pathway in lung cancer cells. Importantly, overexpression of cytosolic Rab37 or treatment of soluble recombinant SFRP1 in CM rescued the increased stemness properties. Moreover, tumor initiation ability was promoted in the xenotransplantation animal model with Rab37 knockdown lung cancer cells, while such a tumor initiation ability-mediated by Rab37 knockdown was attenuated by addition of SFRP1 recombinant protein. Notably, Rab37-mediated anti-stemness ability was confirmed in cells overexpressing wild-type or Q89L (active mutant)-Rab37, while T43N (inactive mutant)-Rab37 expressing cells reduced such an anti-stemness ability. In addition, the collected CM and isolated vesicles from lung cancer cell lines manipulated for Rab37 showed the exocytosis of SFRP1 via Rab37 in a GTPase-dependent manner, which was further confirmed by immunofluorescence images and total internal reflection fluorescence images. Clinically, concordantly low expression of Rab37 and SFRP1 in tumor specimens correlated with high expression of Oct4 stemness marker and the worse clinical outcome of lung cancer patients.
Conclusion: This study provides compelling evidence that Rab37-mediated SFRP1 secretion inhibits Wnt signaling pathway and CSCs properties. Targeting this Rab37-SFRP1-Wnt pathway possesses therapeutic value to treat lung cancer.

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