探討Semaphorin 6A二聚化在誘導肺癌細胞內源性凋亡中的功能

Abstract

肺癌是一種最常見且致死率極高的癌症，在全球被視為最具挑戰性的公共衛生問題。儘管已有新的治療方法被開發出來，但腫瘤的多樣性和對治療的抗性等問題仍有待克服。因此，更深入地了解肺癌細胞中的分子調控機制，將有助於我們更全面地認識疾病的運作及進展，從而為未來的研究提供重要的見解。我們先前的研究顯示，Semaphorin 6A（SEMA6A）在肺癌組織及細胞株中的表現量顯著降低，而過表達 SEMA6A full-length（SEMA6A-FL）可顯著誘導肺癌細胞凋亡。當SEMA6A-FL失去SEMA域時，其胞內區域會與FADD（Fas-associated death domain）結合，進而透過裂解Caspase-8誘導細胞凋亡。然而，即使SEMA6A-FL在肺癌細胞中表現後可觀察到約10%的凋亡比例，其胞內區域卻並未透過FADD和Caspase-8誘導此過程。此外，先前研究發現SEMA6A-FL可誘發內質網壓力，因此我們推測SEMA6A-FL是透過內源性凋亡途徑引發細胞死亡。早期研究也顯示，SEMA6A-FL可在細胞中以單體和二聚體的形式存在，並且SEMA6A-FL透過其SEMA域中第415位胺基酸形成二聚體，進而與Plexin-A2相互作用。這些結果表明，SEMA6A-FL的單體和二聚體可能具有不同的生物學功能。因此，我們假設SEMA6A-FL的結構，尤其是其形成二聚體的能力，可能會顯著影響其誘導內源性細胞凋亡的機制。在本研究中，我們確認了兩個特定的突變——M415C（促進二聚化）和I322E（減少二聚化）——可以有效改變SEMA6A-FL的二聚化能力。為了探討這些結構對細胞功能的影響，我們進行了多項實驗，包括細胞生長測試、集落形成存活分析、細胞凋亡檢測、細胞週期分析以及凋亡相關蛋白的表現量測定。結果顯示，當 SEMA6A-FL的二聚化能力被減弱時，細胞生長或凋亡與對照組相比並無顯著變化。然而，當SEMA6A-FL的二聚化能力增強時，肺癌細胞的生長顯著減少，且凋亡顯著增加。此外，我們製備了更容易形成二聚體的SEMA6A-FL截短版本，並將這些版本過表現於肺癌細胞中，結果顯示細胞生長顯著減少且凋亡增加，進一步強調了 SEMA6A-FL二聚化在誘導細胞凋亡中的重要角色。另外，增加SEMA6A-FL二聚體會增強肺癌細胞內質網壓力的程度，而且，下游Caspase-9的裂解產物也跟著增加。這代表SEMA6A-FL是透過二聚體結構引起內質網壓力，接著誘發肺癌細胞的內源性凋亡訊號。

Lung cancer is a common and deadly disease, representing a significant challenge to public health worldwide. Despite new therapies being developed, problems like tumor diversity and resistance to treatment still need to be overcome. Therefore, to better understand the molecular regulatory mechanisms in lung cancer cells will help us learn more about how the disease works and progress, leading to important insights for future research. Our previous research demonstrated that Semaphorin 6A (SEMA6A) expression is significantly reduced in lung cancer tissues and cell lines, while overexpression of SEMA6A-FL can trigger apoptosis significantly in the lung cancer cell lines. Although the intracellular domain of SEMA6A-FL does not interact with FADD, it has been shown to induce endoplasmic reticulum (ER) stress in lung cancer cells, suggesting that SEMA6A-FL may trigger apoptosis through the intrinsic pathway. Additionally, earlier studies have found that SEMA6A-FL exists in both monomer and dimer forms in the cells. It has also been shown that SEMA6A-FL dimerizes through a specific amino acid (415th position in the SEMA domain), which allows it to interact with another protein called Plexin-A2. These findings suggest that the monomer and dimer forms of SEMA6A-FL might have different biological functions. Based on this, we hypothesize that the structure of SEMA6A-FL, especially its ability to form dimers, may affect how it triggers intrinsic apoptosis. In the study, we confirmed that two specific mutations—M415C (which promotes dimerization) and I322E (which reduces dimerization)—can effectively change how SEMA6A-FL dimerizes. To see how these structures impact cell functions, we carried out several experiments, such as testing cell growth, clonogenic survival, apoptosis detection, cell cycle analysis, and measuring proteins involved in apoptosis. The results showed that when dimerization of SEMA6A-FL was reduced, there was no significant change in cell growth or apoptosis compared to the control group. However, when dimerization of SEMA6A-FL was increased, cell growth was significantly reduced, and apoptosis was greatly increased in the lung cancer cells. We also created truncated versions of SEMA6A-FL that are more likely to dimerize, and overexpressing these versions in lung cancer cells caused a significant reduction in cell growth and an increase in apoptosis, further highlighting the role of SEMA6A-FL dimerization in inducing cell death. Moreover, an increase in SEMA6A-FL dimerization enhances the degree of ER stress, accompanied by an increase in the cleavage products of downstream Caspase-9. This suggests that SEMA6A-FL induces ER stress through its dimeric structure, thereby triggering the intrinsic apoptotic signaling pathway in lung cancer cells.