內質網壓力促進BIK泛素化機制及其治療上的應用

Abstract

BIK (Bcl2-interacting killer)屬於Bcl2家族中BH3-only的促進細胞凋亡蛋白。BIK藉由跟抑制細胞凋亡蛋白包括Bcl-2、Bcl-xL和Mcl-1相互作用進而促使細胞走向細胞凋亡。BIKDD是一個持續活化的BIK變異，能透過和抑制細胞凋亡蛋白有更好的相互作用，來更加增強細胞走向細胞凋亡的功能以達到殺死癌症細胞的目的。然而BIK和BIKDD的半衰期很短，蛋白不穩定性成為BIK在癌症基因治療上的限制。因此，了解BIK和BIKDD的降解機制就變得相當重要。使用dominant-negative形式的Cullin和基因表現降低技術，我們發現Cul5參與在BIK的泛素化和降解中。在所有Cul5的受質連接體中，我們經由泛素化分析確認ASB11可以增加BIK的泛素化，反之，不能連接Cul5的ASB11變異不行。在多株不同的細胞中降低ASB11的表現量會使BIK上升，暗示著ASB11所促進的泛素化會使BIK降解。另外，我們也在泛素化分析中發現，ASB11可將泛素加在BIK K115和K160的位置上並使得BIK被降解。了解了BIK的泛素化降解機制後，接下來，我們想知道怎樣的生理情況可以調控BIK的ASB11-based Cul5降解機制。我們發現ASB11的mRNA和蛋白皆會在加入促進內質網壓力藥物tunicamycin和thapsigargin時會有上升的情形。結果顯示，在許多不同的細胞株中，內質網壓力都可以透過ASB11的上升來加速降解BIK使得BIK的半衰期縮短。另外我們進一步發現，ASB11的上升源自於內質網壓力所導致的UPR之一─IRE1α/XBP1。活化的IRE1會促使轉錄因子XBP1進入細胞核，進而使下游基因的表現，而我們認為ASB11就是XBP1的轉錄目標基因。從染色體免疫沉澱實驗的結果發現，XBP1的確可以結合到ASB11啟動子上。因此，我們提出一個模式，XBP1會和基礎表現的NF-Y形成複合體，並且藉由結合NF-Y，結合到NF-Y在ASB11啟動子上的結合位，以增加ASB11的轉錄。另外，我們也確認了BIKDD也和BIK走相同的機制，在內質網壓力的情況下，ASB11-based Cul5降解機制會被提升使得BIKDD的穩定性下降。因此，我們假設抑制IRE1α/XBP1可以穩定BIKDD，並且增強殺死腫瘤細胞的效果。結果發現，使用IRE1抑制劑STF-083010結合BIKDD，成功的在數個三陰性乳癌(TNBC)細胞株和裸鼠中都比單獨使用BIKDD有更好的治療效果，成功的應證了我們的假設。總結以上，本篇研究找到了BIK的降解機制，並且發現此機制在內質網壓力下會被提升，並且在細胞試驗和老鼠層次中我們發現，結合抑制劑和BIKDD可以在三陰性乳癌有加乘的治療效果。

BIK (Bcl2-interacting killer) is a pro-apoptotic BH3-only protein of Bcl2 family. BIK interacts with the anti-apoptotic proteins, including Bcl-2, Bcl-xL and Mcl-1 to neutralize their function. BIKDD, a constitutively active mutant of BIK with an enhanced interaction with anti-apoptotic proteins, has been shown to elicit an anti-cancer activity. However, BIKDD is of a short half-life, which limits its tumor-killing effect. Thus, it would be important to unravel the degradation mechanism of BIK and BIKDD. Using dominant-negative mutants and knockdown approaches, we identify a role of Cul5-based ubiquitin ligase in mediating BIK degradation. Among the substrate adaptors of Cul5, ASB11, but not its mutant, promotes BIK ubiquitination. Knockdown of ASB11 in multiple cell lines elevates BIK level, indicating that ASB11-mediated ubiquitination leads to BIK degradation. We further identify the critical role of BIK K115 and K160 resides for its ubiquitination and degradation by ASB11. Next, we explore the physiological conditions that could regulate this BIK ubiquitination pathway. Importantly, ER stress inducers tunicamycin and thapsigargin upregulate ASB11 mRNA and protein. As a consequence, ER stress increases BIK protein turnover via proteasome and decreases BIK steady-state level in multiple cell lines. We further show that the IRE1α/XBP1 axis of ER stress-induced unfolded protein responses is responsible for ASB11 upregulation and that ASB11 is a transcriptional target of XBP1. Our findings support a model that XBP1 can form a complex with the basal transcription factor NF-Y. This complex is recruited to the NF-Y binding site in ASB11 promoter to evaluate ASB11 transcription. Finally, we tried to apply our findings to the gene therapy treatment of triple negative breast cancer (TNBC). Similar to BIK, BIKDD is also subjected to ASB11-dependent ubiquitination and degradation, which are also promoted by ER stress. This raises the possibility for targeting the IRE1α/XBP1 axis to enhance the anti-tumor activity of BIKDD. Indeed, MTT assay show that combination of IRE1 inhibitor, STF-083010 with BIKDD has a better killing effect than BIKDD alone on several TNBC cell lines. Furthermore, the combined treatment strategy also has a better tumor killing effect in the mouse model. Thus, our findings identify a BIK ubiquitination pathway, uncover the promotion effect of ER stress on this pathway, and highlight the potential of targeting this pathway combined with active BIK for anti-cancer therapy.