脂多醣受體次單元在結腸上皮細胞凋亡及腫瘤生成中的拮抗作用：分子機制及治療應用之探討

Abstract

研究背景：結直腸癌之特徵在於腸道上皮細胞無限增殖及細胞抗凋亡。細菌脂多醣受體 (LPS receptors，包括CD14/TLR4/MD2) 的訊息傳遞途徑與有上皮細胞增生凋亡及腫瘤癌化過程有關，但其次單元參與之機制仍不清楚。此外，Eritoran為治療嚴重敗血症之研發新藥，因其結構類似脂多醣lipid A官能基，故可作為TLR4抑制劑；然而，eritoran抗癌之療效仍有待證實。目的：(1) 探討脂多醣受體次單元 (CD14和TLR4) 在上皮細胞凋亡與存活反應及腫瘤生成的個別角色，(2) 闡述eritoran抗癌效應之分子機制。結果：研究結果發現人類正常結腸上皮細胞之脂多醣受體次單元表現為 CD14+TLR4-，而腫瘤組織為CD14+TLR4+。利用氧化偶氮甲烷/硫酸葡聚醣鈉 (AOM/DSS) 誘發發炎性結直腸癌模式發現，TLR4 突變小鼠相較於野生型小鼠 (CD14+TLR4+) 在腫瘤形成早期階段有上皮細胞凋亡增加，且後期階段有腫瘤生成降低之現象。此兩種小鼠品系在結直腸癌模式中均可見黏膜層脂多醣含量升高。在結腸內給予中和性抗CD14抗體可抑制脂多醣誘導上皮細胞凋亡。此外，利用小鼠結腸腺窩培養之初代結腸類器官 (organoid) 模式發現，給予脂多醣刺激TLR4 突變小鼠者會引起細胞凋亡，但在野生型小鼠則不會。將野生型小鼠初代結腸類器官之TLR4基因沉默後，脂多醣引起之細胞凋亡量會增加；反之，若將TLR4突變小鼠初代結腸類器官之CD14基因沉默，脂多醣引起之細胞凋亡則會被抑制。體外實驗結果顯示脂多醣刺激會引起人類結直腸癌腺癌細胞株Caco-2 (CD14+TLR4-) 細胞凋亡，但在HT29細胞 (CD14+TLR4+) 則不會；此現象與CD14、磷脂醯膽鹼特異性磷脂酶、鞘磷脂酶或蛋白激酶Cζ (PKCζ)有關。相反地，轉染野生型而非突變型TLR4 (Asp299Gly、Thr399Ile或Pro714His) 可使Caco-2細胞免於凋亡; 反之，TLR4基因沉默後之HT29細胞則無法生存。後續研究發現，經肛門端、經口端及經靜脈給予eritoran可顯著降低發炎性結直腸癌模式小鼠之腫瘤數目、大小及進程。給予eritoran治療可減少腫瘤部位細胞增生及促進細胞凋亡。利用小鼠初代腫瘤細胞培養及人類腺癌細胞株發現，藉由脂多醣/TLR4引起之細胞增生現象可被eritoran及CD14、TLR4基因沉默所抑制。此外，將CD14及PKCζ基因沉默可阻斷由eritoran引起之細胞凋亡，但將TLR4基因沉默則無效。最後，脂多醣及eritoran刺激可使PKCζ在Thr410、Thr560及Tyr位置磷酸化，此現象藉由CD14介導而與TLR4無關。給予Src激酶及PKCζ偽受質可阻止PKCζ活化引起之細胞凋亡。總體而言，eritoran治療可藉由刺激CD14/Src/PKCζ誘導細胞凋亡及阻斷TLR4依賴性細胞增生，來達到抑制結腸癌的目的。結論：本研究指出CD14/TLR4拮抗失衡會導致正常上皮細胞癌化形成腫瘤，eritoran藉由調控CD14/TLR4拮抗的偏倚作用，因此可作為治療結直腸癌之嶄新策略。

Background: Colorectal carcinoma (CRC) is characterized by unlimited proliferation and suppression of apoptosis. Signaling of lipopolysaccharide (LPS) receptors (CD14/TLR4/MD2) is involved in both epithelial homeostasis and tumorigenesis, but the mechanisms remain poorly understood. Furthermore, eritoran is an investigational drug for treatment of severe sepsis as a TLR4 inhibitor based on its structural similarity to lipid A moiety. Potential therapeutic use of eritoran in cancer reduction has yet to be determined. Aims: (1) To characterize individual roles of LPS receptor subunits (CD14 and TLR4) in epithelial death versus survival responses, and tumor development; (2) To clarify the underlying molecular mechanisms for the anticancer effect of eritoran. Results: Our study showed that normal human colonocytes were CD14+TLR4−, whereas cancerous tissues were CD14+TLR4+, by immunofluorescent staining. Using a chemical-induced CRC model, increased epithelial apoptosis and decreased tumor multiplicity and sizes were observed in TLR4-mutant mice compared with wild-type (WT) mice with CD14+TLR4+ colonocytes. Mucosa-associated LPS content was elevated in response to CRC induction. Epithelial apoptosis induced by LPS hypersensitivity in TLR4-mutant mice was prevented by intracolonic administration of neutralizing anti-CD14. Moreover, LPS-induced apoptosis was observed in primary colonic organoid cultures derived from TLR4 mutant but not WT murine crypts. Gene silencing of TLR4 increased cell apoptosis in WT organoids, whereas knockdown of CD14 ablated cell death in TLR4-mutant organoids. In vitro studies showed that LPS challenge caused apoptosis in Caco-2 cells (CD14+TLR4−) in a CD14-, phosphatidylcholine-specific phospholipase C-, sphingomyelinase-, and protein kinase C-ζ (PKCζ)-dependent manner. Conversely, expression of functional but not mutant TLR4 (Asp299Gly, Thr399Ile, and Pro714His) rescued cells from LPS/CD14-induced apoptosis. Further studies demonstrated that eritoran administration via intracolonic, intragastric, or intravenous routes significantly reduced tumor burden and stage in mouse CRC models. Decreased proliferation and increased apoptosis were observed in mouse tumor cells after eritoran treatment. In vitro cultures of mouse primary tumor spheroids and human cancer cell lines displayed augmented cell proliferation and cell cycle progression following LPS challenge, which was inhibited by eritoran and by knockdown of CD14 or TLR4. In contrast, cancer cell apoptosis induced by eritoran was ablated by gene silencing of CD14 or PKCζ, but not TLR4. Finally, LPS and eritoran caused hyperphosphorylation of PKCζ at Thr410, Thr560 and tyrosine sites in a CD14-dependent and TLR4-independent manner. Blockade of PKCζ activation by inhibitors to Src kinase prevented the eritoran-induced apoptosis. In summary, eritoran treatment suppressed colon cancer growth by dual mechanisms, including induction of CD14/Src/PKCζ-mediated cell apoptosis and blockade of TLR4-dependent cell proliferation. Conclusion: Our findings indicate that dysfunction in the CD14/TLR4 antagonism may contribute to normal epithelial transition to carcinogenesis. Eritoran treatment via diversion of functional antagonism of LPS receptors may serve as a novel strategy for intervention against colorectal cancer.