PO.MCB03.02 · 分子与细胞生物学
自分泌淋巴毒素信号通过CD74促进卵巢肿瘤生长
Autocrine lymphotoxin signalling promotes ovarian tumor growth through CD74
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：卵巢癌是全球女性中第八大常见的癌症和癌症死亡原因。晚期卵巢癌的5年总生存率为10-40%。晚期卵巢癌迫切需要新的治疗方法。识别促进肿瘤生长的信号通路对于开发靶向治疗至关重要。淋巴毒素在卵巢癌细胞中过表达，并介导卵巢肿瘤中的肿瘤-基质相互作用。然而，淋巴毒素的转录调控以及自分泌淋巴毒素信号在卵巢癌细胞中的作用仍不清楚。
方法：为研究淋巴毒素的转录调控，在人卵巢癌细胞株中进行了相关性分析和药理学抑制。为研究自分泌淋巴毒素信号，通过CRISPR-cas9敲除在人卵巢癌细胞中编辑淋巴毒素-beta（LTB）。相反地，通过稳定转染在小鼠卵巢癌细胞中过表达淋巴毒素基因（Lta和Ltb）。通过肿瘤球体外模型和临床前转移性肿瘤体内模型检测肿瘤生长。通过药理学抑制研究淋巴毒素-beta受体（LTBR）和非经典NF-κB通路（RelB和p52）的参与情况。为识别NF-κB靶基因，通过NanoString nCounter泛癌通路面板分析差异表达基因。通过相关性分析研究卵巢肿瘤样本（TCGA和CUHK）中淋巴毒素、NF-κB和CD74之间的关系。通过CRISPR-cas9敲除在人卵巢癌细胞中研究CD74的功能。
结果：在人卵巢癌细胞株中发现IL33与LTB表达之间，以及ST2与LTA表达之间存在正相关。抑制IL33和ST2可下调人卵巢癌细胞中LTA的转录。人卵巢癌细胞中LTB敲除诱导细胞周期G2/M阻滞并在体外减少肿瘤生长，而小鼠卵巢癌细胞中淋巴毒素过表达则在体外和体内增加肿瘤生长。抑制LTBR和p52可在体外和体内减少淋巴毒素介导的卵巢肿瘤生长。淋巴毒素过表达和LTBR激活后，小鼠卵巢癌细胞中Cd74上调。在人卵巢癌细胞中，CD74转录受淋巴毒素-LTBR-非经典NF-κB通路调控。在卵巢肿瘤样本中发现淋巴毒素、NFKB2、RELB和CD74 mRNA表达之间存在正相关。人卵巢癌细胞中CD74敲除诱导G2/M细胞周期阻滞并在体外减少肿瘤生长。
结论：我们的结果表明，IL33-ST2通路正向调控卵巢癌中的淋巴毒素表达，而自分泌淋巴毒素信号通过CD74促进卵巢肿瘤生长。这些结果为开发晚期卵巢癌的靶向治疗奠定了基础。
查看英文原文 English abstract
Background: Ovarian cancer is the eighth most common cause of cancer and cancer death in women worldwide. The 5-year overall survival rate for advanced-stage ovarian cancer is 10-40%. A new therapy for advanced ovarian cancer is urgently needed. Identifying signalling pathways which promote tumor growth is crucial to developing targeted therapy. Lymphotoxin is overexpressed in ovarian cancer cells and mediates tumor-stromal interaction in ovarian tumor. However, the transcription regulation of lymphotoxin and the role of autocrine lymphotoxin signalling in ovarian cancer cells are still unclear.
Methods: To investigate transcriptional regulation of lymphotoxin, correlation analysis and pharmacologic inhibitions was performed in human ovarian cancer cell lines. To investigate autocrine lymphotoxin signalling, lymphotoxin-beta (LTB) was edited in human ovarian cancer cells by CRISPR-cas9 knockout. On the contrary, lymphotoxin genes (Lta and Ltb) were overexpressed in mouse ovarian cancer cells by stable transfection. Tumor growth was examined by cancer spheroid in vitro model and preclinical metastatic tumor in vivo model. Involvement of lymphotoxin-beta receptor (LTBR) and noncanonical NF-κB pathway (RelB and p52) were investigated by pharmacological inhibitions. To identify NF-κB target genes, differentially expressed genes were analysed by NanoString nCounter pan-cancer pathways panel. Relationships between lymphotoxin, NF-κB and CD74 in ovarian tumor samples (TCGA and CUHK) were performed by correlation analysis. Function of CD74 was investigated by CRISPR-cas9 knockout in human ovarian cancer cells.
Results: Positive correlations between IL33 and LTB expression, and between ST2 and LTA expression, are found in human ovarian cancer cell lines. Inhibition of IL33 and ST2 downregulates LTA transcription in human ovarian cancer cells. LTB knockout in human ovarian cancer cells induces cell cycle G2/M arrest and reduces tumor growth in vitro, whereas lymphotoxin overexpression in mouse ovarian cancer cells increases tumor growth in vitro and in vivo. Inhibition of LTBR and p52 reduces lymphotoxin-mediated ovarian tumor growth in vitro and in vivo. Cd74 is upregulated in mouse ovarian cancer cells after lymphotoxin overexpression and LTBR activation. In human ovarian cancer cells, CD74 transcription is regulated by lymphotoxin-LTBR-noncanonical-NF-κB pathway. Positive correlations between lymphotoxin, NFKB2, RELB and CD74 mRNA expressions are found in ovarian tumor samples. CD74 knockout in human ovarian cancer cells induces G2/M cell cycle arrest and reduces tumor growth in vitro.
Conclusion: Our results demonstrated that IL33-ST2 pathway positively regulates lymphotoxin expression in ovarian cancer, and autocrine lymphotoxin signalling promotes ovarian tumor growth through CD74. These results are a foundation for developing targeted therapy for advanced ovarian cancer.
利益披露 Disclosure
T. Lau, None..
Y. Wong, None..
M. Trybull, None..
K. Chan, None..
H. J. Lee, None..
C. Wang, None..
J. Kwong, None.