Characterization of Signal Transducer and Activator of Transcription 3 (STAT3) in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a major global health problem and its treatment outcomes are limited by therapeutic resistance, which is largely attributed to cancer stem cells (CSCs). Signal transducer and activator of transcription 3 (STAT3), a transcription factor closely associated with CSCs, has been implicated in tumorigenesis and immune regulation, yet its roles in HCC remain unclear. Natural killer (NK) cells exert cytotoxicity without depending on antigen-specific sensitization and can preferentially target CSCs. Considering the potential for STAT3-targeted therapeutics and NK cell-based immunotherapy, this work aims to investigate the roles of STAT3 in tumorigenesis and NK antitumor activity in HCC.The clinicopathological significance of STAT3 was firstly examined in primary HCC. Immunohistochemistry showed predominant nuclear and cytoplasmic staining of phosphorylated (p)-Y705 and pS727 in hepatocytes respectively, with significantly higher total STAT3 and pS727 expression in tumor than tumor-adjacent normal tissues. Moreover, STAT3 expression in HCC tissues was negatively correlated with NK cell infiltration, implicating the involvement of STAT3 in tumor evasion from NK surveillance. RT-qPCR analysis indicated that elevated STAT3 transcript levels were significantly associated with poor survival and recurrence of HCC patients in Hong Kong cohorts. TCGA HCC datasets showed corroborating results in Asian cohort but not other ethnic groups, suggesting STAT3 may be more important in HCC development among HCC-burdened Asians.The role of STAT3 in HCC was explored using STAT3-specific siRNA and small-molecule STAT3 inhibitor napabucasin. While both reduced viability and induced apoptosis of HCC cells, napabucasin also impaired migration, colony and spheroid formation, and inhibited tumor growth in vivo, accompanied with reduced STAT3 levels, decreased proliferation and enhanced apoptosis. 5-fluorouracil (5-FU) chemoresistant HCC sublines and spheroids showed elevated expression of STAT3 and CSC markers CD133, KLF4 and SOX2. Notably, napabucasin exhibited synergism with 5-FU and targeted chemoresistant HCC cells as effectively as parental counterparts. RNA sequencing results supported the protumorigenic role of STAT3, in which napabucasin altered gene expression in stemness-related Wnt signaling, inflammatory-associated NF-κB signaling, and other growth-regulatory pathways.The role of STAT3 in NK cells in HCC was also investigated using STAT3-specific shRNA and napabucasin in NK92 cell model. While STAT3 knockdown by shRNA did not affect NK cell proliferation, both shRNA silencing and napabucasin treatment of NK and/or HCC cells demonstrated enhanced NK cell-mediated cytotoxicity against HCC cells in co-culture assays. The in vivo antitumor efficacy of NK92 and its combination with napabucasin was manifested as extensive necrosis and apoptosis throughout the tumor mass. RNA sequencing data revealed the regulation of tumor ligands for NK activation including MICA, and the modulation on NK activating receptors and cytotoxic molecules. Detailed mechanistic analysis on the role of STAT3 in tumor and NK cells is warranted.In summary, this study highlighted the clinicopathological significance of STAT3 in Asian HCC, in particular, its role in promoting tumorigenesis with regards to chemoresistance and stemness regulation, as well as suppressing NK cytotoxicity. Importantly, our findings revealed the potential of STAT3-targeted therapeutics napabucasin in combination with chemotherapy and/or NK cell-based immunotherapy to improve treatment outcomes of the highly refractory HCC.肝細胞癌（HCC）是常見的致命腫瘤。其療效通常受抗藥性等問題限制，而癌症幹細胞（CSCs）是主因之一。信號轉導和轉錄激活因子3（STAT3）與CSCs相關，亦在癌變及免疫調控中扮演一定的角色，但它在肝癌的作用仍不清楚。自然殺傷（NK）細胞能靶向CSCs，而且其毒性反應不依賴於特異性抗原的識別。由於STAT3標靶治療和NK細胞療法極具潛力，本研究旨在探討STAT3在肝癌腫瘤形成及NK抗癌反應上的作用。本研究以原發性肝癌樣本探討STAT3在肝癌臨床病理的重要性。免疫組織化學染色法分析可見，肝癌組織的細胞核和質分別呈較顯著的STAT3磷酸（p）-Y705及pS727活化，而腫瘤組織的STAT3表達明顯高於非腫瘤組織。此外，STAT3表達與NK細胞浸潤呈負相關，暗示STAT3牽涉腫瘤的免疫逃逸。即時定量聚合酶連鎖反應結果顯示，在香港隊列中有高量STAT3轉錄表達的肝癌患者存活率明顯較低，復發機會較大。癌症基因組圖譜數據庫（TCGA）中肝癌組織的基因表達數據呼應香港隊列的結果，但僅限於亞洲人而非其他種族，表示STAT3可能對亞裔的肝癌發病更為重要。為探討STAT3在肝癌腫瘤形成的作用，本研究使用了STAT3 siRNA及STAT3抑制劑Napabucasin。兩者都能削減細胞活力及促進細胞凋亡。Napabucasin亦能阻礙細胞遷移﹑細胞群落及團塊的形成，並抑制活體動物的腫瘤生長，降低其STAT3活性﹑細胞增生及誘發細胞凋亡。此外，5-氟尿嘧啶（5-FU）耐藥肝癌細胞株及細胞團塊呈較高STAT3及CSC標誌物CD133﹑KLF4和SOX2的表達。重要地，Napabucasin與5-FU能發揮協同作用，其抑制耐藥細胞的效用亦不亞於親代細胞株。RNA測序顯示Napabucasin改變Wnt信號﹑NF-κB信號及其他與腫瘤生長相關的基因表達，進一步確實STAT3的致癌作用。本研究亦使用了STAT3 shRNA及Napabucasin以探討STAT3在NK細胞中抗肝癌免疫反應的作用。shRNA結果顯示STAT3並不影響NK細胞生長，但兩種STAT3敲降方法都能增強NK對肝癌細胞的毒性反應。在活體動物中，NK92及與Napabucasin的組合治療能促成腫瘤細胞的廣泛壞死及凋亡。RNA測序顯示STAT3可調控腫瘤上激活NK的配體，如MICA，以及NK激活受體和細胞毒性因子等。STAT3對肝癌細胞及NK細胞的調控機制需要進一步研究。總括而言，本研究展示了STAT3在亞裔肝癌病人的臨床病理意義，及其對促進肝癌腫瘤形成和抑制NK抗癌反應的作用。重要地，STAT3抑制劑能靶向肝癌耐藥細胞，與化療藥產生協同作用，並增強NK細胞的抗癌毒性，顯示STAT3是具潛力的肝癌治療靶點。LEE, Carol.Ph.D. Chinese University of Hong Kong 2021.Includes bibliographical references (leaves )Abstracts in English and Chinese.Title from PDF title page (viewed on ...