肝細胞癌（HCC）是肝臟的原發性惡性腫瘤，是具有高死亡率的癌症，同時也發現每年的發病率有不斷攀升的趨勢。目前治療晚期肝癌(HCC)病患的標靶藥物有索拉非尼和瑞戈非尼，這些細胞毒性藥物透過誘導細胞死亡來控制腫瘤生長。然而，越來越多文獻指出當病患接受索拉非尼和瑞戈非尼治療，患者反應率是有限的。因此，迫切需要更好地了解索拉非尼和瑞戈非尼對HCC的低反應率的原因。在我們先前未發表的結果中，我們發現促發炎細胞細胞因子:介白素33 (interleukin-33) 在順鉑處理的肺癌細胞和帶有肺癌腫瘤(Lewis lung cancer, LLC)的小鼠中都有增加的情形。此外，也發現當阻斷IL-33 及其受體 ST2L訊息傳遞,能夠增強順鉑對帶有LLC腫瘤小鼠的抗腫瘤功效。基於上述結果，本研究中我們將會探討索拉非尼和瑞戈非尼是否會誘導 HCC 細胞中IL-33表現量增加，從而降低治療效果。在這裡，我們發現索拉非尼和瑞戈非尼治療不僅會促進細胞內IL-33的表現量和分泌量，同時也發現HCC 細胞上的PD-L1也有上調的情形。有趣的是，我們發現在皮下和原位 HCC 小鼠模型中，當給予IL-33中和性抗體能夠增加小鼠體內CD4+ T細胞、CD8+ T細胞，以及降低腫瘤內部PD-L1的表現來提升索拉非尼的抗腫瘤功效，這表明IL-33的產生會減弱索拉非尼對小鼠的治療效果。接下來，我們試圖研究索拉非尼和瑞戈非尼在 HCC 細胞中誘導IL-33分泌的機制。IL-33 屬於已知通過自噬分泌的 IL-1 家族。因此，我們進一步探討索拉非尼和瑞戈非尼是否可以觸發 HCC 細胞的自噬作用。我們觀察到索拉非尼和瑞戈非尼能夠誘導HCC細胞產生自噬作用，並且是一個不完全自噬作用，這顯示著索拉非尼和瑞戈非尼治療下很有可能是透過自噬作用將IL-33送出細胞外。另一方面，最近的研究顯示衰老細胞能夠產生促發炎細胞因子。因此，我們進一步探討索拉非尼和瑞戈非尼是否可以觸發 HCC 細胞的細胞衰老。從結果中發現索拉非尼和瑞戈非尼治療下能夠在 Huh7 細胞中誘導細胞生長停滯和衰老相關 β-半乳糖苷酶活性的增加。隨後也發現索拉非尼治療會上調 p16 和 p21，而瑞格非尼治療會上調 p21，這表明這兩種藥物都會誘導HCC細胞的細胞衰老。綜合上述，我們的結果表明 IL-33 有助於降低索拉非尼和瑞戈非尼對 HCC 細胞的治療效果。因此靶向IL-33/ST2L axis同時合併標靶藥物,以提升現有標靶藥物有限療效,可成為治療肝癌的新策略。

Hepatocellular carcinoma (HCC) is one of high mortality cancer with an increasing incidence around the world. For the treatment to advanced stage of HCC, the recommended drugs are sorafenib and regorafenib. These cytotoxic drugs are suggested to induce cell death to control tumor growth; however, the response rate of patients is limited. Thus, there is an urgent need to have a better understanding in low response rate of sorafenib and regorafenib to HCC. In our unpublished data, we found that a pro-inflammatory cytokine, interleukin-33 (IL-33), was increased in cisplatin-treated lung cancer cells as well as lung tumor-bearing mice. Moreover, treatment of neutralizing antibodies against IL-33 or its receptor ST2L was able to enhance anti-tumor efficacy of cisplatin in lung tumor-bearing mice. Based on the above results, we aimed to examine whether sorafenib and regorafenib -induce IL-33 expression in HCC cells and hence reduce the treatment efficacy. Here, we found that both sorafenib and regorafenib treatment not only promoted IL-33 upregulation and secretion, but also increase of PD-L1 on the HCC cells. Notably, we showed that treatment of anti-IL-33 neutralizing antibody was able to upregulate infiltrating CD4+ T cells and CD8+ T cells and decrease the PD-L1 expression in TME to enhance the anti-tumor efficacy of sorafenib in subcutaneously inoculated and orthotopic HCC mouse models. This indicates that IL-33 attenuates sorafenib treatment efficacy in mice. Next, we tried to investigate the mechanism of sorafenib and regorafenib-induced IL-33 in HCC cells. IL-33 belongs to IL-1 family in which IL-1β and IL-18 are known to be secreted through autophagy. Therefore, we investigated whether sorafenib and regorafenib could trigger autophagy in HCC cells. Our data revealed that sorafenib and regorafenib were able to induce autophagy in HCC cells, suggesting sorafenib and regorafenib induce autophagy might participate in IL-33 secretion. On other hand, recent studies have suggested that senescent cells are able to produce inflammatory cytokines. Thus, we checked whether sorafenib and regorafenib trigger cellular senescence in HCC cells. We found that sorafenib and regorafenib were able to induce the cell growth arrest and increase of senescence-associated β-galactosidase activity in Huh7 cells. Subsequently, sorafenib treatment was found to upregulate p16 whereas regorafenib treatment upregulated p21, suggesting that both drugs trigger cellular senescence in HCC cells. Together, our results suggest that IL-33 contributes to the reduced therapeutic efficacy of sorafenib and regorafenib to HCC cells. Our observations indicated that targeting the IL-33/ST2L axis combined with targeted therapy may serve as a novel therapeutic strategy to enhance the limited efficacy of sorafenib or regorafenib.

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