肝細胞癌 (Hepatocellular carcinoma, HCC) 是目前最常見的原發性肝癌類型，並且其死亡率佔據全球癌症相關死亡原因的第三名。蕾莎瓦 (Sorafenib) 是一種酪胺酸激酶抑制劑，可以抑制腫瘤的生長及血管新生，並提高晚期HCC患者的存活率。然而，逐漸出現的抗藥性問題使蕾莎瓦的治療效果遇到瓶頸。不幸的是，目前仍未有生物標誌可以有效地預測蕾莎瓦的療效，因此，解決此問題並闡明HCC對於蕾莎瓦抗藥性的相關分子機制便成為本篇研究的重點。前梯度蛋白2 (Anterior gradient 2, AGR2)是一種存在於內質網的蛋白，在我們先前的研究中發現，AGR2於HCC細胞中高度表現並促使細胞對於蕾莎瓦具有抗藥性。同時在過去的文獻中曾指出，蕾莎瓦會誘導細胞走向鐵依賴性細胞死亡。因此，我們假設AGR2透過影響HCC細胞中的鐵依賴性死亡途徑以調控對於蕾莎瓦的抗藥性。在本篇研究中，我們發現蕾莎瓦所造成的鐵離子性死亡在抗藥性細胞株中較低，並且AGR2可以降低藥物處理後的細胞死亡及脂質過氧化並有效保護粒線體的膜電位。從免疫螢光染色及細胞質/粒線體蛋白分離的結果中看到，部分的AGR2會移動到粒線體內。我們也發現AGR2與脂肪酸結合蛋白1 (Fatty acid binding protein 1, FABP1)共同表現，並增加下游油滴的生成。在動物實驗中，AGR2會增加腫瘤的生長及轉移，這樣的現象與抗藥性細胞相似。在臨床HCC患者的檢體中，透過免疫化學染色發現AGR2與鐵依賴性死亡的代表分子4-HNE呈現負相關，這樣的結果與我們所做的細胞及動物實驗結果一致。本篇研究的結果對於AGR2的作用機制有更一步認識，未來可以做為HCC預測、早期診斷及評估預後效果的新型治療標靶。

Hepatocellular carcinoma (HCC) stands as the most prevalent form of primary liver cancer and ranks the third leading cause of cancer-related death worldwide. Sorafenib, a first-line multi-targeted tyrosine kinase inhibitor, inhibits tumor growth and angiogenesis to extend the survival rate of advanced HCC patients. However, the therapeutic efficacy of sorafenib is unsatisfactory due to acquired resistance. Unfortunately, there are no useful biomarkers to predict the efficacy of sorafenib- targeted therapy for advanced HCC patients. Therefore, there is urgent need to identify a useful biomarker for predicting the efficacy of sorafenib-targeted therapy and to elucidate the associated molecular mechanism involved in HCC resistance to sorafenib. Previously, we have demonstrated that anterior gradient 2 (AGR2), an endoplasmic reticulum (ER)-resident protein, is highly expressed in HCC and induces HCC resistance to sorafenib. Sorafenib can induce HCC ferroptosis. Hence, our study sought to explore whether AGR2 plays a role in regulating ferroptosis to induce HCC resistance to sorafenib. Our investigations revealed that sorafenib decreased the levels of ferroptosis-related markers in sorafenib-resistant (SR) cells. AGR2 can reverse the sorafenib-induced cell death and lipid peroxidation, and effectively maintain the mitochondrial membrane potential. The immunofluorescence staining and cytosolic/mitochondrial protein fraction further indicated that AGR2 can partially translocate into mitochondria. And we also found that AGR2 colocalized with fatty acid binding protein 1 (FABP1), which might increase lipid droplet formation. In vivo animal models corroborated that AGR2 augmented tumor growth, and the phenomenon was similar to SR cell-injected conditions compared with parental cells. Analysis of HCC specimens unveiled that AGR2 negative correlated with ferroptosis marker 4-HNE, consistent with our in vitro and in vivo findings. The findings could provide us an understanding the effects and mechanism of AGR2, which can be applied to develop predictive, early diagnostic and prognostic factors for establishing novel therapeutic targets specifically for the treatment of HCC.

1. de Lope, C.R., et al., Management of HCC. Journal of hepatology, 2012. 56: p. S75-S87.
2. Ferenci, P., et al., Hepatocellular carcinoma (HCC): a global perspective. Journal of clinical gastroenterology, 2010. 44(4): p. 239-245.
3. Bosch, F.X., et al., Primary liver cancer: worldwide incidence and trends. Gastroenterology, 2004. 127(5): p. S5-S16.
4. Chakraborty, E. and D. Sarkar, Emerging therapies for hepatocellular carcinoma (HCC). Cancers, 2022. 14(11): p. 2798.
5. Daher, S., et al., Current and future treatment of hepatocellular carcinoma: an updated comprehensive review. Journal of clinical and translational hepatology, 2018. 6(1): p. 69.
6. Zhu, Y.-J., et al., New knowledge of the mechanisms of sorafenib resistance in liver cancer. Acta Pharmacologica Sinica, 2017. 38(5): p. 614-622.
7. Dixon, S.J., et al., Ferroptosis: an iron-dependent form of nonapoptotic cell death. cell, 2012. 149(5): p. 1060-1072.
8. Jiang, X., B.R. Stockwell, and M. Conrad, Ferroptosis: mechanisms, biology and role in disease. Nature reviews Molecular cell biology, 2021. 22(4): p. 266-282.
9. Zhang, C., et al., Ferroptosis in cancer therapy: a novel approach to reversing drug resistance. Molecular cancer, 2022. 21(1): p. 47.
10. Xie, Y., et al., Ferroptosis: process and function. Cell Death & Differentiation, 2016. 23(3): p. 369-379.
11. Chen, X., et al., Ferroptosis: machinery and regulation. Autophagy, 2021. 17(9): p. 2054-2081.
12. Yang, W.S., et al., Regulation of ferroptotic cancer cell death by GPX4. Cell, 2014. 156(1): p. 317-331.
13. Huang, W., et al., ABCC5 facilitates the acquired resistance of sorafenib through the inhibition of SLC7A11-induced ferroptosis in hepatocellular carcinoma. Neoplasia, 2021. 23(12): p. 1227-1239.
14. Gao, M., et al., Role of mitochondria in ferroptosis. Molecular cell, 2019. 73(2): p. 354-363. e3.
15. Wang, H., et al., Mitochondria regulation in ferroptosis. European journal of cell biology, 2020. 99(1): p. 151058.
16. Hay, N., Reprogramming glucose metabolism in cancer: can it be exploited for cancer therapy? Nature Reviews Cancer, 2016. 16(10): p. 635-649.
17. Pedersen, P.L., Warburg, me and Hexokinase 2: Multiple discoveries of key molecular events underlying one of cancers’ most common phenotypes, the “Warburg Effect”, ie, elevated glycolysis in the presence of oxygen. Journal of bioenergetics and biomembranes, 2007. 39: p. 211-222.
18. Bonnet, S., et al., A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth. Cancer cell, 2007. 11(1): p. 37-51.
19. Yagoda, N., et al., RAS–RAF–MEK-dependent oxidative cell death involving voltage-dependent anion channels. Nature, 2007. 447(7146): p. 865-869.
20. DeHart, D.N., et al., Opening of voltage dependent anion channels promotes reactive oxygen species generation, mitochondrial dysfunction and cell death in cancer cells. Biochemical pharmacology, 2018. 148: p. 155-162.
21. Wang, X., et al., RSL3 induced autophagic death in glioma cells via causing glycolysis dysfunction. Biochemical and biophysical research communications, 2019. 518(3): p. 590-597.
22. Ždralević, M., et al., Disrupting the ‘Warburg effect’re-routes cancer cells to OXPHOS offering a vulnerability point via ‘ferroptosis’-induced cell death. Advances in biological regulation, 2018. 68: p. 55-63.
23. Espana, A., et al., Myopathy in hereditary leiomyomatosis and renal cell cancer in an extended family. British Journal of Dermatology, 2015. 172(5): p. 1442-1445.
24. Lawen, A. and D.J. Lane, Mammalian iron homeostasis in health and disease: uptake, storage, transport, and molecular mechanisms of action. Antioxidants & redox signaling, 2013. 18(18): p. 2473-2507.
25. Gao, G. and Y.-Z. Chang, Mitochondrial ferritin in the regulation of brain iron homeostasis and neurodegenerative diseases. Frontiers in Pharmacology, 2014. 5: p. 81247.
26. Wang, Y.-Q., et al., The protective role of mitochondrial ferritin on erastin-induced ferroptosis. Frontiers in aging neuroscience, 2016. 8: p. 308.
27. Lee, H., et al., Energy-stress-mediated AMPK activation inhibits ferroptosis. Nature cell biology, 2020. 22(2): p. 225-234.
28. Blomme, A., et al., 2, 4-dienoyl-CoA reductase regulates lipid homeostasis in treatment-resistant prostate cancer. Nature Communications, 2020. 11(1): p. 2508.
29. Petan, T., E. Jarc, and M. Jusović, Lipid droplets in cancer: guardians of fat in a stressful world. Molecules, 2018. 23(8): p. 1941.
30. Munir, R., et al., Lipid metabolism in cancer cells under metabolic stress. British journal of cancer, 2019. 120(12): p. 1090-1098.
31. Bacci, M., et al., Fat and furious: lipid metabolism in antitumoral therapy response and resistance. Trends in cancer, 2021. 7(3): p. 198-213.
32. Shakya, S., et al., Altered lipid metabolism marks glioblastoma stem and non-stem cells in separate tumor niches. Acta neuropathologica communications, 2021. 9(1): p. 101.
33. Bailey, A.P., et al., Antioxidant role for lipid droplets in a stem cell niche of Drosophila. Cell, 2015. 163(2): p. 340-353.
34. Jarc, E., et al., Lipid droplets induced by secreted phospholipase A2 and unsaturated fatty acids protect breast cancer cells from nutrient and lipotoxic stress. Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids, 2018. 1863(3): p. 247-265.
35. Bezawork-Geleta, A., J. Dimou, and M.J. Watt, Lipid droplets and ferroptosis as new players in brain cancer glioblastoma progression and therapeutic resistance. Frontiers in Oncology, 2022. 12: p. 1085034.
36. Bai, Y., et al., Lipid storage and lipophagy regulates ferroptosis. Biochemical and biophysical research communications, 2019. 508(4): p. 997-1003.
37. Brychtova, V., B. Vojtesek, and R. Hrstka, Anterior gradient 2: a novel player in tumor cell biology. Cancer letters, 2011. 304(1): p. 1-7.
38. Galligan, J.J. and D.R. Petersen, The human protein disulfide isomerase gene family. Human genomics, 2012. 6: p. 1-15.
39. Brychtova, V., et al. Mechanisms of anterior gradient-2 regulation and function in cancer. in Seminars in cancer biology. 2015. Elsevier.
40. Obacz, J., et al., The role of AGR2 and AGR3 in cancer: similar but not identical. European journal of cell biology, 2015. 94(3-4): p. 139-147.
41. Tabas, I. and D. Ron, Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress. Nature cell biology, 2011. 13(3): p. 184-190.
42. Park, S.-W., et al., The protein disulfide isomerase AGR2 is essential for production of intestinal mucus. Proceedings of the National Academy of Sciences, 2009. 106(17): p. 6950-6955.
43. Wang, Z., Y. Hao, and A.W. Lowe, The adenocarcinoma-associated antigen, AGR2, promotes tumor growth, cell migration, and cellular transformation. Cancer research, 2008. 68(2): p. 492-497.
44. Yu, H., et al., Proteomic study explores AGR2 as pro-metastatic protein in HCC. Molecular BioSystems, 2012. 8(10): p. 2710-2718.
45. Li, Z., et al., Anterior gradient 2 is a binding stabilizer of hypoxia inducible factor‐1α that enhances CoCl2‐induced doxorubicin resistance in breast cancer cells. Cancer science, 2015. 106(8): p. 1041-1049.
46. Maslon, M.M., et al., A divergent substrate-binding loop within the pro-oncogenic protein anterior gradient-2 forms a docking site for Reptin. Journal of molecular biology, 2010. 404(3): p. 418-438.
47. Fessart, D., et al., Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties. Elife, 2016. 5: p. e13887.
48. Bu, H., et al., The anterior gradient 2 (AGR2) gene is overexpressed in prostate cancer and may be useful as a urine sediment marker for prostate cancer detection. The Prostate, 2011. 71(6): p. 575-587.
49. Edgell, T.A., et al., Increased plasma concentrations of anterior gradient 2 protein are positively associated with ovarian cancer. Clinical Science, 2010. 118(12): p. 717-725.
50. Liu, Z., et al., Hydrogen peroxide mediated mitochondrial UNG1-PRDX3 interaction and UNG1 degradation. Free Radical Biology and Medicine, 2016. 99: p. 54-62.
51. Thompson, D.A. and R.J. Weigel, hag-2, the human homologue of thexenopus laeviscement gland gene xag-2, is coexpressed with estrogen receptor in breast cancer cell lines. Biochemical and biophysical research communications, 1998. 251(1): p. 111-116.
52. Chevet, E., et al., Emerging roles for the pro-oncogenic anterior gradient-2 in cancer development. Oncogene, 2013. 32(20): p. 2499-2509.
53. Jach, D., et al., From development to cancer-an ever-increasing role of AGR2. American journal of cancer research, 2021. 11(11): p. 5249.
54. Hrstka, R., et al., AGR2 oncoprotein inhibits p38 MAPK and p53 activation through a DUSP10-mediated regulatory pathway. Molecular oncology, 2016. 10(5): p. 652-662.
55. Jung, S.Y., et al., Basic helix-loop-helix transcription factor Twist1 is a novel regulator of anterior gradient protein 2 homolog (AGR2) in breast cancer. Biochemical and biophysical research communications, 2019. 516(1): p. 149-156.
56. Fessart, D., et al., Extracellular AGR2 triggers lung tumour cell proliferation through repression of p21CIP1. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 2021. 1868(3): p. 118920.
57. Milewski, D., et al., FOXM1 activates AGR2 and causes progression of lung adenomas into invasive mucinous adenocarcinomas. PLoS genetics, 2017. 13(12): p. e1007097.
58. Lamichane, B.D., et al., AGR2 is a target of canonical Wnt/β-catenin signaling and is important for stemness maintenance in colorectal cancer stem cells. Biochemical and biophysical research communications, 2019. 515(4): p. 600-606.
59. Tian, S., et al., Secreted AGR2 promotes invasion of colorectal cancer cells via Wnt11-mediated non-canonical Wnt signaling. Experimental cell research, 2018. 364(2): p. 198-207.
60. Gong, W., et al., AGR2-induced glucose metabolism facilitated the progression of endometrial carcinoma via enhancing the MUC1/HIF-1α pathway. Human Cell, 2020. 33: p. 790-800.
61. Yuan, Z., et al., FOXA1 promotes cell proliferation and suppresses apoptosis in HCC by directly regulating miR-212-3p/FOXA1/AGR2 signaling pathway. OncoTargets and therapy, 2020: p. 5231-5240.
62. Liu, Q.-G., Y.-J. Li, and L. Yao, Knockdown of AGR2 induces cell apoptosis and reduces chemotherapy resistance of pancreatic cancer cells with the involvement of ERK/AKT axis. Pancreatology, 2018. 18(6): p. 678-688.
63. THI-THU-TRANG, L., et al., Overexpression of AGR2 Is associated with drug resistance in mutant non-small cell lung cancers. Anticancer Research, 2020. 40(4): p. 1855-1866.
64. Makawita, S., et al., Integrated proteomic profiling of cell line conditioned media and pancreatic juice for the identification of pancreatic cancer biomarkers. Molecular & cellular proteomics, 2011. 10(10).
65. Tohti, M., et al., Serum AGR2 as a useful biomarker for pituitary adenomas. Clinical neurology and neurosurgery, 2017. 154: p. 19-22.
66. Hrstka, R., et al., The pro-metastatic protein anterior gradient-2 predicts poor prognosis in tamoxifen-treated breast cancers. Oncogene, 2010. 29(34): p. 4838-4847.
67. Alavi, M., et al., High expression of AGR2 in lung cancer is predictive of poor survival. BMC cancer, 2015. 15: p. 1-9.
68. Chung, K., et al., Serum AGR2 as an early diagnostic and postoperative prognostic biomarker of human lung adenocarcinoma. Cancer Biomarkers, 2012. 10(2): p. 101-107.
69. Tsai, H.-W., et al., Anterior gradient 2 induces resistance to sorafenib via endoplasmic reticulum stress regulation in hepatocellular carcinoma. Cancer Cell International, 2023. 23(1): p. 42.
70. Sui, X., et al., RSL3 drives ferroptosis through GPX4 inactivation and ROS production in colorectal cancer. Frontiers in pharmacology, 2018. 9: p. 1371.
71. Chen, X., et al., Characteristics and biomarkers of ferroptosis. Frontiers in cell and developmental biology, 2021. 9: p. 637162.
72. Li, Y., et al., Targeting fatty acid synthase modulates sensitivity of hepatocellular carcinoma to sorafenib via ferroptosis. Journal of Experimental & Clinical Cancer Research, 2023. 42(1): p. 6.
73. Wang, Y., et al., Anterior gradient 2 increases long-chain fatty acid uptake via stabilizing FABP1 and facilitates lipid accumulation. International Journal of Biological Sciences, 2021. 17(3): p. 834.
74. Furuhashi, M. and G.S. Hotamisligil, Fatty acid-binding proteins: role in metabolic diseases and potential as drug targets. Nature reviews Drug discovery, 2008. 7(6): p. 489-503.
75. Wang, G., et al., Recent insights into the biological functions of liver fatty acid binding protein 1. Journal of lipid research, 2015. 56(12): p. 2238-2247.
76. Hotamisligil, G.S. and D.A. Bernlohr, Metabolic functions of FABPs—mechanisms and therapeutic implications. Nature Reviews Endocrinology, 2015. 11(10): p. 592-605.