Autophagy Regulators p62/SQSTM1 and NBR1 as new Predictive Biomarkers of Immunotherapy Response in Hepatocellular Carcinoma
DOI:
https://doi.org/10.56919/usci.2543.027Keywords:
Hepatocellular carcinoma (HCC),, New biomarkers for HCC, SQSTM1 (Sequestosome 1), p62 protei, Autophagy, Mallory–Denk bodiesAbstract
Hepatocellular carcinoma (HCC), among the top causes of cancer-associated mortality globally, lacks effective treatment, especially at an advanced stage. While immunotherapy has great promise as an antitumour treatment method, there exist no robust biomarkers for patient response as yet. To evaluate p62 and NBR1 as predictive biomarkers for immunotherapy response. A new biomarker has recently been identified that may be used to predict the efficacy of HCC immunotherapy. Approximately ten articles from Google Scholar and EBSCOhost, selected through a search based on recent mechanistic and preclinical studies, were used for this research. The p62 biomarker enhances STING activation in hepatic stellate cells, and NBR1 (neighbour of BRCA1 gene 1) is an additional key autophagy receptor that plays a comparable role to p62 in sequestering protein aggregates. Early studies suggest that its expression or activation may be used to identify patients who are likely to benefit the most from immune checkpoint inhibitors, thereby producing more effective and personalised treatment approaches. These findings support p62 and NBR1 as promising predictive biomarkers of HCC pending clinical validation.
References
Akinyemiju, T., Abera, S., Ahmed, M., Alam, N., Alemayohu, M. A., & Allen, C. (2017). The burden of primary liver cancer and underlying etiologies from 1990 to 2015 at the global, regional, and national level: Results from the Global Burden of Disease Study 2015. JAMA Oncology, 3(12), 1683–1691. DOI: https://doi.org/10.1001/jamaoncol.2017.3055
Altekruse, S. F., & McGlynn, K. A. (2023). Hepatocellular carcinoma epidemiology. Hepatology, 77(1), 93–111.
Asrani, S. K., Kamath, P. S., & Kim, W. R. (2023). Chronic liver disease and hepatocellular carcinoma. Clinical Gastroenterology and Hepatology, 21(4), 1010–1022.
Bruix, J., Sherman, M., Llovet, J. M., Beaugrand, M., Lencioni, R., Burroughs, A. K., & Christensen, E. (2019). Clinical management of hepatocellular carcinoma. Conclusions of the Barcelona-2000 EASL conference. Journal of Hepatology, 35(3), 421–430. DOI: https://doi.org/10.1016/S0168-8278(01)00130-1
Chen, D. P., Ning, W. R., Jiang, Z. Z., Peng, Z. P., Zhu, L. Y., Zhuang, S. M., Wu, Y., Chen, H., Wei, L. Y., Fang, J. Y., Huang, X. H., & Dong, Z. R. (2019). Glycolytic activation of peritumoral monocytes fosters immune privilege via the PFKFB3-PD-L1 axis in human hepatocellular carcinoma. Journal of Hepatology, 71(2), 333–343. DOI: https://doi.org/10.1016/j.jhep.2019.04.007
Chen, D. S., & Mellman, I. (2013). Oncology meets immunology: The cancer-immunity cycle. Immunity, 39(1), 1–10. DOI: https://doi.org/10.1016/j.immuni.2013.07.012
Chen, L.-T., Martinelli, E., Cheng, A.-L., Pentheroudakis, G., Qin, S., Bhattacharyya, G. S., Srivastava, S., & ESMO Guidelines Committee. (2020). Pan-Asian adapted ESMO clinical practice guidelines for the management of patients with intermediate and advanced/relapsed hepatocellular carcinoma: A TOS-ESMO initiative. Annals of Oncology, 31(3), 334–351. DOI: https://doi.org/10.1016/j.annonc.2019.12.001
Chen, Z., Zhang, X., Zheng, Y., Tao, H., Zhang, W., Ma, L., Liu, H., Wang, G., Qian, G., Cui, X., Huang, L., Huang, W., Wu, B., & Hu, Y. (2021). Response efficacy of PD 1 and PD L1 inhibitors in clinical trials: A systematic review and meta-analysis. Frontiers in Oncology, 11, Article 562315. DOI: https://doi.org/10.3389/fonc.2021.562315
Cheng, Z., Yang, P., Wang, R., Guo, Y., & Zhang, L. (2020). PD-L1 expression and CD8+ T cell infiltration predict a favorable prognosis in advanced gastric cancer. Journal of Immunology Research, 2020, Article 3201908.
Cho, E. J., Leem, S., Kim, S. A., Yang, J., Lee, Y. B., Kim, S. S., Cheong, J. Y., Cho, S. W., Kim, J. W., & Kim, S. (2019). Circulating microbiota-based metagenomic signature for detection of hepatocellular carcinoma. Scientific Reports, 9(1), 44012. DOI: https://doi.org/10.1038/s41598-019-44012-w
Chong, T. T., Natalie, J., Hao-Chun, C., & Victor, C. Y. (2021). p62/SQSTM1 in liver diseases: The usual suspect with multifarious identities. FEBS Letters, 595(13), 1751–1765.
Doroshow, D. B., Bharlia, S., & Beasley, M. B. (2021). PD-L1 as a biomarker of response to immune checkpoint inhibitors. Nature Reviews Clinical Oncology, 18(6), 345–362. DOI: https://doi.org/10.1038/s41571-021-00473-5
Ducreux, M., Abou-Alfa, G., Ren, Z., Edeline, J., Li, Z., Assenat, E., Rimassa, L., Blanc, J., Ross, P., Fang, W., & Hu, S. O. (2023). Management of hepatocellular carcinoma: Current status and future directions. World Journal of Gastroenterology, 29(6), 1054–1075. DOI: https://doi.org/10.3748/wjg.v29.i6.1054
Duran, A., Hernandez, E. D., Reina-Campos, M., Castilla, E. A., Subramaniam, S., Raghunandan, S., Roberts, L. R., Kisseleva, T., Karin, M., Diaz-Meco, M. T., & Moscat, J. (2016). p62/SQSTM1 by binding to vitamin D receptor inhibits hepatic stellate cell activity, fibrosis, and liver cancer. Cancer Cell, 30(4), 595–609. DOI: https://doi.org/10.1016/j.ccell.2016.09.004
El-Khoueiry, A. B., Sangro, B., Yau, T., Crocenzi, T. S., Kudo, M., Hsu, C., Kim, T.-Y., Choo, S.-P., Trojan, J., Welling, T. H., Meyer, T., Kang, Y.-K., Yeo, W., Chopra, A., Anderson, J., Dela Cruz, C., Lang, L., Neely, J., Tang, H., … Melero, I. (2017). Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040): An open-label, non-comparative, phase 1/2 dose escalation and expansion trial. The Lancet, 389(10088), 2492–2502. DOI: https://doi.org/10.1016/S0140-6736(17)31046-2
El-Serag, H. B. (2023). Hepatocellular carcinoma: Epidemiology and risk factors. Nature Reviews Gastroenterology & Hepatology, 20(11), 667–680.
El-Serag, H. B., Marrero, J. A., Rudolph, L., & Reddy, K. R. (2008). Diagnosis and treatment of hepatocellular carcinoma. Gastroenterology, 134(6), 1752–1763. DOI: https://doi.org/10.1053/j.gastro.2008.02.090
El-Serag, H. B., Zhang, H., Kanwal, F., & Kramer, J. (2023). Diabetes and the risk of hepatocellular carcinoma. American Journal of Gastroenterology, 118(1), 123–132.
European Association for the Study of the Liver. (2018). EASL Clinical Practice Guidelines: Management of hepatocellular carcinoma. Journal of Hepatology, 69(1), 182–236. DOI: https://doi.org/10.1016/j.jhep.2018.03.019
Faruk, M. O., Ichimura, Y., & Komatsu, M. (2021). Selective autophagy. Cancer Science, 112(10), 3972–3978. DOI: https://doi.org/10.1111/cas.15112
Finn, R. S., Qin, S., Ikeda, M., Galle, P. R., Ducreux, M., Kim, T.-Y., Kudo, M., Breder, V., Merle, P., Kaseb, A. O., Li, D., Verret, W., Xu, D.-Z., Hernandez, S., Liu, J., Huang, C., Mulla, S., Wang, Y., Lim, H. Y., … Investigators, I. M. (2020). Atezolizumab plus bevacizumab in unresectable hepatocellular carcinoma. New England Journal of Medicine, 382(20), 1894–1905. DOI: https://doi.org/10.1056/NEJMoa1915745
Gonzalez, S. A. (2014). Novel biomarkers for hepatocellular carcinoma surveillance: Has the future arrived? Hepatobiliary Surgery and Nutrition, 3(6), 410–414.
Heimbach, J. K., Kulik, L. M., Finn, R. S., Sirlin, C. B., Abecassis, M. M., Roberts, L. R., Zhu, A. X., Murad, M. H., & Marrero, J. A. (2018). AASLD guidelines for the treatment of hepatocellular carcinoma. Hepatology, 67(1), 358–380. DOI: https://doi.org/10.1002/hep.29086
Kim, H. D., Song, G. W., Park, S., Jung, M. K., Kim, M. H., Kang, H. J., Yoo, C., Yi, K., Kim, K. H., Eo, S., Moon, D. B., Hong, S. M., Ju, Y. S., Shin, E. C., & Lee, K. H. (2018). Association between expression level of PD1 by tumor-infiltrating CD8(+) T cells and features of hepatocellular carcinoma. Gastroenterology, 155(6), 1936–1950.e17. DOI: https://doi.org/10.1053/j.gastro.2018.08.030
Kirkin, V., McEwan, D. G., Novak, I., & Dikic, I. (2009). A role for ubiquitin in selective autophagy. Molecular Cell, 34(3), 259–269. DOI: https://doi.org/10.1016/j.molcel.2009.04.026
Kisseleva, T., & Brenner, D. (2021). Molecular and cellular mechanisms of liver fibrosis and its regression. Nature Reviews Gastroenterology & Hepatology, 18(3), 151–166. DOI: https://doi.org/10.1038/s41575-020-00372-7
Komatsu, M., & Ichimura, Y. (2010). Physiological significance of selective degradation of p62 by autophagy. FEBS Letters, 584(7), 1374–1378. DOI: https://doi.org/10.1016/j.febslet.2010.02.017
Kurebayashi, Y., Ojima, H., Tsujikawa, H., Kubota, N., Maehara, J., Abe, Y., Kitago, M., Shinoda, M., Kitagawa, Y., & Sakamoto, M. (2018). Landscape of immune microenvironment in hepatocellular carcinoma and its additional impact on histological and molecular classification. Hepatology, 68(3), 1025–1041. DOI: https://doi.org/10.1002/hep.29904
Law, K. B., & Kim, P. K. (2022). Autophagy: Cancer, other pathologies, inflammation, immunity, infection, and aging. In M. A. Hayat (Ed.), Autophagy: Cancer, Other Pathologies, Inflammation, Immunity, Infection, and Aging (pp. 1–15). Academic Press.
Li, Z., Li, H., Zhao, Z. B., Zhu, W., Feng, P. P., Zhu, X. W., Gong, J. P., & Huang, S. (2019). SIRT4 silencing in tumor-associated macrophages promotes HCC development via PPARδ signaling-mediated alternative activation of macrophages. Journal of Experimental & Clinical Cancer Research, 38(1), 469. DOI: https://doi.org/10.1186/s13046-019-1456-9
Lindblad, K. E., Ruiz de Galarreta, M., & Lujambio, A. (2021). Tumor-intrinsic mechanisms regulating immune exclusion in liver cancers. Frontiers in Immunology, 12, 642958. DOI: https://doi.org/10.3389/fimmu.2021.642958
Llovet, J. M., Ricci, S., Mazzaferro, V., Hilgard, P., Gane, E., Blanc, J. F., de Oliveira, A. C., Santoro, A., Raoul, J. L., Forner, A., et al. (2018). Lenvatinib versus sorafenib in first-line treatment of patients with unresectable hepatocellular carcinoma: a randomised phase 3 non-inferiority trial. The Lancet, 391(10126), 1163-1173. DOI: https://doi.org/10.1016/S0140-6736(18)30207-1
Makarova-Rusher, O. V., Medina-Echeverz, J., Duffy, A. G., & Greten, T. F. (2015). The Yin and Yang of evasion and immune activation in HCC. Journal of Hepatology, 62(6), 1420–1429. DOI: https://doi.org/10.1016/j.jhep.2015.02.038
Marchesini, G., Aroldi, F., Valenti, L., & Targher, G. (2023). Metabolic syndrome and the risk of hepatocellular carcinoma. Journal of Hepatology, 49(2), 112–136.
Mendeley Data. (2024). Opposing regulation of the STING pathway in hepatic stellate cells by NBR1 and p62 determines the progression of hepatocellular carcinoma [RNAseq] (Version 1) [Data set]. Mendeley. https://data.mendeley.com/datasets/53j3vrhddj/1
Moscat, J., & Diaz-Meco, M. T. (2009). p62 at the crossroads of autophagy, apoptosis, and cancer. Cell, 137(6), 1001–1004. DOI: https://doi.org/10.1016/j.cell.2009.05.023
Nishimura, S., Linares Rodriguez, J. F., L'Hermitte, A., Duran Molina, M. A., Diaz, T., Ordonez, A., Ruiz Martinez, M., Kudo, Y., Marzio, A., Heikenwalder, M., Roberts, L. R., Diaz-Meco, M. T., & Moscat, J. (2024). Opposing regulation of the STING pathway in hepatic stellate cells by NBR1 and p62 determines the progression of hepatocellular carcinoma. Molecular Cell, 84(23), 4660-4676.e10. DOI: https://doi.org/10.1016/j.molcel.2024.09.026
OmicsDI. (2024). GSE254276 - Opposing regulation of the STING pathway in hepatic stellate cells by NBR1 and p62 determines the progression of hepatocellular carcinoma [RNAseq] [Data set]. OmicsDI. https://www.omicsdi.org/dataset/geo/GSE254276
Papaioannou, N. E., Beniata, O. V., Vitsos, P., Tsitsilonis, O., & Samara, P. (2016). Harnessing the immune system to improve cancer therapy. Annals of Translational Medicine, 4(14), 261. DOI: https://doi.org/10.21037/atm.2016.04.01
Patel, S. P., & Kurzrock, R. (2015). PD-L1 expression as a predictive biomarker in cancer immunotherapy. Molecular Cancer Therapeutics, 14(4), 847–856. DOI: https://doi.org/10.1158/1535-7163.MCT-14-0983
Pfister, D., Núñez, N. G., Pinyol, R., Govaere, O., Pinter, M., Szydlowska, M., Gupta, R., Qiu, M., Deczkowska, A., Weiner, A., Müller, F., Sinha, A., Friebel, E., Engleitner, T., Lenggenhager, D., Stirm, K., Kosla, J., Kotsiliti, E., Leone, V., … Brown, M. (2021). NASH limits anti-tumor surveillance in immunotherapy-treated HCC. Nature, 592(7854), 450–456. DOI: https://doi.org/10.1038/s41586-021-03362-0
Piñero, F., Vazquez, M., Baré, P., Rohr, C., Mendizabal, M., Sciara, M., Alonso, C., Fay, F., & Silva, M. (2019). A changing etiologic scenario in hepatocellular carcinoma: A South American experience. Annals of Hepatology, 18(3), 480–487. DOI: https://doi.org/10.1016/j.aohep.2018.10.003
Ponziani, F. R., Nicoletti, A., Gasbarrini, A., & Pompili, M. (2019). Diagnostic and therapeutic potential of the gut microbiota in patients with early hepatocellular carcinoma. Therapeutic Advances in Medical Oncology, 11, 1758835919848184. DOI: https://doi.org/10.1177/1758835919848184
Rehm, J., & Shield, K. D. (2023). Alcohol consumption and liver cancer. The Lancet Gastroenterology & Hepatology, 8(4), 297–298.
Ren, Z., Li, A., Jiang, J., Zhou, L., Yu, Z., Lu, H., Xie, H., Chen, X., Shao, L., Zhang, R., Xu, S., Zhang, H., Cui, G., Chen, Y., & Gao, Y. (2019). Gut microbiome analysis as a tool towards targeted non-invasive biomarkers for early hepatocellular carcinoma. Gut, 68(6), 1014–1023. DOI: https://doi.org/10.1136/gutjnl-2017-315084
Sánchez-Martín, P., & Komatsu, M. (2018). p62/SQSTM1 - steering the cell through health and disease. Journal of Cell Science, 131(21), jcs222836. DOI: https://doi.org/10.1242/jcs.222836
Sanchez-Martin, P., Saito, T., & Komatsu, M. (2019). p62/SQSTM1: 'Jack of all trades' in health and cancer. The FEBS Journal, 286(1), 8–23. DOI: https://doi.org/10.1111/febs.14712
Sangro, B., Sarobe, P., Hervás-Stubbs, S., & Melero, I. (2021). Advances in immunotherapy for hepatocellular carcinoma. Nature Reviews Gastroenterology & Hepatology, 18(8), 525–543. DOI: https://doi.org/10.1038/s41575-021-00438-0
Schütte, K., Schulz, C., Link, A., & Malfertheiner, P. (2015). Current biomarkers for hepatocellular carcinoma: Surveillance, diagnosis and prediction of prognosis. World Journal of Hepatology, 7(2), 139–149. DOI: https://doi.org/10.4254/wjh.v7.i2.139
Shimizu, T., Inoue, K.-I., Hachiya, H., Shibuya, N., Aoki, T., & Kubota, K. (2016). Accumulation of phosphorylated p62 is associated with NF-E2-related factor 2 activation in hepatocellular carcinoma. Journal of Hepato-Biliary-Pancreatic Sciences, 23(8), 467–471. DOI: https://doi.org/10.1002/jhbp.364
Sia, D., Jiao, Y., Martinez-Quetglas, I., Kuchuk, O., Villacorta-Martin, C., Castro de Moura, M., Putra, J., Camprecios, G., Bassaganyas, L., Akers, N., Losic, B., Waxman, S., Thung, S. N., Mazzaferro, V., Esteban, R., Cornella, H., Pinyol, R., Tabrizian, P., Schwartz, M., … Llovet, J. M. (2017). Identification of an immune-specific class of hepatocellular carcinoma, based on molecular features. Gastroenterology, 153(3), 812–826. DOI: https://doi.org/10.1053/j.gastro.2017.06.007
Sung, H., Ferlay, J., Siegel, R. L., Laversanne, M., Soerjomataram, I., Jemal, A., & Bray, F. (2021). Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians, 71(3), 209–249. DOI: https://doi.org/10.3322/caac.21660
Tur, M. K., & Barth, S. (2014). Immunotherapy. In M. Schwab (Ed.), Encyclopedia of Cancer (pp. 320–325). Springer. DOI: https://doi.org/10.1007/978-3-642-27841-9_3014-2
Tzartzeva, K., Obi, J., Rich, N. E., Parikh, N. D., Marrero, J. A., Yopp, A., Waljee, A. K., & Singal, A. G. (2018). Surveillance imaging and alpha fetoprotein for early detection of hepatocellular carcinoma in patients with cirrhosis: A meta-analysis. Gastroenterology, 154(6), 1706–1718.e1. DOI: https://doi.org/10.1053/j.gastro.2018.01.064
Vogel, A., & Saborowski, A. (2020). Current strategy for the treatment of intermediate and advanced hepatocellular carcinoma. Cancer Treatment Reviews, 82, 101946. DOI: https://doi.org/10.1016/j.ctrv.2019.101946
Wang, W., & Wei, C. (2020). Advances in the early diagnosis of hepatocellular carcinoma. Genes & Diseases, 7(3), 308–319. DOI: https://doi.org/10.1016/j.gendis.2020.01.014
Wang, X., Shen, H., Zhangyuan, G., Huang, R., Zhang, W., He, Q., Jin, K., Zhuo, H., Zhang, Z., Wang, J., Sun, B., & Lu, X. (2018). 14-3-3ζ delivered by hepatocellular carcinoma-derived exosomes impaired anti-tumor function of tumor-infiltrating T lymphocytes. Cell Death & Disease, 9(2), 159. DOI: https://doi.org/10.1038/s41419-017-0180-7
Wu, F., & Guo, X. (2023). Aflatoxin and liver cancer: A review. Environmental Toxicology, 38(2), 123–134.
Yang, L., Shao, X., Jia, S., Zhang, Q., & Jin, Z. (2019). Interleukin-35 dampens CD8+ T cells activity in patients with non-viral hepatitis-related hepatocellular carcinoma. Frontiers in Immunology, 10, 1032. DOI: https://doi.org/10.3389/fimmu.2019.01032
Yikudo, T., Sato, T., Matsumoto, Y., & Hirano, K. (2020). PKC loss induces autophagy, oxidative phosphorylation, and NRF2 to promote liver cancer progression. Cancer Cell, 37(2), 143–144. DOI: https://doi.org/10.1016/j.ccell.2020.05.018
Younossi, Z. M., Stepanova, M., Avila, J. E., Younossi, I., Henry, L., & Racila, A. (2023). Global epidemiology of chronic liver diseases. Journal of Hepatology, 78(2), 254–267.
Zhong, C., Li, Y., Yang, J., Jin, S., Chen, G., & Li, D. (2021). Immunotherapy for hepatocellular carcinoma: Current limits and prospects. Frontiers in Oncology, 11, 589680. DOI: https://doi.org/10.3389/fonc.2021.589680
Zhou, D., Luan, J., Huang, C., & Li, J. (2021). Tumor-associated macrophages in hepatocellular carcinoma: Friend or foe? Gut and Liver, 15(4), 500–516. DOI: https://doi.org/10.5009/gnl20223
Zhu, A. X., Finn, R. S., Edeline, J., Cattan, S., Ogasawara, S., Palmer, D., Verslype, C., Zagonel, V., Fartoux, L., Vogel, A., Sarker, D., Verset, G., Chan, S. L., Knox, J., Daniele, B., Webber, A. L., Ebbinghaus, S. W., Ma, J., Siegel, A. B., … KEYNOTE-224 investigators. (2018). Pembrolizumab in patients with advanced hepatocellular carcinoma previously treated with sorafenib (KEYNOTE-224): A non-randomised, open-label phase 2 trial. The Lancet Oncology, 19(7), 940–952. DOI: https://doi.org/10.1016/S1470-2045(18)30351-6
Zongyi, Y., & Xiaowu, L. (2020). Immunotherapy for hepatocellular carcinoma. Cancer Letters, 470, 8–17. DOI: https://doi.org/10.1016/j.canlet.2019.12.002
Zucman-Rossi, J., Villanueva, A., Nault, J.-C., & Llovet, J. M. (2023). Genetic alterations and hepatocellular carcinoma. Nature Reviews Clinical Oncology, 20(1), 12–27.
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