Abstract 5330: Targeting hepatocellular carcinoma through TGF-β pathway E3 ligases
Authors: Rao Shuyun , Levin Heather , Chen Jian , Akbani Rehan , White Jon , Jogunoori Wilma , Gu Shoujun , Ohshiro Kazufumi , Zaidi Sobia , Mishra Bibhuti , Rashid Asif , Li Shulin , Mishra Lopa 
Published: July 2017

Abstract
Hepatocellular carcinoma (HCC) is the 3rd leading cause of cancer deaths worldwide, and rising in the United States at an alarming rate. Multiple E3 ubiquitin ligases such as the SMURFs and RINGH2 proteins have been identified as negative regulators of the TGF-β pathway. However, to our knowledge, there remains a gap in the integration between genomics, underlying mechanisms and the development of targeted therapeutics harnessing these TGF-β-associated E3 ligases for HCC. The aim of this study is to elucidate the role of E3 ligases in HCC, through TCGA analyses and provide mechanistic insight into these as therapeutic targets for HCC. We first analyzed the 488 hepatocellular cancers and screened for alterations in The Cancer Genome Atlas (TCGA). Immunohistochemistry (IHC), Q-PCR, Western blot analysis were used to validate the expression levels of two of the most highly altered E3 ligases, PRAJA and Keap1 in hepatocellular cancer tissues and cell lines in human and in TGF-β-deficient β2SP+/- mouse models. Inhibition studies of PRAJA and Keap1 were performed by lentivirus shRNA in HCC cell lines, and xenograft studies. From the TCGA data, we observe two different signatures (activated and inactivated) for 18 TGF-β pathway genes. While increased levels of TGF-β-related transcripts were associated with activation of hepatic fibrosis/immune microenvironment pathways, decreased levels of TGF-β members were associated with loss of TGF-β tumor suppressor function. HCCs characterized by the “inactivated” TGF-β signature were associated with a significantly poorer survival, compared to HCCs with the “activated” TGF-β signature (p=0.0027). We next analyzed 29 TGF-β-related E3 ligases, and observed raised expression of the following: PRAJA1 (12.7% of HCCs), KEAP1 (6.4%), UCHL5 (16.4%), WWP2 (11.8%), WWP1 (10%), Smurf2 (9.1%), Skp2 (9.1%), and Smurf1 (8.2%). Interestingly, expression patterns corresponded with a few TGF-β signaling members regulated by some of these E3 ligases, namely Smad3 (altered in 54%) and β2SP (27%). We identified that PRAJA1 targets Smad3 and β2SP for ubiquitination and degradation. We further observe raised levels of PRAJA (25%) and KEAP1 (70%) in 176 human liver cancers, by IHC, compared to normal controls. Depletion of PRAJA and KEAP1 with either shRNAs or E3 ligase inhibitors, substantially inhibited growth and induced apoptosis through PRAJA/Smad3/β2SP and KEAP1/Nrf signaling in HCC cell lines and xenografts. These results suggest that E3 ligases such as PRAJA1 and KEAP1 may be valuable therapeutic targets for liver cancer in the context of TGF-β signaling, an important approach given that few effective targeted therapeutics are available for this cancer with poor prognosis.
Citation Format: Shuyun Rao, Heather Levin, Jian Chen, Rehan Akbani, Jon White, Wilma Jogunoori, Shoujun Gu, Kazufumi Ohshiro, Sobia Zaidi, Bibhuti Mishra, Asif Rashid, Shulin Li, Lopa Mishra. Targeting hepatocellular carcinoma through TGF-β pathway E3 ligases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5330. doi:10.1158/1538-7445.AM2017-5330