Abstract 5198: Targeting E3 ligase PRAJA1 in hepatocellular cancer .
Authors: Chen Jian , Chen Jiun-Sheng , Shukla Vivek , Yao Zhixing , Piao Hai-Long , Jogunoori Wilma , Mishra Bibhuti , Mishtra Lopa 
Published: August 2014

Background: Hepatocellular Cancer (HCC) is a rising and lethal disease, that is difficult to treat due to late diagnosis and few viable targeted therapeutics. Recent studies support a key role for TGF-β signaling in suppressing these tumors. Expression of the E3 ubiquitin ligase PRAJA, a negative regulator of TGF-β signaling via Smad3 and its adaptor β2-spectrin (β2SP), is dramatically raised in up to 70% of human HCCs. This indicates that PRAJA1 overexpression suppresses Smad3 and β2SP mediated TGF-β tumor suppressor signaling hence promoting HCC progression.

Hypothesis: Disruption of TGF-β/β2SP tumor suppressor pathway by PRAJA1 leads to uncontrolled activation of chromatin and tumor formation. Therefore, small molecule inhibitors that specifically target PRAJA1 could be very useful in HCC therapy.

Materials & Methods: Cancer patient datasets were retrieved from Gene Expression Omnibus and analyzed with Oncomine analysis tools. We analyzed 3 group-arrays of liver tumor tissues (total 298 cancer tissues vs. 249 normal tissues, p-value&lt;1E-4). We also analyzed 55 arrays for other cancers (total 2850 cancer tissues vs. 779 normal tissues, p-value&lt;1E-4). To validate protein expression, specimens from 13 HCC and normal liver tissues were immunostained with anti-PRAJA1. Whole mount in situ hybridization histochemistry (ISHH) assay was used to determine knock down PRAJA1in zebrafish embryos. Soft agar assay and colony formation assay were performed to elucidate PRAJA1 oncogenic activity in HCC cells. Additionally, we screened a compound library for E3 ligase inhibitors targeting PRAJA1.

Results: (1) PRAJA1 expression is dramatically raised in human HCCs with loss of TGF-β signaling. (2) PRAJA1 interacts with β2SP/Smad3 and inhibits TGF-β tumor suppressor target gene activation. (3) Inhibition of PRAJA1 in the developing Zebrafish embryo and HCCs leads to high levels of apoptosis. (4) Overexpression of PRAJA1 leads to increased HCC growth; conversely knock down of PRAJA1 suppresses its oncogenic activities. (5) TGF-β promotes complex of CTCF and β2SP in nucleus. (6) RTA402/RTA405 inhibits PRAJA1 and restores TGF-β tumor suppressor function in HCC cells. (7) RTA402/RTA405 induces apoptosis and inhibits HCC cell growth and tumorigenesis.

Conclusions: PRAJA1 disrupts the TGF-β tumor suppressor pathway, and its overexpression promotes cancer cell survival and proliferation. We have identified 2 novel triterpenoids that exhibit PRAJA1 inhibitory activity, which have demonstrated that suppression of PRAJA1 by specific small molecule inhibitors restores TGF-β tumor suppressor function and suppresses growth of HCC cells. Our results provide a better understanding of the mechanisms that regulate HCC development. Importantly, this study may lead to new therapeutics targeting this lethal cancer and potentially to a Phase I clinical trial in HCC.

Citation Format: Jian Chen, Jiun-Sheng Chen, Vivek Shukla, Zhixing Yao, Hai-Long Piao, Wilma Jogunoori, Bibhuti Mishra, Lopa Mishtra. Targeting E3 ligase PRAJA1 in hepatocellular cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5198. doi:10.1158/1538-7445.AM2013-5198