Gut Online First, published on March 3, 2016 as 10.1136/gutjnl-2015-310625 Hepatology ORIGINAL ARTICLE The microtubule-associated protein PRC1 promotes Gut: first published as 10.1136/gutjnl-2015-310625 on 3 March 2016. Downloaded from early recurrence of hepatocellular carcinoma in association with the Wnt/β-catenin signalling pathway Jianxiang Chen,1,2 Muthukumar Rajasekaran,1 Hongping Xia,1 Xiaoqian Zhang,2 Shik Nie Kong,1 Karthik Sekar,1 Veerabrahma Pratap Seshachalam,1 Amudha Deivasigamani,1 Brian Kim Poh Goh,3 London Lucien Ooi,3 Wanjin Hong,2 Kam M Hui1,2,4,5 ▸ Additional material is ABSTRACT published online only. To view Objectives Hepatocellular carcinoma (HCC) is the Significance of this study please visit the journal online (http://dx.doi.org/10.1136/ second leading cause of cancer mortality worldwide. gutjnl-2015-310625). Alterations in microtubule-associated proteins (MAPs) have been observed in HCC. However, the mechanisms For numbered affiliations see What is already known on this subject? end of article. underlying these alterations remain poorly understood. ▸ Early recurrence in human hepatocellular Our aim was to study the roles of the MAP protein carcinoma (HCC) remains the major cause of Correspondence to regulator of cytokinesis 1 (PRC1) in hepatocarcinogenesis death after potentially curative liver resection. Professor Kam M Hui, Division and early HCC recurrence. ▸ Protein regulator of cytokinesis 1 (PRC1) of Cellular and Molecular Research, National Cancer Design PRC1 expression in HCC samples was regulates antiparallel microtubule cross-linking Centre, 11 Hospital Drive, evaluated by microarray, immunoblotting and to promote the formation of microtubule Singapore 169610, Singapore; immunohistochemistry analysis. Molecular and cellular architectures to support cell shape and regulate [email protected] techniques including siRNA-mediated and lentiviral cytokinesis. PRC1 has been reported to be vector-mediated knockdown were used to elucidate the Received 25 August 2015 upregulated in breast and bladder tumours. Revised 11 February 2016 functions and mechanisms of PRC1. ▸ The Wnt-β-catenin network is enormously Accepted 12 February 2016 Results PRC1 expression was associated with early complex. Aberrant regulation of the canonical HCC recurrence and poor patient outcome. In HCC, Wnt-β-catenin signalling pathway is associated http://gut.bmj.com/ PRC1 exerted an oncogenic effect by promoting cancer with a variety of diseases, including cancer, proliferation, stemness, metastasis and tumourigenesis. fibrosis and neurodegeneration. There are two We further demonstrated that the expression and types of HCC in which β-catenin signalling are distribution of PRC1 is dynamically regulated by Wnt3a activated. In both cases, nuclear β-catenin is signalling. PRC1 knockdown impaired transcription factor found with a specific transcriptional output. In (TCF) transcriptional activity, decreased Wnt target the first type, this is due to CTNNB1 activating on September 27, 2021 by guest. Protected copyright. expression and reduced nuclear β-catenin levels. mutations and the HCCs are mostly well Mechanistically, PRC1 interacts with the β-catenin differentiated with a rather good prognosis: destruction complex, regulates Wnt3a-induced these tumours overexpress glutamine membrane sequestration of this destruction complex, synthetase, which is a highly specific and inhibits adenomatous polyposis coli (APC) stability and highly sensitive marker of β-catenin mutation. promotes β-catenin release from the APC complex. In The second type of tumours is Wnt-activated vivo, high PRC1 expression correlated with nuclear β- and is mostly not CTNNB1-mutated: they catenin and Wnt target expression. PRC1 acted as a correspond to HCC with a poorer prognosis. master regulator of a set of 48 previously identified Wnt- regulated recurrence-associated genes (WRRAGs) in HCC. Thus, PRC1 controlled the expression and function the poor survival of patients with HCC and is the 3–5 of WRRAGs such as FANCI, SPC25, KIF11 and KIF23 via major obstacle to improving prognosis. Despite Wnt signalling. the recent reports elucidating signalling regulators 67 Conclusions We identified PRC1 as a novel Wnt target related to early HCC recurrence, the underlying that functions in a positive feedback loop that reinforces signalling network remains largely unknown. Wnt signalling to promote early HCC recurrence. Among the growth factor signalling cascades that are deregulated in HCC, evidence suggests that the – canonical Wnt signalling pathway8 11 plays a key 12–20 To cite: Chen J, role in hepatocarcinogenesis. Rajasekaran M, Xia H, et al. INTRODUCTION Microtubules (MTs) play essential roles in cell Gut Published Online First: Hepatocellular carcinoma (HCC) is the second cycle, trafficking, signalling and migration. [please include Day Month leading cause of cancer mortality worldwide.12 Alterations in MT-associated proteins (MAPs) have Year] doi:10.1136/gutjnl- 2015-310625 Early HCC recurrence is primarily responsible for been demonstrated in cancers, and these changes Chen J, et al. Gut 2016;0:1–13. doi:10.1136/gutjnl-2015-310625 1 Copyright Article author (or their employer) 2016. Produced by BMJ Publishing Group Ltd (& BSG) under licence. Hepatology were provided by the SingHealth Tissue Repository. Written fi informed consent was obtained from the participating patients, Signi cance of this study Gut: first published as 10.1136/gutjnl-2015-310625 on 3 March 2016. Downloaded from and relevant clinical and histopathological data provided to the researchers were anonymised. The antibodies for this study are fi listed in online supplementary table S1. Microarray was per- What are the new ndings? 7 ▸ PRC1 is upregulated in HCC, especially in early recurrent formed as described previously. HCC. ▸ PRC1 has an oncogenic function in promoting cancer proliferation, stemness, metastasis and tumourigenesis. Animal studies ▸ PRC1 is a direct Wnt signalling target, and its cytoskeletal Stably transfected HCCLM3 cells were resuspended in phos- localisation is dynamically regulated by Wnt3a activity. phate-buffered saline (PBS) and subcutaneously implanted into ▸ PRC1 is necessary for Wnt signalling to mediate the left and right flanks (5×106 cells per flank) of male BALB/c oncogenesis and cancer stem cells self-renewal. nude mice. Tumour volume was monitored and calculated as ▸ PRC1 potentiates Wnt signalling by promoting the described previously.25 membrane sequestration of the Axin1 destruction complex, Additional materials and methods are provided in online sup- thereby reducing APC stability but stabilising β-catenin. plementary information. ▸ Wnt-regulated recurrence-associated genes (WRRAGs) were identified in HCC, and PRC1 regulated the expression of WRRAGs via Wnt signalling. RESULTS How might it impact on clinical practice in the PRC1 is upregulated in HCC and its expression correlates foreseeable future? with early HCC recurrence ▸ The PRC1-coordinated WRRAG cluster might be useful as a Based on the results from data mining of our microarray data- predictor of the risk of early HCC recurrence and as an base previously established,7 PRC1 expression was significantly indicator of Wnt signalling activities. upregulated in HCC tumours compared with histologically ▸ Abundant clinical evidence has implicated a critical role of normal liver (NN) from colorectal cancer patients with liver aberrant canonical Wnt/β-catenin signalling in HCC initiation metastases or matched adjacent histologically normal liver (MN) and development, underscoring the potential of targeting tissues (p<0.001; figure 1A, left panel). PRC1 expression in a this pathway for the therapeutic intervention of this typically subset of these samples was further validated by real-time PCR difficult-to-treat cancer. In this study, PRC1 emerged as a (see online supplementary figure S1A). In addition, PRC1 new therapeutic target, and its microtubule-binding domain expression was markedly upregulated in tumours from patients could be targeted using small molecule inhibitors. Specific with early (<2 years) recurrent disease (HCC-R) compared with small molecule inhibitors or activators with defined targets samples from patients with no demonstrable evidence of recur- rence within 5 years (HCC-NR) (p<0.001; figure 1A, right and mechanisms would provide therapeutic leads for HCC http://gut.bmj.com/ and research tools to manipulate the Wnt pathway in panel). PRC1 expression in a subset of these HCC-NR samples precise temporal and spatial ways. was also validated (see online supplementary figure S1B). Furthermore, data mining of RNA-seq data from HCC and adja- generally correlate with poor prognosis. A role for a divergent cent matched tissues that are publically available in The Cancer fi canonical Wnt pathway in regulating the MT cytoskeleton to Genome Atlas database con rmed that PRC1 expression is sig- fi promote cell movement has been suggested,21 implying that ni cantly elevated in tumours compared with matched normal fi fi on September 27, 2021 by guest. Protected copyright. MAPs may be involved in Wnt signalling by interacting with tissues (p<0.001; gure 1B). Signi cantly higher PRC1 expres- Wnt components. This suggestion is supported by the observa- sion levels were detected in tumours exhibiting more frequent fi tion that many Wnt pathway components, including APC, vascular invasion (see online supplementary gure S1C) and in Axin1 and GSK3β, are associated with MTs.21 However, little is those scored as high-grade cancer (see