AUTHOR=Lin Shuye , Xu Hanli , Pang Mengdi , Zhou Xiaomeng , Pan Yuanming , Zhang Lishu , Guan Xin , Wang Xiaoyue , Lin Bonan , Tian Rongmeng , Chen Keqiang , Zhang Xiaochen , Yang Zijiang , Ji Fengmin , Huang Yingying , Wei Wu , Gong Wanghua , Ren Jianke , Wang Ji Ming , Guo Mingzhou , Huang Jiaqiang 

TITLE=CpG Site-Specific Methylation-Modulated Divergent Expression of PRSS3 Transcript Variants Facilitates Nongenetic Intratumor Heterogeneity in Human Hepatocellular Carcinoma

JOURNAL=Frontiers in Oncology

VOLUME=12

YEAR=2022

URL=https://www.frontiersin.org/journals/oncology/articles/10.3389/fonc.2022.831268

DOI=10.3389/fonc.2022.831268

ISSN=2234-943X

ABSTRACT=<sec><title>Background</title><p>Hepatocellular carcinoma (HCC) is one of the most lethal human tumors with extensive intratumor heterogeneity (ITH). Serine protease 3 (PRSS3) is an indispensable member of the trypsin family and has been implicated in the pathogenesis of several malignancies, including HCC. However, the paradoxical effects of <italic>PRSS3</italic> on carcinogenesis due to an unclear molecular basis impede the utilization of its biomarker potential. We hereby explored the contribution of <italic>PRSS3</italic> transcripts to tumor functional heterogeneity by systematically dissecting the expression of four known splice variants of <italic>PRSS3</italic> (<italic>PRSS3-SVs, V1~V4</italic>) and their functional relevance to HCC.</p></sec><sec><title>Methods</title><p>The expression and DNA methylation of <italic>PRSS3</italic> transcripts and their associated clinical relevance in HCC were analyzed using several publicly available datasets and validated using qPCR-based assays. Functional experiments were performed in gain- and loss-of-function cell models, in which <italic>PRSS3</italic> transcript constructs were separately transfected after deleting <italic>PRSS3</italic> expression by CRISPR/Cas9 editing.</p></sec><sec><title>Results</title><p><italic>PRSS3</italic> was aberrantly differentially expressed toward bipolarity from very low (<italic>PRSS3<sup>Low</sup></italic>) to very high (<italic>PRSS3<sup>High</sup></italic>) expression across HCC cell lines and tissues. This was attributable to the disruption of <italic>PRSS3-SVs</italic>, in which <italic>PRSS3-V2</italic> and/or <italic>PRSS3-V1</italic> were dominant transcripts leading to <italic>PRSS3</italic> expression, whereas <italic>PRSS3-V3</italic> and <italic>-V4</italic> were rarely or minimally expressed. The expression of <italic>PRSS3-V2</italic> or <italic>-V1</italic> was inversely associated with site-specific CpG methylation at the <italic>PRSS3</italic> promoter region that distinguished HCC cells and tissues phenotypically between hypermethylated low-expression (m<italic>PRSS3-SV<sup>Low</sup></italic>) and hypomethylated high-expression (um<italic>PRSS3-SV<sup>High</sup></italic>) groups. <italic>PRSS3-SVs</italic> displayed distinct functions from oncogenic <italic>PRSS3-V2</italic> to tumor-suppressive <italic>PRSS3-V1</italic>, <italic>-V3</italic> or <italic>PRSS3-V4</italic> in HCC cells. Clinically, aberrant expression of <italic>PRSS3-SVs</italic> was translated into divergent relevance in patients with HCC, in which significant epigenetic downregulation of <italic>PRSS3-V2</italic> was seen in early HCC and was associated with favorable patient outcome.</p></sec><sec><title>Conclusions</title><p>These results provide the first evidence for the transcriptional and functional characterization of <italic>PRSS3</italic> transcripts in HCC. Aberrant expression of divergent <italic>PRSS3-SVs</italic> disrupted by site-specific CpG methylation may integrate the effects of oncogenic <italic>PRSS3-V2</italic> and tumor-suppressive <italic>PRSS3-V1</italic>, resulting in the molecular diversity and functional plasticity of <italic>PRSS3</italic> in HCC. Dysregulated expression of <italic>PRSS3-V2</italic> by site-specific CpG methylation may have potential diagnostic value for patients with early HCC.</p></sec>