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

Abstract

BackgroundHepatocellular 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 PRSS3 on carcinogenesis due to an unclear molecular basis impede the utilization of its biomarker potential. We hereby explored the contribution of PRSS3 transcripts to tumor functional heterogeneity by systematically dissecting the expression of four known splice variants of PRSS3 (PRSS3-SVs, V1~V4) and their functional relevance to HCC.MethodsThe expression and DNA methylation of PRSS3 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 PRSS3 transcript constructs were separately transfected after deleting PRSS3 expression by CRISPR/Cas9 editing.ResultsPRSS3 was aberrantly differentially expressed toward bipolarity from very low (PRSS3Low) to very high (PRSS3High) expression across HCC cell lines and tissues. This was attributable to the disruption of PRSS3-SVs, in which PRSS3-V2 and/or PRSS3-V1 were dominant transcripts leading to PRSS3 expression, whereas PRSS3-V3 and -V4 were rarely or minimally expressed. The expression of PRSS3-V2 or -V1 was inversely associated with site-specific CpG methylation at the PRSS3 promoter region that distinguished HCC cells and tissues phenotypically between hypermethylated low-expression (mPRSS3-SVLow) and hypomethylated high-expression (umPRSS3-SVHigh) groups. PRSS3-SVs displayed distinct functions from oncogenic PRSS3-V2 to tumor-suppressive PRSS3-V1, -V3 or PRSS3-V4 in HCC cells. Clinically, aberrant expression of PRSS3-SVs was translated into divergent relevance in patients with HCC, in which significant epigenetic downregulation of PRSS3-V2 was seen in early HCC and was associated with favorable patient outcome.ConclusionsThese results provide the first evidence for the transcriptional and functional characterization of PRSS3 transcripts in HCC. Aberrant expression of divergent PRSS3-SVs disrupted by site-specific CpG methylation may integrate the effects of oncogenic PRSS3-V2 and tumor-suppressive PRSS3-V1, resulting in the molecular diversity and functional plasticity of PRSS3 in HCC. Dysregulated expression of PRSS3-V2 by site-specific CpG methylation may have potential diagnostic value for patients with early HCC.

Serine Protease 3 Promotes Progression of Diffuse Large B-Cell Lymphoma and Serves as a Novel Prognostic Predictor

“…Ghilardi et al implied that TFPI-2 can directly interact with and was degraded by active PRSS3, thus promoting of tumor vascular migration [23]. Expression of PRSS3 can be modulated by methylation regulation [24] and upstream microRNAs such as miR-217 [25]. Whether these signaling pathways are involved in DLBCL remain further investigations.…”

Garlic‐derived compounds: Epigenetic modulators and their antitumor effects

“…such as Azacitidine and its deoxycytidine analog 5-Aza-2 0 -deoxycytidine (5-Aza-CdR), have been approved as therapies for acute myeloid leukemia and myelodysplastic syndrome and are also in ongoing clinical trials for multiple solid cancers in combination with other anticancer agents to increase treatment efficacy and reduce considerable side effects and non-specificity (Koch et al, 2018;Nervi et al, 2015). where hypermethylation impairs gene transcription and is difficult to be erased by either 5-Aza-CR or DATS alone (Lin et al, 2019;Lin et al, 2022;Pan et al, 2016). DATS increased methylation at hypomethylated sites in the PRSS3 iCpGI.…”

“…5‐Aza‐CdR/DAC generally showed extensive CpG site demethylation but did not work well on constitutively methylated CpG sites. These CpG sites are usually in important regulatory regions (e.g., DMRs) for tissue‐specific gene expression, such as the transcription factor MZF1 motifs within PRSS3 iCpGI (intragenic CpG island), where hypermethylation impairs gene transcription and is difficult to be erased by either 5‐Aza‐CR or DATS alone (Lin et al, 2019; Lin et al, 2022; Pan et al, 2016). DATS increased methylation at hypomethylated sites in the PRSS3 iCpGI.…”

“…Garlic‐derived compounds may be more specifically effective in cancer cells with epigenetic dysregulation, differing from the current chemotherapeutic agents with both strong antitumor and toxic effects (C. Lv et al, 2021). The combination of garlic‐derived compounds with epigenetic inhibitors or chemotherapeutic drugs thus may be a more efficient （related to 5‐Aza‐CdR）(Lin et al, 2022; Lin et al, 2023) and safer (compared to first‐line chemotherapy agents） (Pan et al, 2016) therapeutic option in some cancer subtypes (i.e., personalized treatment).…”