Functional and mechanistic characterization of PRMT6 in regulating the diverse cellular properties implicated in the pathogenesis of hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) accounts for 75%-80% of primary malignancy of the liver. HCC is the fifth most common cancer and the second leading cause of cancer-related mortality worldwide. HCC is also the third leading cause of mortality among Southeast Asian and local population. The major challenges for treating HCC patients mainly attribute to high incidence of metastasis, chemotherapy resistance and tumor relapse, which can be partially explained by the cancer stem cells (CSCs) model. The CSC model suggests the presence of subsets of stem-like cells that reside at the apex of a cellular hierarchy and are endowed with unique ability to self-renew, differentiate, initiate tumor (re)generation and resist conventional chemo-and radiotherapy. Previous studies in our group and others have identified and characterized 〖CD133〗^+cells in HCC as liver CSCs. Despite our increasing recognition of the impact of 〖CD133〗^+ liver CSCs in promoting HCC pathogenesis, the underlying mechanisms governing the stem-cell like properties of this subset of cells have yet to be sufficiently elucidated. Therefore, the current study aims at identifying novel mediators and their mechanisms in regulating HCC development such that to discover new therapeutic target to improve treatment efficacy for this mortal disease. By adopting a PCR array encompassing diverse human chromatin modifiers, protein arginine methyltransferase 6 (PRMT6) was found to be differentially down-regulated in 〖CD133〗^+ human HCC cells. 55.8% (43/77) of clinical HCC tumor tissues showed down-regulated expression of PRMT6and patients with reduced PRMT6expression were at significant risk of disrupted tumor encapsulation and manifested vascular invasion. Negative correlation between the expression levels ofPRMT6 and CD133 was identified at both transcriptional and proteomic levels. The abundance of CD133+ subsets in HCC cells were also negatively regulated by PRMT6 levels. Subsequent functional characterization by lentiviral-based stable knockdown or overexpression of PRMT6 in HCC cells showed that PRMT6 regulate diverse cancer and stem cell-like properties including proliferation, clonogenicity, invasion, migration, angiogenesis, chemoresistance and self-renewal. Morphological alterations in HCC cells upon PRMT6 knockdown were observed, where extensive cytoplasmic stretching and filopodia-like projections were detected. In vivo experiments indicated PRMT6 to regulate the tumorigenicity and disease progression to metastasis of HCC cells. Further, we found that PRMT6 reprogrammed the energy metabolism of HCC cells, where metabolic dysregulation are currently recognized as a cancer hallmark. Mechanistically, despite previous study indicated asymmetric dimethylation of nuclear histone H3R2 as a prominent epigenetic mechanism of PRMT6 in mediating cellular transcriptomic activity, we observed remarkable cytoplasmic localizationofPRMT6 in HCC cells. Thus, tandem affinity purification and mass spectrometry were adopted to identify PRMT6-interacting proteins. We found that PRMT6 bind with and methylated CRAF, a regulator of the ERK pathway. CRAF methylation suppressed its kinase activity and hence inhibited the ERK signaling. Rescue experiments through using MEK inhibitor U0126 reversed the oncogenic effects of HCC cells induced by PRMT6 silencing. Taken together, PRMT6 was identified as a novel tumor suppressor of HCC. It regulated diverse cancer and stem cell-like properties of HCC cells through regulating the ERK pathway via asymmetric arginine dimethylation of CRAF.