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MenSCs inhibit HCC growth through oncogenic pathway suppression via regulating 5-hmC in enhancer elements

ABSTRACT: MenSCs inhibit HCC growth through oncogenic pathway suppression via regulating 5-hmC in enhancer elements

PROVIDER: PRJNA491745 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR7874020.fastq.gz	Fastqsanger.gz
	SRR7874030.fastq.gz	Fastqsanger.gz
	SRR7874040.fastq.gz	Fastqsanger.gz
	SRR7874130.fastq.gz	Fastqsanger.gz
	SRR7874021.fastq.gz	Fastqsanger.gz

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Project description:BackgroundEpigenetic alteration is an important indicator of crosstalk between cancer cells and surrounding microenvironment components including mesenchymal stem cells (MSC). Human menstrual blood-derived stem cells (MenSCs) are novel source of MSCs which exert suppressive effects on cancers via multiple components of microenvironmental paracrine signaling. However, whether MenSCs play a crucial role in the epigenetic regulation of cancer cells remains unknown.MethodsEpigenetic alterations of hepatocellular carcinoma (HCC) mediated by MenSCs were examined by immunofluorescence, ELISA, and RT-PCR assays. The suppressive impact of MenSCs on HCC was investigated in vitro using CCK8, apoptosis, wound healing, and invasion assays and in vivo using a xenograft mice model. MeDIP-seq, hMeDIP-seq, and RNA-seq were used to identify the genome-wide pattern of DNA methylation and hydroxymethylation in HCC cells after MenSC therapy.ResultsWe show that HCC cells display distinct genome-wide alterations in DNA hydroxymethylation and methylation after MenSC therapy. MenSCs exert an inhibitory effect on HCC growth via regulating 5-hmC and 5-mC abundance in the regulatory regions of oncogenic pathways including PI3K/AKT and MAPK signaling, especially in enhancers and promoters. FOXO3 expression is rescued via reversal of 5-hmC and 5-mC levels in its enhancers and contributes to the activation of downstream apoptosis. Inactivation of the MAPK pathway further disrupts c-myc-mediated epithelial-mesenchymal transitions (EMT). Additionally, chemotherapy resistance-associated genes including ID4 and HMGA1 are suppressed via amending 5-hmC and 5-mC abundance at their regulatory regions. HMGA1 and BYSL might be potential targets for gene-modified MSC therapy.ConclusionsOur results confirm that MSCs could regulate the epigenetic mechanism of HCC cells and provide a novel concept for a modified MSC strategy or combination therapy with chemotherapeutics based on epigenetics.

Project description:The HMC-1.1 and 1.2 human mast cell (huMC) lines are often employed in the study of attributes of neoplastic huMCs as found in patients with mastocytosis and their sensitivity to interventional drugs in vitro and in vivo. Both cell lines express mutated and constitutively active KIT, an essential growth factor receptor for huMC survival and function, with one KIT mutation in HMC-1.1 cells (V560G-KIT) and two in HMC-1.2 cells (D816V-KIT and V560G-KIT). D816V-KIT is commonly associated with systemic mastocytosis, while V560G-KIT appears more frequently in other human neoplasms. The functional consequences of the coexisting KIT mutations in HMC-1.2 cells are unknown. We used CRISPR/Cas9 engineering to reverse the V560G-KIT mutation in HMC-1.2 cells, resulting in a subline (HMC-1.3) with a single monoallelic D816V-KIT mutation. Transcriptome analyses of differentially expressed genes in HMC1.3 compared to HMC-1.2 predicted reduced activity in pathways involved in survival, cell-to-cell adhesion, and neoplasia, with differences in expression of molecular components and cell surface markers. Consistently, subcutaneous inoculation of HMC-1.3 into mice produced significantly smaller tumors than HMC-1.2 cells, and in colony assays, HMC-1.3 formed less numerous and smaller colonies than HMC-1.2 cells. However, in liquid culture conditions, the growth of HMC-1.2 and HMC-1.3 cells was comparable, albeit increased compared to HMC-1.1 cells. Phosphorylation levels of ERK1/2, AKT and STAT5, representing pathways associated with constitutive oncogenic KIT signaling, were also similar between HMC-1.2 and HMC-1.3 cells but increased compared to HMC-1.1 cells. Despite these similarities, survival of HMC-1.3 cells in liquid culture was diminished in response to various pharmacological inhibitors, including tyrosine kinase inhibitors used clinically for treatment of advanced systemic mastocytosis, and JAK2 and BCL2 inhibitors, making HMC-1.3 more susceptible to these drugs than HMC-1.2 cells. Our study reveals that the additional V560G-KIT variant in HMC-1.2 cells modifies transcriptional programs induced by D816V-KIT, confers a survival advantage, alters sensitivity to interventional drugs, and increases the tumorigenicity, suggesting that engineered huMCs with a single D816V-KIT variant may represent an improved preclinical model for mastocytosis.

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MenSCs inhibit HCC growth through oncogenic pathway suppression via regulating 5-hmC in enhancer elements

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