Hypoxia-induced myeloid derived growth factor promotes hepatocellular carcinoma progression through remodeling tumor microenvironment

Abstract

Purpose: Exploring and studying the novel target of hepatocellular carcinoma (HCC) has been extremely important for its treatment. The principal objective of this project is to investigate whether myeloid derived growth factor (MYDGF) could accelerate the progression of HCC, and how it works. Methods: Cell proliferation, clonal formation, sphere formation and xenograft tumor experiments were used to prove the critical role of MYDGF in HCC progression. Tumor angiogenesis, immune cell infiltration, macrophage chemotaxis and inflammatory cytokines detection were utilized to clarify how MYDGF remodeled the tumor microenvironment (TME) to accelerate the progress of HCC. Results: Here, we reported a secretory protein MYDGF, which could be induced by hypoxia, was significantly upregulated in HCC and associated with poor clinical outcomes. Using bioinformatics and experimental approaches, we found that MYDGF promotes cell proliferation in vitro and in vivo through a mechanism that might involve enhanced self-renewal of liver CSCs. Furthermore, MYDGF can also promote tumor angiogenesis, induce macrophages to chemotaxis into tumor tissue, and then release various inflammatory cytokines, including IL-6 and TNF-α, which ultimately aggravate inflammation of tumor microenvironment and accelerate HCC progression. Conclusions: We provided evidence that MYDGF could directly affect the self-renewal of liver CSCs, and indirectly aggravate the inflammatory microenvironment to accelerate the progression of HCC.

Keywords: Cancer stem cell; Hepatocellular carcinoma; MYDGF; Tumor inflammatory microenvironment; Tumor-associated macrophages.

Figure 1

MYDGF is highly expressed in HCC and correlates with poor prognosis. (A) MYDGF gene expression in Roessler's liver datasets from Oncomine database. (B) MYDGF gene expression analysis in GSE36376 datasets from GEO database containing 193 non-tumor samples and 240 HCC tumor samples. (C) MYDGF gene expression in 50 paired normal and HCC samples from TCGA database. (D) Protein level of MYDGF in normal liver and HCC sample from ProtienAtlas database. (E) 5-year overall survival rate between MYDGF high and low expression groups. (F) mRNA expression of MYDGF in human normal tissue and primary HCC samples by qPCR (n = 41). (G) Protein level of MYDGF in normal and primary HCC samples, 3 samples in each group was showed. (H) Level of MYDGF protein in paracancerous and primary HCC tumor samples as detected by immunohistochemistry. (I) Protein level of MYDGF in normal mouse liver tissues and DEN-induced mouse HCC samples as detected by immunohistochemistry.

Figure 2

MYDGF promotes cell proliferation in vitro. (A) Single gene GSEA enrichment analysis indicating that four proliferation related signatures were significantly enriched in MYDGF high expression group. Normalized enrichment score (NES), nominal P-value were shown in the plot. (B) The expression pattern of MYDGF in the high and low groups of one enriched signature “MYC_UP. V1_UP”. (C) The relative proliferation rate of murine Hepa1-6 and human BEL-7404 HCC cell lines cultured with 100 ng/mL recombinant murine and human MYDGF protein, respectively for 24 h by SRB. (D) The effects of MYDGF knockdown on the proliferation rate in Hepa1-6 cells by SRB. (E) The effect of MYDGF knockdown on clone formation of Hepa1-6 cells. (F) Clone numbers and (G) area fraction of MYDGF knockdown counted. (H) The clone formation assay based on MYDGF overexpressed cells. (I-J) Clone numbers and area fraction of MYDGF overexpression were counted.

Figure 3

MYDGF promotes proliferation in vivo. (A) Tumor growth curve of MYDGF knockdown in C57/BL6 xenograft mice models. Tumor volume was calculated by the formula: 1/2 length*width². (B) Tumor picture at the end of the experiment. Tumor volume (C) and weight (D) measured at the end of the experiment. Quantitative results of Ki67 (E) and PCNA (F) positive staining area percentage by immunohistochemistry. Immunohistochemistry staining of Ki67 (G) and PCNA (H).

Figure 4

MYDGF enhances self-renewal of liver CSCs. (A-D) GSEA of stem cell related signatures in MYDGF high versus low samples from TCGA database. Normalized enrichment score (NES), nominal P-value is shown in each plot. (E) MYDGF mRNA expression in non-sphere and sphere human HCC cell lines. (F) MYDGF protein expression in non-sphere and sphere Hepa1-6 cell. (G) The effect of MYDGF knockdown on the sphere formation ability of Hepa1-6 cells. (H) Quantitatively count sphere numbers of Hepa1-6 after knocking down MYDGF. (I) Sphere formation and statistics of Hepa1-6 cells treated with murine recombinant MYDGF. (J) Expression of stemness-related proteins after MYDGF knocking down.

Figure 5

MYDGF indirectly promoted HCC development by remodeling TME. (A) The proportion of macrophage infiltration in tumor tissue detected by flow cytometry. (B) Quantitative of macrophage infiltration percentage. (C) Immunohistochemistry of F4/80 positive cells in tumor tissues. (D) Quantitative analysis of F4/80 positive cells in tumor tissues. (E) Transwell experiments to assess chemotaxis of MYDGF on macrophages. (F) CCL2 protein level in BMDM and M2-like macrophages cultured with 100 ng/mL MYDGF recombinant protein. IL-6 mRNA (G) and protein (H) level in BMDM and M2 type macrophages cultured with 100 ng/mL MYDGF recombinant protein. TNF-α mRNA (I) and protein (J) level in BMDM and M2 type macrophages cultured with 100 ng/mL MYDGF recombinant protein. (K) Expression of inflammation-related proteins in macrophages after culturing with 100 ng/mL murine MYDGF recombinant protein.

Figure 6

Hypoxia induces MYDGF expression in HCC. (A) Single gene GESA revealed that hypoxia and its related signaling pathways were enriched in the high MYDGF expression group. The color bars indicate that genes are ranked according to the high and low expression of MYDGF (Red represents high MYDGF expression, Blue represents low MYDGF expression). The vertical line indicates genes in the feature set, and the position of the vertical line indicates genes that appears in the ranking gene list position, the height of the bars indicates the running GSEA enrichment score. (B) Gene expression heat map of “CELLULAR RESPONSE TO HYPOXIA” signature in MYDGF high and low groups. (C) Correlation between MYDGF and hypoxia metagenes in TCGA liver cancer dataset. (D) MYDGF mRNA expression during normoxia and hypoxia. (E) MYDGF protein level in two HCC cell lines under hypoxia condition. (F) MYDGF mRNA expression in cell treated with HIF inhibitor Bay 87-2243 and exposed to hypoxia for 48 and 72 hours.

Figure 7

Schematic illustration of the mechanisms by which MYDGF accelerates the progression of HCC. At the initial state of tumor formation, tumor cells under a normal oxygen environment, the expression of MYDGF is relatively low. Under hypoxia condition, triggering the upregulation and release of MYDGF from the tumor cells, which could directly enhance self-renewal of hepatoma cells and promote proliferation of HCC through autocrine secretion. Meanwhile, MYDGF could also promote tumor angiogenesis. Through paracrine action, it could chemotactic macrophages and promote macrophages releasing various inflammatory cytokines, such as IL-6 and TNF-α. In this way, it may indirectly remodel the tumor microenvironment and accelerate the malignant progression of HCC.