Chondroitin Polymerizing Factor (CHPF) promotes cell proliferation and tumor growth in human osteosarcoma by inhibiting SKP2's ubiquitination while activating the AKT pathway

Abstract

Osteosarcoma is a common malignant tumor occurring in children and young adults. Chondroitin sulfate (CS) participates in cell adhesion, cell division, and the formation of neural networks in the body, the biosynthesis of which requires the participation of glycosyltransferases. CHPF, a glycosyltransferase, plays a role in the extension of CS. Recently, CHPF's biological roles and functional importance in human diseases including malignant tumors have been widely discussed. However, whether CHPF is involved in osteosarcoma development and growth has not been revealed. The present work aimed to investigate the expression levels, functional significance and molecular mechanism of CHPF in osteosarcoma progression. Our results revealed that CHPF is strongly expressed in osteosarcoma tissues and cells. Furthermore, CHPF serves as a tumor promoter in the development and progression of osteosarcoma through enhancing cell proliferation and migration while suppressing apoptosis. Exploration of the mechanism by which CHPF promotes osteosarcoma indicated that CHPF promotes osteosarcoma through counteracting SKP2's ubiquitination and activating the Akt signaling pathway. For the first time, we clarified the roles of CHPF in osteosarcoma, and our results suggested that CHPF might be a novel therapeutic target in the treatment strategies for osteosarcoma.

Keywords: Akt signaling pathway; CHPF; Osteosarcoma; SKP2; Ubiquitination.

Figure 1

CHPF was abundantly expressed in osteosarcoma. (A) The protein expression of CHPF in osteosarcoma and normal tissues was detected by IHC staining. (B) The IHC staining score of CHPF in osteosarcoma and normal tissues was quantified. (C) The mRNA expression of CHPF in osteosarcoma cell lines was detected by qRT-PCR.

Figure 2

CHPF knockdown inhibited cell proliferation, colony formation and migration, induced cell apoptosis. (A) MTT assay was used to detect the effects of CHPF knockdown on cell proliferation of MNNG/HOS and U-2OS cells. (B) The abilities of MNNG/HOS and U-2OS cells to form colony after infection were assessed. (C, D) The effects of CHPF knockdown on MNNG/HOS and U-2OS cell migration capacities were detected by Transwell assay (C) and wound-healing assay (D). (E, F) Flow cytometry was performed to evaluate the effects of CHPF knockdown on cell cycle (E) and apoptosis (F) of MNNG/HOS and U-2OS cells. (G, H) The changes in apoptosis-related proteins were analyzed in MNNG/HOS cells following infection by a Human Apoptosis Antibody Array. Protein level was visualized by R studio (G) and presented in gray value (H). The data were expressed as mean ± SD. ∗P < 0.05, ∗∗∗P < 0.001.

Figure 3

CHPF knockdown inhibited tumor growth of osteosarcoma in vivo. (A) A nude mice model of CHPF knockdown was constructed. (B) The photos of tumors removed from mice models were collected. (C–E) The fluorescence (C), volume (D) and weight (E) of xenograft tumors were measured. (F) The patterns of CHPF and Ki-67 were detected by IHC analysis in tumor sections from the mice models. The data were expressed as mean ± SD (n ≥ 3). ∗P < 0.05.

Figure 4

SKP2 was identified as the downstream target of CHPF regulating osteosarcoma. (A) PrimeView human gene expression array (3 v 3) was performed to identify differentially expressed genes in MNNG/HOS cells with or without CHPF knockdown. (B) The enrichment of the DEGs in canonical signaling pathways was analyzed by IPA. (C) The IPA analysis was performed to produce the CHPF-related interaction network. (D, E) The expression of several most significantly down-regulated DEGs was further determined via qRT-PCR (D) and Western blotting (E). (F) Celigo cell counting assay was performed to detect the inhibition of cell proliferation of MNNG/HOS cells after infecting shCCND2, shCCNE2, shRHOU or shSKP2 corresponding lentiviruses. (G) Co-IP assay was used to verify whether there was protein interaction between CHPF and SKP2. (H) The level of SKP2 protein in MNNG/HOS cells with CHPF depletion was detected following 50 μg/mL CHX treatment for indicated times. (I) After MG-132 treatment, the level of SKP2 protein in MNNG/HOS cells with CHPF depletion was examined. (J) The lysate of MNNG/HOS cells with CHPF depletion was immunoprecipitated using SKP2 and IgG antibodies, and the Western blotting was performed to examine the ubiquitination of SKP2. (K) There was a positive correlation between CHPF level and SKP2 expression through analyzing the RNA-seq data collected from TARGET database. (L, M) The protein level of SKP2 in osteosarcoma/normal tissues and the mRNA level of SKP2 in osteosarcoma cell lines were detected via IHC staining (L) and qRT-PCR (M), respectively.

Figure 5

SKP2 knockdown alleviated the promotion of osteosarcoma from CHPF overexpression. (A) MTT assay was performed to examine the effects of CHPF overexpression, SKP2 downregulation as well as CHPF overexpression combining with SKP2 downregulation on MNNG/HOS cell proliferation. (B) Flow cytometry was performed to detect the effects of CHPF overexpression, SKP2 downregulation as well as CHPF overexpression combining with SKP2 downregulation on MNNG/HOS cell apoptosis. (C, D) The effects of CHPF overexpression, SKP2 downregulation as well as CHPF overexpression combining with SKP2 downregulation on MNNG/HOS cell migration were evaluated by Transwell (C) and wound healing (D) assays. (E) The expression of some well-known cancer-associated elements was detected by Western blotting in MNNG/HOS cells after silencing CHPF. (F) Western blotting revealed protein expression of AKT, p-AKT, mTOR and p-mTOR in shCtrl or shCHPF infected MNNG/HOS cells treated with or without an AKT activator: SC79. (G) Flow cytometry experiment showed the changes in shCtrl or shCHPF infected MNNG/HOS cell apoptosis after treatment with or without SC79. The data were expressed as mean ± SD (n ≥ 3). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.