PRP‑1 significantly decreases the ALDHhigh cancer stem cell population and regulates the aberrant Wnt/β‑catenin pathway in human chondrosarcoma JJ012�cells

Abstract

Chondrosarcomas are malignant bone tumors refractory to chemotherapy and radiation treatment; thus, novel therapeutic strategies are required. Proline-rich polypeptide 1 (PRP-1) has previously demonstrated antitumor properties in chondrosarcoma. To further investigate the role of PRP-1 in chondrosarcoma cells, its effects on cancer stem cell (CSC) populations were determined by analyzing aldehyde dehydrogenase (ALDH) activity, an established marker of CSCs, in association with regulation of the Wnt/β-catenin signaling. A significant decrease in ALDH high CSCs was observed following treatment of chondrosarcoma JJ012 cells with PRP-1. For RT 2 profiler PCR array analysis of Wnt/β-catenin signaling genes, cells were sorted into: i) Bulk JJ012 cells; ii) ALDH high cells sorted from untreated JJ012 cells (ALDH high-untreated ); and iii) ALDH low cells sorted from PRP-1-treated JJ012 cells (ALDH low-PRP-1 ). The expression levels of Wnt/β-catenin signaling genes were determined to be downregulated in the ALDH high-untreated cells and upregulated in ALDH low-PRP-1 cells when compared to the bulk JJ012 cells. Additionally, two important oncogenes involved in this pathway, MMP7 and CCND2, were found to be downregulated in the ALDH low-PRP-1 cells. Immunocytochemistry demonstrated the localization of β-catenin in the nuclei of the PRP-1-treated cells. Western blotting indicated increased β-catenin expression in the ALDH low-PRP-1 cells compared with the bulk JJ012 cells. Analysis of the cytoplasmic and nuclear fractions of cells treated with increasing concentrations of PRP-1 and β-catenin nuclear translocation inhibitor CGP57380, suggested the nuclear translocation of β-catenin following PRP-1 treatment. In addition, treatment of JJ012 cells with a specific ALDH inhibitor, diethylaminobenzaldehyde, and PRP-1 resulted in a significant decrease in cytoplasmic β-catenin protein expression. This indicated that ALDH inactivation may be associated with the nuclear translocation of β-catenin. Derivation of sarcomas from mesenchymal stem cells via inactivation of the Wnt pathway has been previously documented. The findings of the present study support the notion that Wnt/β-catenin activation may serve a differential role in sarcomas, limiting tumor progression in association with decreased CSC activity.

Cancer stem cells as a therapeutic target in 3D tumor models of human chondrosarcoma: An encouraging future for proline rich polypeptide‑1

“…The antitumorigenic effects of PRP-1 on bulk, a term which designates tumor stromal chondrosarcoma cell monolayers, has previously been established, including the antineoplastic regulation of the Wnt/β-catenin pathway ( 30 ); this prompted further investigation into the role of PRP-1 on anchorage independent growth. To ensure the specific investigation of anchorage independent growth, cell colonies were cultivated using an agarose colony formation assay known to involve anchorage independence and necessitate CSC involvement as a prerequisite ( 31 ).…”

“…Aldefluor ® assay and fluorescence-activated cell sorting (FACS). For the following experiment, we followed the methods of our previous study, Hoyt et al (30). To measure cells with ALDH activity, the Aldefluor ® assay was carried out as described according to manufacturer's protocol (Aldefluor kit, cat.…”

“…Given the lack of a dependable treatment and an overwhelmingly poor prognosis, identification of novel therapies for chondrosarcoma is a critical clinical priority. The efficiency by which PRP-1 eliminates CSCs has been previously described in a monolayer, and was flow cytometrically determined to result from the aldehyde dehydrogenase (ALDH) high population of the chondrosarcoma monolayer, in association with the antineoplastic regulation of aberrant Wnt/β-catenin signaling ( 30 ). Likewise, PRP-1 was also found to exert cytostatic properties in a chondrosarcoma monolayer by promoting cell cycle arrest in the S phase ( 26 ).…”

Proline‑rich polypeptide‑1 decreases cancer stem cell population by targeting BAFF chromatin‑remodeling complexes in human chondrosarcoma JJ012 cells

“…1A). JJ012 fractions treated with PRP-1 were labeled as ALDH low-PRP-1 (21). These gates were used in the analysis of surface staining of well-established CSC markers for sarcoma.…”

Biological Heterogeneity of Chondrosarcoma: From (Epi) Genetics through Stemness and Deregulated Signaling to Immunophenotype

“…Highly upregulated ALDH has been found in established human ChS cell lines with enhanced invasiveness, tumorigenicity, and the ability to grow as tumor spheres. Since ALDH is one of the detoxifying enzymes contributing to the oxidation of aldehydes [ 51 ], and therefore involved in the scavenging of reactive oxygen species (ROS), ALDH + ChS cells demonstrated a significantly lower ROS level than ALDH − cells, which is characteristic for CSCs [ 52 , 53 ]. Furthermore, ALDH-positive, but not ALDH-negative, cells showed an expression of the Krüppel-like factor 4 (KLF4) protein, which is one of the so-called Yamanaka factors of pluripotency.…”