PRP-1, a toll-like receptor ligand, upregulates the unfolded protein response in human chondrosarcoma cells.

BACKGROUND: Previous studies showed that proline-rich polypeptide (PRP-1) is a ligand for innate immunity toll-like receptors (TLR), and an inhibitor of the mammalian target of rapamycin complex 1 (mTORC1) which induces the death of chondrosarcoma cancer stem cells (CSC). The aim of this study was to investigate the effect of PRP-1 on the regulation of unfolded protein response (UPR) in human chondrosarcoma cells. MATERIALS AND METHODS: Lysates were prepared from a monolayer (bulk or ALDHhigh population), or spheroids chondrosarcoma cell cultures and treated with PRP-1 or control, followed by protein levels quantification by western blotting and mRNA expression by RT-qPCR of protein-RNA-like endoplasmic reticulum kinase (PERK), eukaryotic translation initiation factor 2α (eIF2α), activating transcription factor 4 (ATF4), CCAAT-enhancer-binding protein homologous protein (CHOP), activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1α), and X-box binding protein (XBP1). RESULTS: The PRP-1 has been shown to increase the expression of PERK, eIF2α, ATF4, CHOP, ATF6, IRE1α, and XBP1, on both protein and mRNA levels. CONCLUSION: PRP-1 activated UPR branches in monolayer, spheroid, and stem cell populations of human chondrosarcoma.

Cancer stem cells as a therapeutic target in 3D tumor models of human chondrosarcoma: An encouraging future for proline rich polypeptide­1.

Chondrosarcoma is a malignant bone neoplasm that is refractory to chemotherapy and radiation. With no current biological treatments, mutilating surgical resection is the only effective treatment. Proline rich polypeptide 1 (PRP­1), which is a 15­amino acid inhibitor of mammalian target of rapamycin complex­1 (mTORC1), has been indicated to exert cytostatic and immunomodulatory properties in human chondrosarcoma cells in a monolayer. The aim of the present study was to evaluate the effects of PRP­1 on an in vitro 3D chondrosarcoma tumor model, known as spheroids, and on the cancer stem cells (CSCs) which form spheroids. JJ012 cells were cultured and treated with PRP­1. An ALDEFLUOR™ assay was conducted (with N,N­diethylaminobenzaldehyde as the negative control) to assess aldehyde dehydrogenase (ALDH) activity (a recognized CSC marker), and bulk JJ012, ALDHhigh and PRP­1 treated ALDHlow cells were sorted using flow cytometry. Colony formation and spheroid formation assays of cell fractions, including CSCs, were used to compare the PRP­1­treated groups with the control. CSCs were assessed for early apoptosis and cell death with a modified Annexin V/propidium iodide assay. Western blotting was used to identify mesenchymal stem cell markers (STRO1, CD44 and STAT3), and spheroid self­renewal assays were also conducted. A clonogenic dose­response assay demonstrated that 20 µg/ml PRP­1 was the most effective dose for reducing colony formation capacity. Furthermore, CSC spheroid growth was significantly reduced with increasing doses of PRP­1. Annexin V analysis demonstrated that PRP­1 induced CSC cell death, and that this was not attributed to apoptosis or necrosis. Western blot analysis confirmed the expression of mesenchymal markers, and the spheroid self­renewal assay confirmed the presence of self­renewing CSCs. The results of the present study demonstrate that PRP­1 eliminates anchorage independent CSC growth and spheroid formation, indicating that PRP­1 likely inhibits tumor formation in a murine model. Additionally, a decrease in non­CSC bulk tumor cells indicates an advantageous decline in tumor stromal cells. These findings confirm that PRP­1 inhibits CSC proliferation in a 3D tumor model which mimics the behavior of chondrosarcoma in vivo.

Targeting Mitochondrial Proline Dehydrogenase with a Suicide Inhibitor to Exploit Synthetic Lethal Interactions with p53 Upregulation and Glutaminase Inhibition.

Proline dehydrogenase (PRODH) is a p53-inducible inner mitochondrial membrane flavoprotein linked to electron transport for anaplerotic glutamate and ATP production, most critical for cancer cell survival under microenvironmental stress conditions. Proposing that PRODH is a unique mitochondrial cancer target, we structurally model and compare its cancer cell activity and consequences upon exposure to either a reversible (S-5-oxo: S-5-oxo-2-tetrahydrofurancarboxylic acid) or irreversible (N-PPG: N-propargylglycine) PRODH inhibitor. Unlike 5-oxo, the suicide inhibitor N-PPG induces early and selective decay of PRODH protein without triggering mitochondrial destruction, consistent with N-PPG activation of the mitochondrial unfolded protein response. Fly and breast tumor (MCF7)-xenografted mouse studies indicate that N-PPG doses sufficient to phenocopy PRODH knockout and induce its decay can be safely and effectively administered in vivo Among breast cancer cell lines and tumor samples, PRODH mRNA expression is subtype dependent and inversely correlated with glutaminase (GLS1) expression; combining inhibitors of PRODH (S-5-oxo and N-PPG) and GLS1 (CB-839) produces additive if not synergistic loss of cancer cell (ZR-75-1, MCF7, DU4475, and BT474) growth and viability. Although PRODH knockdown alone can induce cancer cell apoptosis, the anticancer potential of either reversible or irreversible PRODH inhibitors is strongly enhanced when p53 is simultaneously upregulated by an MDM2 antagonist (MI-63 and nutlin-3). However, maximum anticancer synergy is observed in vitro when the PRODH suicide inhibitor, N-PPG, is combined with both GLS1-inhibiting and a p53-upregulating MDM2 antagonist. These findings provide preclinical rationale for the development of N-PPG-like PRODH inhibitors as cancer therapeutics to exploit synthetic lethal interactions with p53 upregulation and GLS1 inhibition.