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Sarcomas remain to be very dangerous, challenging tumors in oncology. Chondrosarcomas, the cartilage cancer tumors are particularly refractory to chemotherapy and radiation. To investigate potential novel therapies, more must be understood about the cellular mechanisms driving chondrosarcoma proliferation and metastasis. PRP-1 neuropeptide has previously been shown by Dr. Galoian to have significant potential as a novel therapy for chondrosarcoma as a cytostatic, antiproliferative, and tumor-suppressive agent, leading to 90% inhibition of sarcoma growth and even triple-negative breast cancer cells. Interestingly, PRP-1 was proven for the first time by the team to be the ligand of innate immunity pattern recognition Toll-like receptors 1/2 and 6. The injured tumor cells have the ability to release danger-associated molecular patterns (DAMPs) and contribute to the activation of the innate immune system. This neuropeptide can upregulate most of the tumor suppressors, depending on the pathway they are involved in, and downregulate embryonic stem cell factors such as Nanog, polycomb protein Bmi-1, and c Myc. The resilience to chemotherapy and radiation of these tumors is a property bestowed by self-renewing and stroma-perpetuating cancer stem cells (CSCs). Lack of dependable treatment and poor prognosis warrants the search for novel therapies is critical. Most recently, Dr. Galoian’s team demonstrated the mechanism of such inhibition. PRP-1 showed the ability to almost eliminate CSCs and sarcosphere formation. Currently, Dr. Galoian’s laboratory is focused on understanding the epigenetic mechanisms underlying such effects while targeting cancer stem cells, responsible for tumor growth renewal and drug resistance. This peptide also targets the bulk of tumor cells by slowing down the cell cycle. Unlike many toxic chemotherapies, PRP-1 is a natural compound also with antimicrobial, anti-neurodegenerative, and immunomodulatory properties. Galoian’s laboratory is also involved in the search for potential biomarkers for disease prognosis and early detection. For the past years, Dr. Galoian is mentoring medical students at the University of Miami, who are determined to dedicate their careers to orthopedic surgery and cancer research. Our Lab performs a varied number of experiments including activity antiproliferative in tumor cell culture, proteomic and genomic assays. As a result, many new manuscripts have been published with our student’s active participation, leading to their research distinction awards and residency in orthopedics.
Proline-rich polypeptide (PRP-1) targets cancer stem cells (CSC) in chondrosarcoma
PRP-1, cytokine neuropeptide manifested itself as a strong antiproliferative peptide (Galoian at al., Neurochem Res., 2009 and 2011; Galoian at al., Tumour Biol. and Neurochem Res., 2011) inhibited mTOR serine/threonine-protein kinase (mTORC-1) (Galoian et al., Neurochem. Res. 2011). Also, PRP-1 treatment showed 80% inhibition in breast carcinoma cell line, MDA 231 estrogen negative breast cancer cell line (Galoian et al., Tumour Biol. 2011). Our laboratory was the first to demonstrate the presence of innate immune toll-like receptors (TRL) on the surface of chondrosarcoma cells, moreover, PRP-1 is a ligand for TLR-1,2 and TLR-6 (Galoian et al., Int J Oncol., 2018).
The CSC theory states that tumor growth is similarly triggered by small numbers of tumor stem cells. This population is responsible for the inevitable recurrence of tumors after initially successful chemotherapy and/or radiation therapy CSCs, sometimes called tumor-initiating cells are responsible for the phenomenon of tumor dormancy, and metastasis
In sorted by flow cytometry cell fractions, the PRP-1 inhibited the expression of ALDH1, the only stem cell marker for chondrosarcoma. The western blot experiments confirmed the results. The elimination of CSCs population corresponding to the ALDH1high pool was observed when treated with PRP-1 (Moran et al., Oncology Reports, 2020).
This data led us to the isolation of 3D spheroids from human chondrosarcoma cells which correspond to the CSCs model. Using colony formation and spheroid assays, we observed PRP-1 to target the specific CSC population in a powerful inhibitory dose-response manner (Granger et al., Molecular Medicine Reports, 2020).
This study won the first place in the SAMA Grand Rounds competition organized by Orthopaedic Surgery Department, Miller School of Medicine of the University of Miami. The presentation was accepted by the American Academy of Orthopedic Surgery Congress for the podium presentation in 2020.
CSCs have powerful tumorigenesis ability, self-renewal capacity, as well as chemotherapy and radiotherapy resistance. CSCs may be a critical driving force for several types of cancer. Selectively targeting CSCs with various agents may be a novel and promising therapeutic strategy against cancer.
Our future goals include creating bone and soft tissue sarcoma xenograft mice models to study the ability of PRP-1 and its analogs to shrink and destroy the primary tumor as a subsequent metastatic spread. Unlike many conventional chemotherapy drugs which do not target CSCs (A, B), we expect that PRP-1 will be able to eradicate CSCs and prevent disease relapse (C). Moreover, PRP-1 (being a natural brain neuropeptide) does not affect normal benign cells, while inhibiting malignant ones (Galoian et al., Neurochem Res, 2009) guaranteeing less toxicity associated with conventional chemotherapy side effects.
PRP-1 is a powerful effector of Endoplasmic Reticulum (ER) Stress and unfolded protein response (UPR)
Cancer can lead to the accumulation of toxic unfolded proteins resulting in excessive cellular cargo that alter endoplasmic reticulum (ER) functioning, a state known as ER stress. The cell responds to ER stress through a process known as “unfolded protein response” (UPR). Depending on cellular context UPR can be pro or anti oncogenic. In chondrosarcoma, UPR protein machinery is suppressed, (Galoian et al., Oncology Reports submitted, 2022) thus manifesting tumor-suppressive properties. The treatment with PRP-1 showed upregulation of UPR proteins such as IRE1α, PERK, ATF6, ATF4, EIF2α, XBP-1, and CHOP on both protein and mRNA levels (Galoian et al., Oncology Reports submitted, 2022).
We hypothesize that PRP-1 leads to activation of the PERK- peIF2α -ATF4-CHOP, which in turn activates IRE1α -XBP1, ATF6 branch.
This is the first indication that innate receptors ligand PRP-1 is able to regulate the branches of the UPR. Since the activation of the PERK- eIF2α -ATF4-CHOP and IRE1α -XBP1 branches we have observed in ALDHhigh and in spheroid corresponding fractions to cancer stem cells, we assume that this activation has an important role in the ability of PRP-1 drastically decrease the CSC. Our future efforts will be directed to understanding the link between UPR pathways, TLR signaling, and its epigenetic regulation which leads to CSC targeting with PRP-1 treatment.
PRP-1 as epigenetic modulator leading to inhibition of sarcoma growth
Downregulation of oncogenes and upregulation of tumor suppressors was reported by our group (Galoian et al., Neurochem. Res., 2009 and (Galoian et al., Int. J Oncol. 2015).
Upregulation of desmosomal proteins is attributed to PRP-1 ability to upregulate most of the tumor suppressors in human chondrosarcoma (Galoian et al., Mol Clin Oncol. 2015), depending on the molecular pathway they represent, for example, it did not inhibit Hedgehog signaling pathway tumor suppressors (Galoian et al., Mol Clin Oncol. 2016).
On the epigenectic level, PRP-1 was able to down regulate onco (miR509-3p, miR589, miR490-3p, and miR 550) and up regulate tumor suppressor miRNAs (miR20a, miR125b, and miR192) (Galoian et al., Tumour Biol. 2014). Also, the treatment with PRP-1 reduced the expression of the miR302-367, which targets the embryonic stem cell marker Nanog and Bmi-1 (Galoian et al., Int. J Oncol. 2015)
The H3K9-specific histone demethylase is an enzyme that represses tumor suppressor genes and contributes to cancer progression. PRP-1 also showed the ability to inhibit H3K9 (Galoian et al., Mol Clin Oncol. 2015).
Continuing our epigenetic studies, PRP-1 is shown to be an inhibitor of SWI/SNF(BAF) chromatin remodeling complexes (Moran et. al., Oncol Rep. 2020).