We examined the effects of ALOS4, a cyclic peptide discovered previously by phage library selection against integrin αvβ3, on a human melanoma (A375) xenograft model to determine its abilities as a potential anti-cancer agent. We found that ALOS4 promoted healthy weight gain in A375-engrafted nude mice and reduced melanoma tumor mass and volume. Despite these positive changes, examination of the tumor tissue did not indicate any significant effects on proliferation, mitotic index, tissue vascularization, or reduction of αSMA or Ki-67 tumor markers. Modulation in overall expression of critical downstream αvβ3 integrin factors, such as FAK and Src, as well as reductions in gene expression of c-Fos and c-Jun transcription factors, indirectly confirmed our suspicions that ALOS4 is likely acting through an integrin-mediated pathway. Further, we found no overt formulation issues with ALOS4 regarding interaction with standard inert laboratory materials (polypropylene, borosilicate glass) or with pH and temperature stability under prolonged storage. Collectively, ALOS4 appears to be safe, chemically stable, and produces anti-cancer effects in a human xenograft model of melanoma. We believe these results suggest a role for ALOS4 in an integrin-mediated pathway in exerting its anti-cancer effects possibly through immune response modulation.

Interferon (IFN) signaling resulting from external or internal inflammatory processes initiates the rapid release of cytokines and chemokines to target viral or bacterial invasion, as well as cancer and other diseases. Prolonged exposure to IFNs, or the overexpression of other cytokines, leads to immune exhaustion, enhancing inflammation and leading to the persistence of infection and promotion of disease. Hence, to control and stabilize an excessive immune response, approaches for the management of inflammation are required. The potential use of peptides as anti-inflammatory agents has been previously demonstrated. Our team discovered, and previously published, a 9-amino-acid cyclic peptide named ALOS4 which exhibits anti-cancer properties in vivo and in vitro. We suggested that the anti-cancer effect of ALOS4 arises from interaction with the immune system, possibly through the modulation of inflammatory processes. Here, we show that treatment with ALOS4 decreases basal cytokine levels in mice with chronic inflammation and prolongs the lifespan of mice with acute systemic inflammation induced by irradiation. We also show that pretreatment with ALOS4 reduces the expression of IFN alpha, IFN lambda, and selected interferon-response genes triggered by polyinosinic-polycytidylic acid (Poly I:C), a synthetic analog of viral double-stranded RNA, while upregulating the expression of other genes with antiviral activity. Hence, we conclude that ALOS4 does not prevent IFN signaling, but rather supports the antiviral response by upregulating the expression of interferon-response genes in an interferon-independent manner.

Tumors are heterogeneous, consisting of different cell subpopulations to allow tumor survival in variable conditions. We observed the survival of a small percentage of HCT116 cells under glucose starvation. We hypothesized that these cells represent a previously undescribed subpopulation able to adjust their metabolism in the absence of glucose. Here we aimed to characterize the metabolic and molecular properties allowing these cells to survive. We propagated individual clones from HCT116 cells and further analyzed a single clone descendants were able to survive glucose starvation. Biochemical and molecular analyses were performed under standard glucose conditions, under glucose starvation during adaptation period, and under glucose starvation after resumption of proliferation. Metabolic analysis by Seahorse revealed that surviving cells completely shifted their metabolism from glycolysis to oxidative phosphorylation. LC-MS metabolomics analysis revealed that during senescence-like adaptation the cells accumulate metabolites required for NAD synthesis. FAD, a co-factor involved in the TCA cycle, was not found in the dormant cells, while higher levels of carnosine, essential for ROS elimination, were observed. The metabolic shift toward enhanced oxidative phosphorylation was accompanied by increased production of ROS leading to DNA damage, reflected in an upregulation of the genes NUPR, ST6GAL1, and EGRF as observed by RNAseq. Cells that resumed proliferation were also characterized by enhanced expression of DNA damage repair genes and upregulated subunits of ATP-synthase, a key enzyme in oxidative phosphorylation. We suggest that cells predisposed to effective DNA repair have more time and capabilities to adjust their metabolism and to survive. Citation Format: Elimelech Nesher, Bar Levi, Hadas Levy, Raichel Cohen-Harazi, Igor Koman. Identification and molecular characterization of cancer cells survived under glucose starvation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6044.