Salicylates from plant sources have been used for centuries by different cultures to treat a variety of ailments such as inflammation, fever and pain. A chemical derivative of salicylic acid, aspirin, was synthesised and mass produced by the end of the 19th century and is one of the most widely used drugs in the world. Its cardioprotective properties are well established; however, recent evidence shows that it can also act as a chemopreventive agent. Its antithrombotic and anti-inflammatory actions occur through the inhibition of cyclooxygenases. The precise mechanisms leading to its anticancer effects are not clearly established, although multiple mechanisms affecting enzyme activity, transcription factors, cellular signalling and mitochondrial functions have been proposed. This review presents a brief account of the major COX-dependent and independent pathways described in connection with aspirin's anticancer effects. Aspirin's unique ability to acetylate biomolecules besides COX has not been thoroughly investigated nor have all the targets of its primary metabolite, salicylic acid been identified. Recent reports on the ability of aspirin to acetylate multiple cellular proteins warrant a comprehensive study to investigate the role of this posttranslational modification in its anticancer effects. In this review, we also raise the intriguing possibility that aspirin may interact and acetylate cellular molecules such as RNA, and metabolites such as CoA, leading to a change in their function. Research in this area will provide a greater understanding of the mechanisms of action of this drug.

Data emerging from the past 10 years have consolidated the rationale for investigating the use of aspirin as a chemopreventive agent; however, the mechanisms leading to its anti-cancer effects are still being elucidated. We hypothesized that aspirin’s chemopreventive actions may involve cell cycle regulation through modulation of the levels or activity of cyclin A2/cyclin dependent kinase-2 (CDK2). In this study, HT-29 and other diverse panel of cancer cells were used to demonstrate that both aspirin and its primary metabolite, salicylic acid, decreased cyclin A2 (CCNA2) and CDK2 protein and mRNA levels. The down regulatory effect of either drugs on cyclin A2 levels was prevented by pretreatment with lactacystin, an inhibitor of proteasomes, suggesting the involvement of 26S proteasomes. In-vitro kinase assays showed that lysates from cells treated with salicylic acid had lower levels of CDK2 activity. Importantly, three independent experiments revealed that salicylic acid directly binds to CDK2. Firstly, inclusion of salicylic acid in naïve cell lysates, or in recombinant CDK2 preparations, increased the ability of the anti-CDK2 antibody to immunoprecipitate CDK2, suggesting that salicylic acid may directly bind and alter its conformation. Secondly, in 8-anilino-1-naphthalene-sulfonate (ANS)-CDK2 fluorescence assays, pre-incubation of CDK2 with salicylic acid, dose-dependently quenched the fluorescence due to ANS. Thirdly, computational analysis using molecular docking studies identified Asp145 and Lys33 as the potential sites of salicylic acid interactions with CDK2. These results demonstrate that aspirin and salicylic acid down-regulate cyclin A2/CDK2 proteins in multiple cancer cell lines, suggesting a novel target and mechanism of action in chemoprevention. Implications Biochemical and structural studies indicate that the anti-proliferative actions of aspirin are mediated through cyclin A2/CDK2.

Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first reaction in the pentose phosphate pathway, and generates ribose sugars, which are required for nucleic acid synthesis, and nicotinamide adenine dinucleotide phosphate (NADPH), which is important for neutralization of oxidative stress. The expression of G6PD is elevated in several types of tumor, including colon, breast and lung cancer, and has been implicated in cancer cell growth. Our previous study demonstrated that exposure of HCT 116 human colorectal cancer cells to aspirin caused acetylation of G6PD, and this was associated with a decrease in its enzyme activity. In the present study, this observation was expanded to HT-29 colorectal cancer cells, in order to compare aspirin-mediated acetylation of G6PD and its activity between HCT 116 and HT-29 cells. In addition, the present study aimed to determine the acetylation targets of aspirin on recombinant G6PD to provide an insight into the mechanisms of inhibition. The results demonstrated that the extent of G6PD acetylation was significantly higher in HCT 116 cells compared with in HT-29 cells; accordingly, a greater reduction in G6PD enzyme activity was observed in the HCT 116 cells. Mass spectrometry analysis of aspirin-acetylated G6PD (isoform a) revealed that aspirin acetylated a total of 14 lysine residues, which were dispersed throughout the length of the G6PD protein. One of the important amino acid targets of aspirin included lysine 235 (K235, in isoform a) and this corresponds to K205 in isoform b, which has previously been identified as being important for catalysis. Acetylation of G6PD at several sites, including K235 (K205 in isoform b), may mediate inhibition of G6PD activity, which may contribute to the ability of aspirin to exert anticancer effects through decreased synthesis of ribose sugars and NADPH.

Aspirin's ability to inhibit cell proliferation and induce apoptosis in cancer cell lines is considered to be an important mechanism for its anti-cancer effects. We previously demonstrated that aspirin acetylated the tumor suppressor protein p53 at lysine 382 in MDA-MB-231 human breast cancer cells. Here, we extended these observations to human colon cancer cells, HCT 116 harboring wild type p53, and HT-29 containing mutant p53. We demonstrate that aspirin induced acetylation of p53 in both cell lines in a concentration-dependent manner. Aspirin-acetylated p53 was localized to the nucleus. In both cell lines, aspirin induced p21(CIP1). Aspirin also acetylated recombinant p53 (rp53) in vitro suggesting that it occurs through a non-enzymatic chemical reaction. Mass spectrometry analysis and immunoblotting identified 10 acetylated lysines on rp53, and molecular modeling showed that all lysines targeted by aspirin are surface exposed. Five of these lysines are localized to the DNA-binding domain, four to the nuclear localization signal domain, and one to the C-terminal regulatory domain. Our results suggest that aspirin's anti-cancer effect may involve acetylation and activation of wild type and mutant p53 and induction of target gene expression. This is the first report attempting to characterize p53 acetylation sites targeted by aspirin.

Epidemiological studies have demonstrated a significant correlation between regular aspirin use and reduced colon cancer incidence and mortality; however, the pathways by which it exerts its anti-cancer effects are still not fully explored. We hypothesized that aspirin's anti-cancer effect may occur through downregulation of c-Myc gene expression. Here, we demonstrate that aspirin and its primary metabolite, salicylic acid, decrease the c-Myc protein levels in human HCT-116 colon and in few other cancer cell lines. In total cell lysates, both drugs decreased the levels of c-Myc in a concentration-dependent fashion. Greater inhibition was observed in the nucleus than the cytoplasm, and immunofluorescence studies confirmed these observations. Pretreatment of cells with lactacystin, a proteasome inhibitor, partially prevented the downregulatory effect of both aspirin and salicylic acid, suggesting that 26S proteasomal pathway is involved. Both drugs failed to decrease exogenously expressed DDK-tagged c-Myc protein levels; however, under the same conditions, the endogenous c-Myc protein levels were downregulated. Northern blot analysis showed that both drugs caused a decrease in c-Myc mRNA levels in a concentration-dependent fashion. High-performance liquid chromatography (HPLC) analysis showed that aspirin taken up by cells was rapidly metabolized to salicylic acid, suggesting that aspirin's inhibitory effect on c-Myc may occur through formation of salicylic acid. Our result suggests that salicylic acid regulates c-Myc level at both transcriptional and post-transcription levels. Inhibition of c-Myc may represent an important pathway by which aspirin exerts its anti-cancer effect and decrease the occurrence of cancer in epithelial tissues.

Epidemiological studies have demonstrated a significant correlation between regular aspirin use and reduced colon cancer incidence and mortality. Although a consensus is emerging on the view that aspirin has significant anti-cancer properties, it is not yet clear which pathways or molecular targets might be involved. We hypothesized that aspirin's anti-cancer effect may occur through acetylation of a panel of protein targets, including novel candidates with modulated functional activity, as a result of aspirin-mediated acetylation. In the present study, using anti-acetyl lysine antibody, we determined aspirin-mediated protein acetylation profiles in three different colon cancer cell lines namely HCT-116, HT-29 and GC3/c1, by treating cells with aspirin at different concentration, and analyzing them in Western Blot. We observed that in all cell lines tested, aspirin induced acetylation of multiple proteins in a concentration dependent fashion spanning a molecular weight range from 20 to 200 kDa. Aspirin's acetylation targets included the tumor suppressor protein p53, and glucose 6 phosphate dehydrogenase (G6PD), an enzyme in the pentose phosphate pathway. Mass spectrometry analysis of the recombinant G6PD, treated with aspirin in vitro, revealed acetylation of up to 14 distinct lysine residues, including lysine 235 at its active site. In vitro G6PD assay in lysates isolated from the cancer cell lines showed that aspirin caused a 70% decrease in the G6PD enzyme activity in HCT116 cells; however, the reduction in the enzyme activity was less in HT-29/GC3/c1 cells (up to 30%). Inhibition of G6PD activity leading to a reduction in ribonucleotide synthesis and cell proliferation may represent an important mechanism by which aspirin exerts its anticancer effects. Citation Format: Guoqiang Ai, Fred K. Hagen, Jayarama B. Gunaje. Aspirin acetylates glucose 6 phosphate dehydrogenase and inhibits its activity in colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3681. doi:10.1158/1538-7445.AM2013-3681

4034 Background: EpCAM is highly expressed in various cancer types of the GI system and at metastatic sites. It serves as a promising prognostic/predictive marker (CTC) and therapeutic target. IMC001 is an EpCAM-targeted CAR-T cell that showed promising anti-tumor activities in preclinical studies. Here, we report the results of the low and middle dosage groups in advanced gastric cancers (CT03) and GI cancers (CT04). Methods: This first-in-human, open-label trial involved two separate single-site trials. Both followed a classic 3+3 design with dose escalation of 0.3, 1 or 3 million CAR-T cells/kg after lymphodepletion chemotherapy. CT03 was an IMC001 monotherapy trial (Stage 1) for gastric cancer, while CT04 had Stage 1 and the combination with RFA or microwave ablation (Stage 2) for GI cancers. Eligible patients were those with EpCAM-positive (more than 10%) cancers who had no further standard treatment options and were ECOG 0 or 1. The objective was to assess the safety, PK/PD profile and preliminary efficacy of IMC001. Results: As of January 30, 2023, 12 patients with 6 colorectal and 6 gastric cancers had enrolled, with half receiving 0.3 million and the other half receiving 1 million cells/kg IMC001 infusion. No patient experienced DLT within the 4-week follow-up visits after infusion. All patients experienced ≥Grade 3 hematologic toxicity. One patient in the low-dose group had a SAE of immune hepatitis (Grade 3), which might have been related to cell therapy, and occurred around 11 days after the CAR-T infusion, prolonging the patient's hospitalization. Manageable CRS (Grade 1 to 3) and no ICANS were observed. Other adverse events related to cell therapy were CTCAE Stage 1-2 nausea, vomiting, asthenia, or pruritus, and these recovered quickly. Analyses of the CAR-T cells in peripheral blood revealed robust engraftment in all patients, with the peak number of CAR+ cells reaching on day 5-7 after infusion. CTC remained under the detection limit for more than 40 weeks after cell infusion. Significant elevations of serum levels of IL-6, IP-10, IFN-γ, IL-15, and MCP-1 were observed in most patients. Preliminary efficacy data showed that 2 out of 6 advanced gastric cancer patients were evaluated as PR and 3 remained SD by RECIST 1.1 at the low and middle dosages (CT03). The first PR patient received a second IMC001 infusion on week 50 and had survived for more than 60 weeks. The second PR patient underwent successful surgical removal of the stomach 28 weeks after IMC001 infusion. The CT04 trial is ongoing. Conclusions: This is the first CAR-T therapy ever tested in humans targeting the EpCAM. IMC001 showed a favorable safety profile and reasonable anti-tumor activities at the low and middle dosage levels in patients with advanced EpCAM+ GI cancers, especially in gastric cancer. In addition, our trial has successfully provided a surgical treatment opportunity after CAR-T therapy downstaging of unresectable gastric cancers. Updated data from open cohorts will be presented. Clinical trial information: NCT05028933 .

Aspirin's ability to inhibit cell proliferation and induce apoptosis in cancer cell lines is considered to be an important mechanism for its anticancer effects. We previously demonstrated that aspirin acetylated the tumor suppressor protein p53 at lysine 382 in MDA-MB-231 human breast cancer cells. Here, we extended these observations to human colon cancer cells, HCT 116 harboring wild type p53, and HT-29 containing mutant p53. Aspirin induced acetylation of p53 in both cell lines in a concentration-dependent manner. Aspirin-acetylated p53 was localized to the nucleus. In both cell lines, aspirin induced p21CIP1. Aspirin also acetylated recombinant p53 (rp53) in vitro suggesting that it occurs through a non-enzymatic chemical reaction. Mass spectrometry analysis and immunoblotting identified 10 acetylated lysines on rp53, and molecular modeling showed that all lysines targeted by aspirin are surface exposed. Five of these lysines are localized to the DNA-binding domain, four to the nuclear localization signal domain, and one to the C-terminal regulatory domain. Our results suggest that aspirin's anticancer effect may involve acetylation and activation of wild type and mutant p53 and induction of target gene expression. This is the first report attempting to characterize p53 acetylation sites targeted by aspirin. Citation Format: Guoqiang Ai, Rakesh Dachineni, Ramesh D. Kumar, Srinivasan Marimuthu, Lloyd F. Alfonso, Jayarama G. Bhat. Aspirin acetylates wild type and mutant p53 in colon cancer cells: Identification of aspirin acetylated sites on recombinant p53. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3699.