Are platelets the primary target of aspirin’s remarkable anti-cancer activity?

Abstract

Aspirin, when administered at low-doses, has emerged as a powerful anti-cancer drug due to both chemopreventive activity against many forms of cancer and its ability to block metastases when administered post-diagnosis. Platelets, which are often elevated in circulation during the latter stages of cancer, are known to promote epithelial-mesenchymal transition, cancer cell growth, survival in circulation, and angiogenesis at sites of metastases. Low-dose aspirin has been demonstrated to block these pro-carcinogenic action of platelets. In this perspective, we present evidence that aspirin’s unique ability to irreversibly inhibit platelet cyclooxygenase-1 is a key mechanism by which aspirin exerts anti-cancer activity.

Introduction:

One of the oldest and least appreciated observations in the field of oncology, is that of Armand Trousseau in 1865 that late stage cancer patients frequently develop thrombosis/thrombophlebitis – which is now a syndrome bearing his name (1). This pioneering observation predated the discovery of platelets in 1882, that are anucleated blood cells originating from megakaryocytes. Platelets are best known to promote hemostasis and thrombosis and have gained attention in oncology due to their role in cancer progression (2). Patients at the latter stage of cancer affecting the breast, colon, gastric, lung, ovary and prostate often have thrombocytosis (elevated platelet counts) (2). Furthermore, the efficacy of chemotherapy is attenuated in cancer patients with high platelet counts. Tumor educated platelets display altered RNA profiles and their potential value as biomarkers using analytics called “liquid biopsies” is currently being explored (3). Thus, the fundamental question is the importance of platelets to the progression and metastatic spread of cancer vs their elevation in circulation being a secondary consequence of the disease itself.

Aspirin, the first nonsteroidal anti-inflammatory drug (NSAID) discovered (in 1897) and widely used to reduce fever, pain, and platelet activity, has emerged as a powerful agent in secondary prevention of cancer including colorectal cancer (4, 5). Aspirin has the unique capability of inhibiting both isoforms of cyclooxygenase (COX-1 and COX-2) to reduce the synthesis of eicosanoids which will be subsequently converted via cell-specific synthases into biologically active prostaglandins (PGE_2, PGF_2α, PGI₂) and thromboxane A₂ (TXA₂). Of particular importance to both its cardiovascular and cancer indications are the drug’s unique ability to irreversibly inhibit platelet COX-1 (via acylation of serine 530), preventing platelet activation over the remaining lifetime of the affected platelets. Aspirin also shares the ability with other NSAIDs to non-specifically inhibit COX-2, which is overexpressed in many cancers as reported by DuBois and associates (6) Since PGE₂ promotes tumor proliferation, differentiation and angiogenesis, it is widely considered that the anti-cancer effect of aspirin and related NSAIDs resides primarily in their COX-2 inhibitory activity, which is supported by preclinical and clinical evidence that COX-2 selective inhibitors (coxibs) possess chemopreventive activity.

In this article, we will present our perspective that the primary target of low-dose aspirin’s anti-cancer activity is platelet COX-1, thereby preventing platelets from inducing epithelial-mesenchymal transition (EMT) and the downstream pro-carcinogenic responses including the induction of COX-2. Thus, we feel this action of low dose aspirin should be studied more broadly to further elucidate its contribution and that of related anti-platelet agents, in cancer prevention/therapy. Our perspective of highlighting the anti-platelet action of aspirin should not be interpreted as a dismissal of the evidence supporting the contribution of COX-2 to cancer progression and the potential utility of coxibs in cancer prevention/therapy, as an appreciation of both COX isoforms is important.

Evidence that platelets promote cancer cell proliferation, migration, survival and metastasis:

A compelling body of evidence can be found in the scientific literature which supports the role of platelet-cancer cell interaction in the progression of cancer from its induction at sites of inflammation to the development and growth of metastases, which will be reviewed below. A bidirectional signaling crosstalk occurs between platelets and tumor cells in both the peripheral circulation and in the tumor microenvironment (2, 7). Tumor cells can induce platelet aggregation, promote secretion of platelet granular contents and generate platelet microparticles. Tumor-derived interleukin-6 (IL-6) stimulates megakaryopoiesis that support thrombocytosis. Conversely, platelets promote cancer cell growth, EMT and help evade immune attack (2, 7). Intravasation of circulating platelets into tissues within the tumor microenvironment undergoing inflammation and dysplasia can redirect cell properties to support pro-carcinogenic changes. Both cell culture and animal experiments suggest, eicosanoids like PGE₂ (generated by tumor COX-2) and TXA₂ (generated by platelet COX-1) (8) promotes cancer cell growth and proliferation. Platelets facilitate angiogenesis by secreting pro-angiogenic factors like vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), basic fibroblast growth factor (bFGF) from their α granules. Platelet-derived TGF-β/SMAD signaling induces EMT in a diverse group of tissues and in doing so, increase migration and invasive activity required for tumor metastasis. Platelet-derived chemokine CCL-5/RANTES upregulates interleukin-8 (IL-8) in breast cancer cells to promote tumor invasion, (9). VEGF also promotes immunosuppression of cancer cells by facilitating the recruitment of tumor associated macrophages, myeloid-derived suppressor cells and T regulatory cells to the tumor micro-environment. Upon gaining access into circulation, tumor cells rely on platelets to overcome detachment induced apoptosis (anoikis) by activating Yes Associated Protein 1 (YAP1) signaling in tumor cells (2). Furthermore, platelets protect tumor cells from immune attack by suppressing T cells via the platelet TGFβ/GARP (glycoprotein A repetitions predominant) axis (10). Platelets contribute to neutrophil extracellular traps (NETS) that can engulf surrounding circulating cancer cells and thereby prevent attack by NK cells and macrophages in blood. Finally, platelet receptors facilitate cancer cell attachment to distal endothelium and mediate their arrest in the vasculature for metastasis. Thus, blood platelets can induce major changes in target tissues by promoting a transformation that can lead to dysplasia, cancer growth and metastatic spread. These pre-clinical observations suggest fertile opportunities for anti-platelet agents in cancer prevention and treatment.

Aspirin as a powerful anti-cancer agent:

It is well established that aspirin inhibits platelet reactivity by blocking COX-1 activity irreversibly and reducing TXA₂ production and because of this relationship low-dose aspirin drug is widely employed, in the management of cardiovascular disease. Consistent with the potential role of platelets in various stages of cancer progression in murine models, inhibition of platelet reactivity by aspirin attenuated: platelet-mediated proliferation of tumor cells (8), adhesion of tumor cells to the endothelium (11), aggregation of platelets on tumor cells (11), invasion of tumor cells facilitated by tumor IL-8 (9), and translocation and EMT of tumor cells (8) (12). Aspirin also inhibits angiogenesis which promotes cancer growth and metastases by markedly reducing the production/secretion of platelet pro-angiogenic factors like 15(S)-hydroxyeicosatetraenoic acid (HETE) and VEGF. Lastly, aspirin has been reported to enhance cancer immunotherapy by blocking the immunosuppressive action of platelets on T-cell function (10).

A large number of observational studies in human have strongly indicated that regular aspirin consumption is associated with a significant 20–40% reduction in the incidence of as many 20 different types of solid cancers with the strongest evidence for a survival advantage of chronic low-dose aspirin users against deaths attributable to colorectal cancer (CRC), ovarian, and breast cancer (4, 5, 13). These conclusions are primarily based on long-term data from the Nurses Health Studies (NHS) I-III. This compelling association between aspirin use and CRC, led the US Preventive Services Task Force to officially recommend in 2016 that older subjects (>50 years for males and >60 years of age for females) who have the greatest risk of cardiovascular disease should take low dose aspirin on a regular basis to reduce the risk of both myocardial infarcts and CRC) (14), under the proviso that they consult their physicians due to the fact that aspirin will cause GI ulceration and bleeding in sensitive individuals. It is also important to note that not all the studies have obtained positive data on aspirin’s chemo-preventive activity, notably recent findings of the Aspirin in Reducing Events in the Elderly (ASPREE) trial that subjects >70 years of age who took low-dose (100 mg) enteric coated aspirin on a daily basis showed no clinical benefit and in fact had a modestly higher death rate overall, which unexpectedly was attributed mostly to cancer-related mortality, that even surpassed deaths due to major hemorrhage (15) The reasons for these conflicting findings in the literature are uncertain, but may in part be explained by differences among the studies, with regard to: whether the study was designed as a Primary (ASPREE) or Secondary Prevention trial; the age of the subjects at the initiation of the study (ranging from 30 years in NHS to 70 years in ASPREE), the regularity of taking aspirin (once or twice/weekly vs 5–7 times/week); the aspirin dose consumed which ranged from 75 mg to 600mg/daily: and the dose form (enteric-coated vs immediate-release aspirin).

The life time of platelets is ~7 days and this may be further shortened during conditions of inflammation. In this context, if the major target of aspirin is platelets, then we argue that the consumption of NSAID only once/twice a week may lead to an increase in the subpopulation of platelets in circulation that are not inhibited by aspirin. In a recently published study, Lucotti et al. (11) provided compelling evidence using transgenic mouse models that, similar to the COX-1 selective inhibitor SC-560, aspirin reduced lung metastases in mice primarily by inhibiting platelet COX-1, the generation of thromboxane and the activation of platelet/cancer cell cross-talk. In contrast, inhibition of COX-2 did not prevent metastatic colonization. Furthermore, aspirin has a short half-life in circulation of <15 minutes, as it is rapidly metabolized to salicylic acid. Unlike aspirin, salicylic acid is a weak inhibitor of COX (requires millimolar range). Based on the pharmacokinetic studies, lower doses (75–100 mg) of orally ingested aspirin should be sufficiently effective to selectively inhibit platelet activation, as platelets cannot resynthesize COX-1. On the other hand, cancer cells can resynthesize COX-2, and therefore it is likely that the dose of circulating drug may not mount sustained inhibition of COX-2 (16). It therefore will be of interest to test if chronic consumption of low dose aspirin results in sustained concentrations of acetylsalicylic acid in circulation, which are sufficient to induce long-term repression of COX-2 in cancer cells as well. However, it should be noted that high doses of the drug in addition to inhibiting COX-2 may also have unanticipated targets due to non-specific protein acylation, which is a characteristic of aspirin.

The central and most provocative question for oncologists is whether aspirin administration post-cancer diagnosis has significant efficacy as a Secondary Preventive anti-cancer agent in reducing the growth, recurrence and metastatic spread of cancer. Building on the above and other pre-clinical studies that aspirin treatment was effective in reducing tumor size and metastases, a number of outcome studies have focused on post-diagnostic use of aspirin, in most cases as an adjunctive therapy. Most studies have demonstrated encouraging results, that the recurrence and/or metastatic spread of a number of cancers (colorectal, gastro-esophageal ovarian, breast and prostate cancer) were reduced in subjects who took aspirin post-diagnosis alone or together with other chemotherapeutic agents (17). In addition, consumption of aspirin has been reported to be significantly associated with a decrease in cancer-associated mortality and a prolongation of life (4, 17, 18). Based upon these observational findings, several prospective clinical trials are now recruiting patients that have an arm, where patients take aspirin either alone or as an adjunctive therapy after they were diagnosed with: breast cancer (Alliance trial); four solid cancers (CRC, breast, gastroesophageal, or prostate cancer, (ADD-ASPIRIN) (19); or subjects with Lynch Syndrome who have a genetic risk of developing colonic polyps or CRC (CAPP2). Although these studies are directing cancer patients to take aspirin at different doses ranging from 100 mg (ADD-ASPIRIN) to 300 mg (ADD-ASPIRIN and Alliance) to 600 mg (CAPP2), it is our recommendation that future studies focus on low dose immediate-release aspirin, as enteric coating appears to reduce the bioavailability of the drug. In support of using aspirin at low dose, Rothwell et al. who first reported the efficacy of aspirin to reduce cancer metastases and mortality post-diagnosis (17) has recently reported that low dose aspirin (75 mg) was most effective at reducing CRC – related mortality in non-obese patients (18). Thus, the aspirin dose most selective to irreversibly inhibit platelet activity appears to be the most effective anti-cancer dose. Lastly, in support of platelets being the primary target of the drug’s anti-cancer action, pre-clinical and clinical studies have reported that platelet ADP P2Y12 receptor antagonist, clopidogrel/Plavix^™ possesses chemopreventive properties (20). Furthermore, anti-platelet agents such as DG-041[PGE2 EP3 receptor blocker] and ticagrelor (a P2Y12 receptor antagonist) have been reported to block EMT (12). However, use of aspirin in cancer management is not without challenge. Variable resistance to aspirin in cancer patients could potentially confound the outcome. The effective dose of the drug should be able to balance the ability to block cancer growth with minimal changes in hemostasis.

Conclusion:

In our opinion, there is emerging evidence to support platelets as a key factor in the progression of many cancers, and the major target of low dose aspirin is platelet COX-1 as opposed to cancer cell COX-2. Based on these considerations, oncologists should be closely monitoring patients with Stage-2 or −3 cancers for a spike in circulating platelet number over the course of their illness, as potential biomarkers of metastases. We also feel a compelling case can be made for investigators to monitor COX-1 expression in tumor tissue/cells as diligently as they have for monitoring the expression of COX-2, as several studies have indicated that both COX isoforms are expressed in a number of cancers, though the majority of studies have focused only on COX-2 (16). Lastly, we recommend that consideration should be given by oncologists to include aspirin in the management of patients diagnosed with various forms of cancer for secondary prevention of cancer recurrence and metastases.