Aspirin in hepatocellular carcinoma

Abstract

Pre-clinical and clinical studies provide evidence for aspirin as a preventative agent for cancer. Compelling direct evidence supports a chemopreventive effect of aspirin in individuals at high risk of developing colorectal cancer (CRC) due to Lynch syndrome, while indirect evidence indicates that aspirin may reduce the risk of and mortality from sporadic CRC. There is weaker evidence for a protective effect of aspirin against all cancers taken as a group. Nevertheless, the results of recent retrospective cohort studies consistently indicate a beneficial effect of aspirin as a chemopreventive or adjuvant chemotherapeutic agent in hepatocellular carcinoma (HCC). Epidemiological studies conducted in the general population or in selected populations at higher risk for HCC reveal that regular aspirin use is associated with reduced HCC incidence. In addition, aspirin may act as an adjuvant to other therapies in reducing HCC recurrence. According to studies in animal models, the cancer-preventative effect of aspirin may be related to its antiplatelet and anti-inflammatory activities. Prospective studies are warranted to determine whether aspirin should be recommended to diverse populations of patients at risk for HCC.

Introduction

Aspirin is a nonsteroidal anti-inflammatory drug (NSAID) which inhibits the activity of cyclooxygenase (COX)-1 and/or COX-2, resulting in decreased prostanoid biosynthesis. In contrast to other NSAIDs, aspirin at low doses (75–100 mg) functions as an antiplatelet drug by covalently modifying COX-1 expressed in mature platelets. Higher doses of aspirin (650–1,300 mg) are required to cause COX-2 acetylation in inflammatory cells and therefore its analgesic and anti-inflammatory effects. Nowadays, aspirin is most commonly used at low daily doses for the secondary prevention of cardiovascular diseases (CVD), while its use in primary prevention of CVD is still unclear (1).

In addition to aspirin’s cardioprotective benefits, experimental and clinical evidence indicates chemopreventive and/or chemotherapeutic properties against cancer. The chemopreventive effect of aspirin in reducing the risk of hereditary colorectal cancer (CRC) is supported by strong evidence from a placebo-controlled randomized clinical trial (RCT) in patients with Lynch syndrome (2, 3) and to a lesser extent in patients with Familial Adenomatous Polyposis (4, 5). Epidemiological data deriving from large CV prevention trials provide moderate evidence for a beneficial effect of aspirin on sporadic CRC incidence and mortality (6). Indeed, the use of low-dose aspirin is recommended by international societal guidelines to prevent CRC in high-risk patients or in certain patients who are already likely to benefit from aspirin for CV prevention (1). There is weaker evidence for a protective effect of aspirin against other cancers taken as a group or from cancers other than CRC. However, recent retrospective cohort studies support a chemoprotective and/or adjuvant chemotherapeutic role of aspirin in hepatocellular carcinoma (HCC). As for CRC (7–9), the inverse association between aspirin use and HCC risk or mortality appears to depend on the dose and/or duration of treatment.

Here, we review the evidence supporting the effect of aspirin in HCC and discuss the molecular basis of aspirin’s chemopreventive and adjuvant chemotherapeutic effects.

Aspirin and HCC

The investigation of the chemopreventive effect of aspirin in HCC started in the late 1990s with retrospective studies in the general population including all causes of HCC (Table 1). Further investigations were carried out in individuals at higher risk for HCC, namely chronic hepatitis B virus (HBV), chronic hepatitis C virus (HCV) or alcoholic cirrhosis (Table 1). Additional studies were performed to investigate the effect of adjuvant aspirin therapy on cancer recurrence in patients with HCC after locoregional or systemic treatment.

Table 1.

Population studies of aspirin in hepatocellular carcinoma (HCC)

Study (Publication Year, Reference)	Study Type	Comparison	Type of participants	Number of participants	Males (%)	Age (years)	Follow-up	Cancer outcome	Side effects
NIH-AARP Diet and Health Study (2012)23	Population-based cohort study	Aspirin users versus nonusers (self-reported)	Participants in the NIH-AARP Diet and Health Study	300,504 (HCC=250)	58.4	50–71 range (mean, 62.8)	10–12 years	HCC incidence (RR= 0.59; 95% CI= 0.45–0.77); mortality due to chronic liver disease (RR= 0.55; 95% CI= 0.45–0.67)	NR
Liver Cancer Pooling Project (2015)24	Pooled data from ten U.S.-based prospective cohort studies	Aspirin users versus nonusers (self-reported)	Individuals taking aspirin for prevention purpose	1,084,133 (HCC= 679)	35.9	NR	11.9 years	HCC incidence (RR = 0.68; 95% CI= 0.57–0.81)	NR
Simon TG et al. (2018)25	Pooled data from two U.S.-based prospective cohort studies	Aspirin users versus nonusers (self-reported)	Individuals taking aspirin for headache, pain, and CV prevention	133,371 (HCC= 108)	38.6	30–75 (mean, 62 for women and 68 for men)	More than 26 years	HCC incidence (Adjusted HR= 0.51; 95% CI= 0.34–0.77)	NR
National Cohort Study of Korean Adults (2018)26	Population-based cohort study	Aspirin users versus nonusers (prescription database)	Individuals ≥40 years of age who went to the Korean national health-examination service at least once between 2002–2006	460,755 (HCC= 2,336)	53.6	50 (median, 44–59)	6 years	HCC incidence (RR= 0.87; 95% CI= 0.77–0.98)	NR
Seoul National University Hospital Study (2017)29	Population-based cohort study	Antiplatelet [Aspirin (100 mg, 61.5% of the treated group), clopidogrel (75 mg; 16%), dual therapy (22.5 %)] users versus nonusers	Chronic HBV patients with suppressed HBV by antiviral treatment taking antiplatelet drugs for CVD prevention	1,674 (HCC= 63)	64.2 (control group) and 60.8 (antiplatelet group)	50.3±10.8 (mean ± SD, control group) and 55.2 ± 11.0 (mean ± SD, antiplatelet group)	57 months (median)	HCC incidence: in time-varying Cox Proportional analyses (HR, 0.44; 95% CI, 0.23–0.85); in propensity score–matched pairs, HR, 0.34; 95% CI, 0.15–0.77)	Bleeding events: antiplatelet drugs HR, 3.28; 95% CI, 1.98–5.42; only aspirin HR, 1.11; 95% CI, 0.48–2.54
Taiwan National Health Insurance Research Study (2019)30	Retrospective population-based cohort study	Aspirin (≤100 mg) users versus nonusers (Prescription database). Aspirin was taken for CV prevention	HBV-related HCC patients	10,615	72.4	58.7 (median)	From 180 days after starting aspirin to up to 5 years	HCC occurrence (HR, 0.71; 95%CI, 0.58–0.86)	Cumulative incidence of Peptic Ulcer Bleeding (P=0.09)
Taiwan Longitudinal Health Insurance Study (2020)31	Retrospective population-based cohort study	Aspirin users versus nonusers	HCV-related HCC patients	3,822 (HCC=278)	47.0%	64.6±14.4 (mean ± SD, non-aspirin group) 64.3±13.5 (mean ± SD, aspirin group)	Up to 12 years	HCC occurrence (Adjusted HR, 0.56, 95% CI, 0.43–0.72)	NR
Taiwan National Health Insurance Research Study (2020)32	Retrospective population-based cohort study	Aspirin (≤100 mg) users versus nonusers (prescription database). Aspirin was taken for CV prevention	HCV-related HCC patients	7,434	44.3%	64.1 (median)	4 years (median)	HCC occurrence (HR, 0.78, 95% CI, 0.64–0.95); 10-year cumulative incidences of HCC (11.23%, 95% CI: 9.31–13.15 in aspirin group versus 14.89%, 95% CI: 12.32–17.46 in untreated group; P< 0.001)	10-year cumulative incidence of Peptic Ulcer Bleeding (4.85%, 95% CI: 3.58–6.12 in aspirin group versus 3.71%, 95% CI: 2.73–4.69 in untreated group; P= 0.369)
Kangwon National University Hospital Study (2020)33	Retrospective population-based cohort study	Aspirin (100 mg; prescribed to prevent CV disease) users versus nonusers	Patients with alcoholic cirrhosis who abstained from alcoholic drinking	949 (HCC= 133)	72.3% (non-aspirin group); 67.0% (aspirin group)	57.9±12.2 (mean±SD, non-aspirin group) 64.6±10.8 (mean±SD, aspirin group)	3.1 years (median)	HCC development: in time-varying Cox Proportional analyses (aHR, 0.13; 95% CI, 0.08–0.21); in propensity score–matched pairs, HR, 0.14; 95% CI, 0.09–0.22)	GI bleeding: (aHR: 0.81; 95% CI: 0.45–1.44)
Nationwide Swedish registries study (2020)34	Retrospective population-based cohort study	Aspirin (≤ 160 mg; prescribed to prevent CV disease) users versus nonusers	HBV and HCV-related HCC patients	50,275 (HCC= 1,612)	64.1%(non-aspirin group); 71.6 % (aspirin group)	Age at notification of HBV or HCV: 39.6±13.5 (mean± SD, non-aspirin group) 50.5±13.0 (mean± SD, aspirin group	7.9 years (median)	HCC incidence (aHR: 0.69; 95% CI, 0.62 to 0.76); Liver-related mortality (aHR: 0.73; 95% CI, 0.67 to 0.81).	Major GI bleeding (HR:1.06; 95% CI: 0.94–1.18)
Li JH et al. (2016)39	Retrospective matched-based study	Aspirin full dose users (100 mg/day for more than 3 months), aspirin non-full dose users (100 mg intermittently for more than 3 months) versus nonusers	Patients with unresectable HCC treated with transarterial chemoembolization	120 (46 patients were full dose aspirin users)	80	67.2 ± 15.5 (mean ± SD, aspirin users); 66.0 ± 15.6 (mean ± SD, aspirin non-users);	25.4 (median range 4–82.2)	Overall survival (months) after transarterial chemoembolization: 32.5 (median, non-aspirin users) versus 20.3 (median, aspirin users); P=0.05	Deaths due to GI bleeding: NS
Boas FE et al. (2018)40	Retrospective study	Aspirin (81–325 mg)	HCC patients after transarterial embolization	304 (42 patients on aspirin)	76	69	12 months	Overall survival (months, median) after transarterial embolization: 57 (non-aspirin users) versus 23 (aspirin users); p= 0.008	NR
Taiwan National Health Insurance Research Study (2016)41	Population-based cohort study	Antiplatelet [Aspirin (100 mg; 88 % of the treated cohort); clopidogrel (75 mg; 5%) or dual therapy (7%)] users versus nonusers (prescription database)	HBV-related HCC patients who had undergone liver resection	2,210	83.9	62.3 ± 9.8 (mean± SD, cases) 62.2 ± 9.9 (mean ± SD, controls)	1.27 ± 1.31 years (mean)	HCC recurrence (HR, 0.73; 95 % CI 0.63–0.85); risk of mortality after resection (HR, 0.57; 95 % CI 0.45–0.71)	Upper GI bleeding (OR 1.91; 95 % CI 1.49–2.44)
Taipei Veterans General Hospital Study (2020)42	Retrospective population-based cohort study	Aspirin (3.5 % of the subjects). Aspirin was taken for CV prevention.	HBV-related HCC patients who had undergone liver resection	430 (10.9% had recurrence)	85.1	58.4 ± 11.9 (mean± SD)	50.3 months (median)	Recurrence-free survival (HR, 0.18; 95% CI 0.05–0.73); overall survival	NR
Casadei-Gardini A et al. (2020)44	Retrospective cohort study	Sorafenib alone or in combination with aspirin (≤ 100 mg)	Advanced or intermediate-stage HCC patients	304 (93 on aspirin)	89.1	Median 70 years (range 25–88 years)	39 months	Overall survival: HR 0.57; P < 0.0001	All grade of toxicity: NS

CI= confidence interval; CV= cardiovascular; GI= gastrointestinal; HR= hazard ratio; NR= not reported; NS= not statistically significant; OR=odds ratio; RR= relative risk.

Epidemiology and prevention of HCC

Liver cancer is the fourth most common cause of cancer mortality in the world (10). HCC is the main histologic type of primary liver cancer and accounts for approximately >80% of cases (11). HCC develops in most cases as a result of underlying chronic liver disease (CLD), such as HBV, HCV, excessive alcohol consumption, smoking, aflatoxin, non-alcoholic steatohepatitis, metabolic syndrome, obesity and diabetes (12).

HCC has a very poor overall 5-year survival rate of less than 20% (13). The highest rates of survival in patients with HCC are with those who have a single tumor or a small number of small tumors and who undergo definitive therapy with liver transplantation, which is associated with 5-year survival of greater than 80% (14). Surgical resection is the standard treatment for early-stage HCC in patients without major comorbidity or extrahepatic metastasis; however, the rate of recurrence of HCC or development of new tumors at 5-years is as high as 75% (14–16). Local or systemic pharmaceutical treatments for HCC have only limited efficacy.

Since at least 60% of HCC can be attributed to underlying HBV or HCV, effective control of the viral infection reduces the risk of HCC (17). HBV can be prevented by immunization, and antiviral treatment of HBV infection is highly effective at blocking viral replication, but it seldom leads to elimination of the virus. Therefore, HBV treatment often must be taken indefinitely (18).

HBV universal vaccination programs started in the nineties and were proven to be safe and efficacious, providing greater than 98% protection with the three-dose series (https://www.who.int/news-room/fact-sheets/detail/hepatitis-b). However, HBV vaccine is still not universally accessible and vaccine escape mutations may reduce the success of the existing prevention strategy. Consequentially, HBV infection is still endemic in areas of sub-Saharan Africa and South-East Asia and these populations are developing HCC at high rates (17).

Antiviral therapy consists of nucleos(t)ide analogues (NAs), such as entecavir and tenofovir, which are effective in preventing or delaying HCC development in some HBV carriers with cirrhosis and/or active hepatitis (19, 20) and in reducing the risk of recurrence among patients with HBV-related HCC undergoing hepatic resection (20, 21). However, therapy with NAs cannot completely eliminate the risk of HCC in patients who have cirrhosis, and their high cost limits the access to these drugs (22).

An HCV vaccine is not yet available and HCV infection is mostly prevented by avoiding other people’s blood (17). Therapy to cure chronic HCV infection is now possible with an 8–12 week course of direct acting anti-virals (17). Similar to treatment of HBV, elimination of HCV greatly decreases the risk of incident HCC, but in persons with cirrhosis significant residual risk persists for up to a decade or more (20).

Other than treatment of chronic HBV or HCV, there are no approved chemopreventive agents that can reduce the risk of recurrent HCC in patients undergoing surgical resection with a curative intent.

Aspirin and primary prevention of HCC in the general population

The first suggestion of a chemopreventive effect of aspirin in HCC arose from the NIH-AARP (National Institutes of Health – American Association of Retired Persons) Diet and Health Study, which reported that recall of aspirin use was associated with a 41% lower risk of HCC and 45% lower risk of mortality due to CLD as compared to nonuse (23). The Liver Cancer Pooling Project reported that aspirin use, but not ibuprofen, was associated with a 32% reduced risk of HCC (24). The reduction in HCC incidence was stronger with daily aspirin use and at lower doses (<163 mg), but was independent of treatment duration (24).

The pooled analysis of the Nurses’ Health Study (NHS) and the Health Professionals Follow-up Study (HPFS) reported that regular aspirin use was associated with a 49% reduction in HCC incidence, in both men and women (25). Aspirin provided a protective effect in patients with or without cirrhosis (25). The benefit from aspirin in HCC risk was dose (P trend = 0.006) and duration dependent (P trend = 0.03), appearing after 5 or more years of use of 1.5 or more standard-dose (325 mg) aspirin tablets per week, but the beneficial effect was lost 8 years beyond the last use (25).

Consistent with the results of the previous studies in patients from United States, the National Cohort Study of Korean Adults, which is considered a population at high risk for HCC due to endemic HBV infection in Korea, reported that aspirin use was associated with a dose related 13% reduction of HCC risk versus nonuse (P trend= 0.002) (26). In contrast, aspirin was not associated with a reduction in HCC mortality. The combination of aspirin with non-aspirin NSAIDs amplified the chemopreventive effect of aspirin alone, with a reduction of HCC risk by 35% versus non-users (26).

These retrospective studies carried out in the general population share a number of limitations including: non-randomized allocation of treatment, variable dosage and duration of aspirin therapy (self-reported or derived from a prescription database), variable causes of liver disease, incomplete adjustment for confounding factors, such as age, sex, cirrhosis, HBV/HCV status, alcohol use, use of HBV antiviral therapy or other potential chemopreventive drugs (i.e. statins, metformin, non-aspirin NSAIDs). Moreover, aspirin and other NSAIDs are generally not recommended in patients with cirrhosis. These drugs can increase GI bleeding from esophageal varices or portal hypertensive gastropathy (27) and trigger hepatorenal syndrome by inducing renal vasoconstriction and reducing glomerular filtration rate (28). Consequently, this may have caused a potential selection bias.

Since the incidence of HCC is very low in the general population, more recent investigations were carried out in patients with specific liver disease-related risk factors for HCC.

Aspirin and primary prevention of HCC in persons at risk

The first retrospective analysis of populations at risk for HCC assessed whether aspirin (61.5% of all patients in the antiplatelet group) or clopidogrel use was associated with incident HCC in Korean HBV patients (29). Antiplatelet use was associated with a 54% reduction in HCC risk as compared to nonuse. Aspirin use alone was associated with a 37% to 56% lower risk of HCC incidence compared to nonuse, but had no effect on HCC-related mortality (29).

Similarly, a retrospective population-based cohort study of HBV patients from Taiwan reported that low-dose use aspirin was independently associated with a 29% reduction in HCC risk (30). However, in a multivariable stratified analysis, aspirin use was not significantly associated with a reduction of HCC risk in patients with underlying cirrhosis, or in patients taking nucleos(t)ide analogs. The beneficial chemopreventive effect in lowering the 5-year incidence of HCC was evident after 2 years of aspirin therapy (30).

A cohort study based on the Taiwan’s Longitudinal Health Insurance database reported that aspirin reduced HCC risk in HCV carriers by about 50% in both sexes (31). Similarly, a cohort study based on Taiwan’s National Health Insurance Research Database reported that the 5-year and 10-year cumulative incidence of HCC in patients with HCV taking aspirin was significantly lower than those in those without aspirin (P < 0.001), after adjusting for competing risk (32).

A retrospective cohort study of Korean patients with alcoholic cirrhosis reported that low-dose aspirin was associated with an 87% lower risk of HCC compared to nonuse (33).

A recent retrospective cohort study based on nationwide Swedish patient registries reported that treatment with low-dose aspirin in patient with HBV or HCV was associated with a 31% lower risk of HCC and 27% lower adjusted risk of liver-related mortality (34). The inverse relationship between aspirin use and risk of HCC appeared to be duration dependent, with a significantly lower risk of HCC after 3 to 5 years of use (35).

Consistent with the previous studies, a recent meta-analysis of pooled data from contemporary observational studies carried out in the general population or in patients with specific etiologies of liver disease reported a 41% reduction in HCC risk amongst aspirin users (36). Similarly, a meta-analysis of five epidemiological HCC primary prevention studies revealed that aspirin significantly decreases the risk of HCC occurrence by 46%. A similar effect was not observed with non-aspirin NSAIDs (37). Lately, a meta-analysis of seven cohort studies of patients with HBV or HCV infection reported that aspirin use was independently associated with a 27% reduction in HCC risk (38).

In some of the contemporary studies mentioned above, the chemopreventive effect of aspirin in HCC was assessed after adjusting for possible confounding factors based on detailed clinical and pathological characteristics. However, some studies lacked information regarding dose and the duration of aspirin therapy, HBV viral load or antigen status, HCV titer or genotype, alcohol consumption, smoking habit, fibrosis stage, HCC stage, use of other potential chemopreventive drugs (i.e. statins, metformin, non-aspirin NSAIDs) or GI side-effects.

Aspirin as an adjuvant treatment in established HCC

In addition to the emerging evidence in support of a chemopreventive effect of aspirin in HCC, epidemiological studies also revealed its potential as an adjuvant agent after locoregional or systemic treatment.

A small retrospective study reported that low-dose aspirin was associated with an improved overall survival compared to nonuse in patients with unresectable HCC who underwent transarterial chemoembolization (39). Similarly, aspirin use was associated with improved liver function and survival in HCC patients after transarterial embolization (40).

Among patients with HBV-related HCC undergoing liver resection in Taiwan, aspirin and/or clopidogrel use (88% of the cases received only low-dose aspirin) was associated with a 23% reduced risk of HCC recurrence and 43% reduction in overall mortality as compared to nonuse (41). However, antiplatelet use was also associated with a 90% increased risk of upper GI bleeding as compared to nonuse (41).

The Taiwanese Veterans study reported that aspirin use for CV prevention for at least 30 days before tumor resection was associated with an 82% reduction in HCC recurrence but did not change overall survival in HBV-infected patients undergoing curative resection (42).

Additionally, a meta-analysis of two epidemiological HCC studies showed that adjuvant aspirin therapy in patients with HCC significantly decreased 2-year and 4-year mortality (37), and a recent meta-analysis of six retrospective cohort studies in patients with HCC undergoing liver resection or transarterial chemoembolization reported that aspirin reduced HCC recurrence by 26% and all-cause mortality by 41% (43).

The benefit of aspirin as an adjuvant to systemic therapy is also being explored. A recent retrospective study revealed that aspirin in combination with sorafenib, an FDA-approved protein kinase inhibitor, prolonged overall survival in patients with advanced HCC (44).

These retrospective studies present several limitations, including restricted patient populations; incomplete adjustment for confounding factors; variable dose and duration of aspirin therapy; and lack of information on GI side effects. An ongoing clinical trial is investigating aspirin in combination with the NA lamivudine in HBV patients after surgical resection (NCT01936233).

Additional data on aspirin in HCC prevention or treatment

In contrast to the aforementioned studies, secondary analysis of retrospective studies did not report a clear beneficial effect of aspirin on liver cancer risk (45) or HCC recurrence (46) in the general population or on HCC recurrence in patients with HBV (47). Chiu et al reported a neutral effect of aspirin (7% of the population) on liver cancer risk in a population-based case – control study conducted to investigate the effect of statins (45). Yew et al. reported that aspirin alone (3.8% of the population), non-aspirin NSAIDs or NSAID selective for COX-2 inhibition display a trend toward reduced risk of early recurrent HCC after liver resection (46). In contrast, any NSAID was associated with a 20% lower risk of HCC recurrence (46). Rungsakulkij et al. reported that the use antiplatelet drugs (aspirin and/or clopidogrel; about 8% of the population) had a neutral effect on HCC recurrence in patients with HBV-related HCC who underwent hepatic resection, (47). The study did not examine the effect of aspirin alone. Other studies reported inconsistent beneficial effects of aspirin in addition to statins on HCC risk in HBV patients (48) or in diabetic patients (49).

A meta-analysis of twelve studies also appeared to find a neutral effect of aspirin on HCC recurrence (50). The study found that non-aspirin NSAID use, but not aspirin, was associated with a 20% lower risk of HCC recurrence in a random-effects model, but had no effect on HCC mortality. The neutral effect of aspirin use was largely driven by data from the two retrospective studies mentioned above, however, in which aspirin was not the main study target (46, 47).

Side-effects associated with the use of aspirin in HCC

An increased risk of bleeding is the major side-effect that may limit the use of aspirin for HCC prevention or treatment, particularly in cirrhotic patients. The toxicity of aspirin was assessed only in some of the more recent retrospective studies of aspirin in primary prevention or as adjuvant treatment for HCC and they revealed divergent results (Table 1). Similar mixed resulted emerged from the few meta-analyses of aspirin studies in HCC which reported on the bleeding risk associated with aspirin use (37, 38, 43).

Non-aspirin NSAIDs and HCC

Conflicting results have emerged from the few epidemiological studies of non-aspirin NSAIDs in HCC. Retrospective studies having NSAIDs as the main drug study target have reported that their use is associated with a reduction in HCC risk (26) and recurrence (46, 51) in the general population and HCC recurrence in HBV patients (41). However, other prospective studies reported no effect of NSAID use on HCC risk (23, 25) in the general population. In some of these studies, aspirin treatment was included in the NSAID group. Thus, additional studies are required to understand fully the impact of NSAIDs on HCC.

Mechanism of action of aspirin in HCC prevention

Aspirin’s antiplatelet and anti-inflammatory effects afford biological plausibility to the associations between aspirin use and HCC prevention or recurrence described here (Table 1). In fact, regardless of the etiology, HCC is an inflammation-associated cancer. It occurs as a result of vicious cycles of hepatocyte damage, inflammation, necrosis, and regeneration which cause chronic inflammation, fibrosis and cirrhosis and genomic instability (20, 52). During this necro-inflammatory process in the liver, platelets play a key role by promoting accumulation of inflammatory and immune cells, facilitating immune-mediated liver injury and carcinogenesis, and favoring a fibrogenic microenvironment (53–55). A higher platelet-to-lymphocyte ratio has been associated with poor prognosis in patients with advanced HCC (56).

Indeed, the use of other antiplatelet drugs, like clopidogrel, has been associated with a reduction in HCC incidence, progression and/or mortality (29, 41, 57). Aspirin and clopidogrel have been reported to prevent fibrosis (58, 59) and HCC through platelet glycoprotein Ibα-mediated inhibition of intrahepatic platelet activation, degranulation, and immune cell trafficking (59).

Furthermore, aspirin has also been reported to reduce advanced fibrosis in rodents by suppressing platelet-derived growth factor-β, inactivating hepatic stellate cells and thereby improving fibrosis grade and hepatic regenerative activity (60, 61). Indeed, the use of aspirin has been associated with a significantly lower index of liver fibrosis in patients with CLD (62) and a reduced risk of progression of fibrosis in patients with nonalcoholic fatty liver disease (63)

Preclinical studies using cell lines and mouse models of cancer have identified several molecular targets involved in the putative anti-cancer effects of aspirin (Table 2).

Table 2.

Potential targets of aspirin in HCC

Drug concentration/dose	Experimental model	Drug target	Functional effect	Reference
2.5 and 5 mM aspirin	HepG2 cells	↓ NFκB-ACSL1 signaling ↓ ACSL1 expression	↓ triglyceride and cholesterol	64
2 and 4 mM aspirin	HepG2 cells	↓ Wnt/β-catenin signaling pathway ↓TCF4 and LEF1	↓ cell proliferation/ ↓Colony formation	65
2 mM aspirin or 50 μM NS-398	HGF treated HepG2 cells	↓ ERK1/2 pathway	↓ Cell invasion	66
5 and 10 mM aspirin	HepG2 cells	metabolic (↓NADH-ubiquinone oxidoreductase, cytochrome c oxidase, aconitase) and oxidative stress (↑ ROS ↓GSH)	↑ apoptosis G0/G1 cell cycle arrest	67
0.63–10 mM aspirin	Hep3B, HepG2, or SMMC-7721 cells	↑ LC3II/LC3I ratio, ↓ p62 expression, ↑ autophagic flux via disruption of the interaction between Bcl-2 and Beclin-1	↑ autophagy	68
5–15 mM aspirin 100 mg/Kg aspirin, orally	Nude mice transplanted with HepG2 cells	↑ Bax/Bcl-2 ratio, ↑ caspases activities ↑ expression apoptotic proteins	↑ apoptosis Tumor growth	69
2.5 mM aspirin 60 mg/Kg aspirin, ip, for 12 days in mice bearing tumors (>3 mm)	HLE, HLF, Huh-7, PLC/PRF/5, Hep-3B, Li-7 and Hep-G2 cell lines Nude mice transplanted with Huh-7 cells	miRNA-137/EGFR pathway ↓ clusterin and HSP70	↓ cell proliferation ↑ apoptosis G0/G1 cell cycle arrest ↓Tumor volume and tumor weight	70
5 mg/ Kg diet (1 mg/Kg body weight per day) aspirin 5 mg/ Kg diet (1 mg/Kg body weight per day) aspirin + 10 μg/ml drinking water (1 mg/Kg body weight per day) clopidogrel	HBV transgenic mouse model	↓ platelet aggregation ↓ platelet aggregation	↓ intrahepatic HBV-specific CD8+ T cells ↓ HBV-nonspecific inflammatory cells ↓ intrahepatic HBV-specific CD8+ T cells ↓ HBV-nonspecific inflammatory cells ↓ fibrosis ↓ HCC development ↑ survival	71
1 mM aspirin 15 mg/Kg aspirin or 15 mg/Kg aspirin + 7.5 × INF-α 106 U/Kg	INF-α treated Bel-7402 or MHCC97L cells Nude mice transplanted with Bel-7402 or MHCC97L cells	↑ JAK1/STAT1 pathway: ↑ Bax expression ↑ caspase-3 and caspase-9 activation	↑ Apoptosis induced ↓ Cell proliferation ↓Tumor volume ↑ apoptosis ↑ STAT 1 expression	72
2 and 4 mM aspirin 75 mg/Kg aspirin by gavage for 3 weeks in mice bearing tumors (100 mm)	HepG2 cell line Nude mice transplanted with HepG2 cells	↓ NF-κB and LncRNA LMCD1-AS1/let-7g axis ↓ P4HA2 ↓ P4HA2	↓ cell proliferation/ ↓Colony formation ↓ tumor growth, ↓ collagen deposition	73
2–8 mM aspirin	Huh7 HCV replicon cells	↓ COX-2 expression ↓ MEK1/2/p38 MAPK signaling	↓ viral replication	74
4 mM aspirin	Huh7 HCV replicon cells	↓ ROS production ↑ Cu/Zn-SOD expression	↓ viral replication	75
4 mM aspirin	Huh7 HCV replicon cells	↓ Inos ↓ nitrosylated protein levels ↓ transcription factor C/EBP-β binding to iNOS promoter	↓ viral replication	76
2–8 mM aspirin	Huh 7.5.1 cells	↓ Claudin-1 (an HCV receptor)	↓ HCV entry	77
2.5–5 mM aspirin 15 mg/kg sorafenib, 100 mg/kg aspirin, or 15 mg/kg sorafenib and 100 mg/kg aspirin	HepG2 cells HepG2 xenograft nude mice	↑ AMPK-Akt/ERK1/2- myeloid leukemia cell differentiation protein (MCL)-1 axis	↓ cell proliferation ↑ apoptosis ↓ cell proliferation ↑ apoptosis ↓tumor volume	92
0.36–0.72 mg/ml aspirin 1.10 mg/kg (b.i.d.) sorafenib, and/or 6.75 mg/kg aspirin	Hep3B and HuH7 cells HuH-7-Luc2 xenograft nude mice	↓ Long-chain-fatty-acid—CoA ligase 4 (ACSL4) ↑ Growth arrest and DNA-damage-inducible, beta (GADD45B)	↓ cell viability ↑ apoptosis ↓ tumor volume	93
1–4 mM aspirin and/or 5 sorafenib 15 mg/kg sorafenib and/or 100 mg/kg aspirin mg/kg aspirin	SMMC-7721 cells SMMC-7721 orthotopic model in nude mice	↓ mammalian target of rapamycin complex 1 (mTORC1) signaling ↓ PI3K/AKT, MAPK/ERK pathway	↓ cell proliferation ↑ apoptosis ↓ tumor volume	94
2.5 mM aspirin and/or 500 ng/ml doxorubicin	MHCC-97L cells	↑ miR-491 expression	↓ cell viability	95
0.1–0.5 mM aspirin 30 mg/kg sorafenib, 15 mg/kg aspirin, or 30 mg/kg sorafenib and 15 mg/kg aspirin	HCCLM3 cell line HepG2 xenograft nude mice	↓ COX-2 expression ↑ HIV-1 Tat Interactive Protein 2 (HTATIP2) expression ↑ HTATIP2 expression	↓ invasiveness ↓ mesenchymal-to-epithelial transition ↓ invasiveness ↓ metastatic potential	96
0.5 mM aspirin 30 mg/kg sorafenib, 15 mg/kg aspirin, or 30 mg/kg sorafenib and 15 mg/kg aspirin	HepG2 and HCCLM3 cells HCCLM3 orthotopic tumor model	↓ stromal-derived factor 1-alpha (SDF1-α) ↓ SDF1-α	↓invasiveness	97
ASA 5 mM aspirin and/or sorafenic 10 mMsorafenib (10 mg/kg) and or and aspirin (20, 50 and 100 mg/kg)	Huh7-R and HCC-LM3 cells xenograft mouse model	↓ 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) ↓ PFKFB3	↓ cell proliferation ↑ apoptosis↑ apoptosis ↓ tumor volume	98

Aspirin may prevent HCC progression through diverse COX-dependent and independent mechanisms including inhibition of inflammation or cell proliferation, induction of apoptosis or autophagy, reduction of fibrosis, and suppression of platelet function (64–73). In addition, an antiviral activity of aspirin against HCV or other viruses has been reported (74–77). Up to now, only five studies have reported an effect of aspirin in mouse models of HCC (69–73) and three of them attempted to examine the mechanism of chemoprevention seen with aspirin (71–73).

In a mouse model of chronic HBV immune-mediated HCC, Sitia et al. reported that platelet activation promotes the accumulation of virus-specific CD8⁺ T cells and virus-nonspecific inflammatory cells in the liver, and that treatment with aspirin and/or clopidogrel reduced hepatic inflammation and immune infiltration (71). Moreover, the combination of aspirin and clopidogrel reduced liver fibrosis, HCC progression, and increased mouse survival, without being associated with increased bleeding. These beneficial effects were not seen in hepatitis induced by carbon tetrachloride (71).

Li et al. reported that aspirin enhanced IFNα-induced antiproliferative and apoptotic effects in HCC cell lines by inducing the phosphorylation of STAT1. Moreover, in a nude mouse xenograft model, aspirin alone or in combination with IFNα, reduced tumor growth and increased the number of apoptotic cells in tumor tissue expressing STAT1 (72).

Wang et al. reported that aspirin reduced the expression of collagen prolyl 4-hydroxylase α2 (P4HA2) in a HCC cell line and in a nude mouse xenograft model by dampening the NFκB and the LncRNA LMCD1-AS1/let7g axis (73). In vivo, aspirin decreased collagen deposition in the liver and delayed liver tumor growth. In contrast, mice overexpressing P4HA2 presented increased collagen deposition in the liver and bigger tumor size compared to wild-type xenograft mice (73).

Since some of the epidemiological and the pre-clinical studies have used anti-inflammatory doses of aspirin, and since NSAIDs have also shown some efficacy in HCC chemoprevention (26), COX-2 inhibition by high-dose aspirin may also be contributing to its anti-cancer effects. COX-2 has been reported to be highly expressed in injured livers and in the well-differentiated histological subtype of human HCC (78–80). COX-2 overexpression promoted tumor initiation, differentiation, invasion, and metastasis in a HCC cell line (81) and induced spontaneous HCC in mice (82). Moreover, high COX-2 expression in HCC has been associated with poor overall survival (83). Indeed, in vitro and in vivo studies indicate that COX-2 inhibition reduces liver cancer cellular growth by reducing inflammation and inducing cell apoptosis (84–89). Celecoxib, a COX-2 inhibitor, potentiates the antitumor effect of other drugs in a human HCC cell line and in rats (90, 91). Currently, celecoxib, alone or in combination with metformin, is under clinical investigation as adjuvant treatment after liver resection in HCC patients (NCT03184493).

Aspirin may also synergize with other systemic treatments for HCC. Pre-clinical studies showed that aspirin can increase the sensitivity to various anticancer drugs, including sorafenib and doxorubicin, (92–95) reduce the pro-metastatic effect of sorafenib (96, 97) and overcome sorafenib resistance (98) in vitro and in vivo. Several molecular targets have been proposed to mediate the adjuvant chemotherapeutic effect of aspirin in HCC (Table 2). These targets are mainly involved in cell metabolism [AMP-activated protein kinase (AMPK); long-chain-fatty-acid—CoA ligase 4 (ACSL4); mammalian target of rapamycin complex 1 (mTORC1); 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3)] or in inflammatory response [COX-2; stromal cell-derived factor 1 (SDF1)-α].

Conclusion and Future perspectives

Aspirin has emerged as a promising chemoprotective agent for CRC. In contrast, convincing evidence for the efficacy of aspirin as a chemoprotective agent in other cancers is lacking, with the exception of HCC. Recent retrospective studies in patients at high risk of HCC confirmed the inverse association between aspirin use and HCC incidence reported earlier in the general population (Table 1). However, no evidence emerged from the meta-analyses of RCTs of aspirin in CV prevention that had shown protection from CRC, possibly because these trials did not have enough power to detect an effect of aspirin on HCC in populations with a low annual incidence of the disease.

In addition, small, retrospective population-based studies indicate a potential role of aspirin as an adjuvant agent in patients undergoing local or systemic therapy for HCC.

These findings highlight the need for adequately powered prospective RCTs to determine the place of aspirin in HCC prevention, particularly in populations at high risk of HCC development. Such trials should include racially and ethnically diverse populations, have prospectively updated data on medication adherence, frequent monitoring of viral parameters, co-adjustment for the type and severity of underlying liver disease, concomitant use of potential chemopreventive agents and control for risk factors for HCC. Such trials might also control for aspirin dose and duration of treatment and monitor the durability of response.

Should aspirin prove to be effective, the precise molecular mechanism by which its efficacy occurs in HCC remains to be elucidated. Initial pre-clinical studies have implicated several molecular targets in the beneficial effect of aspirin in HCC progression (Table 2). The most plausible mechanism likely involves aspirin’s antiplatelet and anti-inflammatory effects since: 1) HCC is a type of inflammation-associated cancer; 2) platelets play an important role in HCC initiation and development; 3) aspirin is reported to be effective in HCC prevention also at low-doses; 4) NSAIDs have also shown some efficacy in HCC chemoprevention. Thus, aspirin may reduce HCC progression through additional mechanisms distinct from those of non-aspirin NSAIDs, such as immunologic effects related to its anti-platelet properties (59, 71).

However, most of these molecular targets/metabolic pathways have been identified only in in vitro experiments, often using high concentrations of aspirin (in the millimolar range) which cannot be, even transiently, clinically achievable (99, 100). Moreover, the cancer models used in the in vivo studies of aspirin only allowed investigation of its effect on cancer progression and not on prevention.

Further experimental studies at clinically relevant levels of drug exposure in more appropriate models are needed to determine the mechanisms linking aspirin use and HCC and the role of platelets on this setting.

In conclusion, should clinicians start to consider recommending a baby aspirin to their patients with cirrhosis as a chemo-preventive agent for HCC? The answer, for now, is ‘no’, as more evidence is needed from prospective studies. The potential benefits of aspirin must be weighed against the potential for bleeding in patients with CLD and HCC, as they may already be at risk of life-threatening hemorrhages due to portal hypertension (101). However, if patients have other reasons to take aspirin, such as for the secondary prevention of CVD, this may widen the therapeutic index and tip the scales.