INTRODUCTION
Hepatocellular carcinoma (HCC) represents the predominant form of primary liver malignancy and constitutes a significant public health burden globally. It is the sixth most frequently diagnosed cancer and ranks third in cancer-related mortality worldwide[1]. HCC typically emerges within a milieu of chronic liver pathology, including cirrhosis, predominantly driven by chronic hepatitis infections, alcohol consumption, and other etiologic agents such as aflatoxin contamination. The prognosis for individuals diagnosed with HCC remains dire, especially for those identified at advanced stages, with survival rates significantly diminished in regions lacking early detection and comprehensive healthcare services. Despite progress in diagnostic methodologies and therapeutic interventions, the clinical outcomes for HCC patients continue to be suboptimal, highlighting the critical need for enhanced screening programs and innovative treatment modalities.
Aflatoxin B1 (AFB1), a notably toxic member of the broader category of mycotoxins known as aflatoxins and produced by Aspergillus fungi, is distinguished by its potent carcinogenic effects on the liver[2]. It is frequently found in food supplies that have been stored improperly, making it a critical public health issue[3]. This toxin has been definitively classified as a group 1 carcinogen by the International Agency for Research on Cancer, highlighting its role in causing HCC, a prevalent liver cancer[2]. The occurrence of AFB1 is especially concerning in regions where chronic hepatitis infections are common, further elevating the risk of HCC[4]. Its carcinogenicity arises from the ability of its metabolites to bind to DNA, initiating cellular processes that lead to cancer[2]. However, beyond direct DNA damage, how AFB1’s multifaceted actions - including its known capacity to induce epigenetic alterations - might intersect with and subvert key tumor suppressor pathways, such as those involving tissue inhibitor of metalloproteinase (TIMP)-3, remains a critical area for exploration in the context of AFB1-related HCC (AHCC). Therefore, controlling AFB1 exposure is vital to reducing the global incidence of liver cancer, particularly in developing countries where exposure levels are highest.
TIMP-3, a key member of the tissue inhibitor of metalloproteinases family, plays a crucial role in regulating the extracellular matrix (ECM) and inhibiting enzymes involved in cancer progression[5,6]. In HCC, TIMP-3 expression is often reduced due to promoter methylation, highlighting its often-overlooked importance in tumor biology[5]. This observation raises a pivotal question in aflatoxin-endemic regions: Could AFB1, with its established epigenetic modifying effects, be a direct driver of TIMP-3 silencing in the liver, thereby uniquely shaping the landscape of AHCC? Studies have shown that increasing TIMP-3 Levels in HCC cells can inhibit tumor growth, reduce invasiveness, and limit metastatic potential by targeting the enzymes necessary for cancer cell invasion[7,8]. This also promotes cell cycle arrest and apoptosis, underscoring TIMP-3’s potential as a therapeutic target in HCC[5]. Despite these promising findings, the specific role and the mechanistic underpinnings of TIMP-3 dysregulation in the unique pathogenic context of AHCC remain poorly understood and represent a significant knowledge gap. Further research is needed to fully explore its therapeutic potential and underlying mechanisms in this context. This editorial aims to synthesize current knowledge to propose specific mechanistic links between AFB1 exposure and TIMP-3 modulation, and to highlight key research questions concerning the AFB1-TIMP-3 axis in AHCC, thereby offering a perspective on potential avenues for both biomarker development and therapeutic intervention.
GLOBAL TRENDS AND DETERMINANTS IN HCC: FOCUS ON AFB1
HCC remains a significant global health concern, representing the predominant form of primary liver cancer with 75% to 85% of cases. In 2022, it was recorded as the third leading cause of cancer death worldwide after lung and colorectal cancers, and the sixth most commonly diagnosed cancer, accounting for approximately 865000 new cases and 757948 deaths[1]. Geographically, HCC incidence and mortality rates are notably higher among men, with a disparity of two to three times that seen in women in most regions of the world. These rates are particularly elevated in transitioning countries, marking it as the leading cause of cancer death among men in several diverse regions including Eastern Asia, South-Eastern Asia, Northern and Western Africa, and Central America. Primary risk factors for HCC vary by region; chronic infections of hepatitis B virus (HBV) and hepatitis C virus contribute significantly, alongside lifestyle factors such as aflatoxin exposure, heavy alcohol consumption, excess body weight, type 2 diabetes, and smoking[9]. The diverse oncogenic pressures exerted by these risk factors are often counteracted or modulated by host cellular defense mechanisms, including tumor suppressor proteins such as those in the TIMP family. Understanding how specific carcinogens, notably AFB1, might overwhelm or dysregulate these protective pathways is crucial for developing targeted prevention and treatment strategies.
AFB1, a potent liver carcinogen produced by Aspergillus fungi, is a critical environmental factor associated with the development of HCC, especially in high-risk areas such as China and Eastern Africa where it frequently contaminates food supplies like grains and nuts[10]. Chronic infection with HBV, combined with exposure to AFB1, significantly increases the risk of HCC, underlining a synergistic interaction between viral infection and environmental carcinogen exposure[11]. Efforts to mitigate AFB1 exposure have played a key role in the observed decline of liver cancer rates in parts of Asia since the late 1970s[12-14]. Nevertheless, the enduring risk posed by AFB1 highlights the need for ongoing surveillance and proactive prevention strategies, especially in areas where HBV prevalence remains high and dietary aflatoxin exposure is prevalent. The global hepatitis strategy by World Health Organization, which emphasizes vaccination and reductions in AFB1 exposure, aims to significantly lower the incidence and mortality of liver cancers linked to these risk factors by 2030[1].
MECHANISMS OF AFB1 CARCINOGENICITY IN HCC
AFB1 is a potent hepatocarcinogen implicated in the pathogenesis of HCC through its multifaceted influence on cellular and molecular pathways. AFB1 undergoes metabolic biotransformation in the liver, primarily mediated by cytochrome P450 enzymes, namely CYP1A2, CYP3A4, CYP3A5, and CYP3A7[15]. This metabolic activation converts AFB1 into AFB1-8,9-epoxide, a highly reactive metabolite[16]. AFB1-8,9-epoxide preferentially binds to the N7 position of guanine in DNA, forming AFB1-N7-guanine adducts, which are pivotal in the induction of mutagenic changes, notably G to T transversions[16-18]. These mutations are critical in oncogene activation and tumor suppressor gene inactivation, specifically highlighting the mutation at codon 249 of the TP53 gene, a frequent alteration observed in AHCC[19,20]. The relevance of this to TIMP-3 Lies in the potential for such genetic instability to indirectly affect TIMP-3. For instance, while direct mutation of the TIMP-3 gene by AFB1 is not commonly reported, the characteristic AFB1-induced TP53 R249S mutation could alter p53’s transcriptional regulatory network, potentially impacting TIMP-3 expression if p53 is involved in its regulation, or by affecting pathways that converge on TIMP-3 function. This warrants specific investigation in AHCC models harboring this mutation.
The resultant DNA adducts and mutations interfere with normal cellular processes and genetic stability. This mutagenic impact is complemented by AFB1-induced oxidative stress, characterized by the production of reactive oxygen species (ROS)[21,22]. These ROS not only augment DNA damage but also lead to lipid peroxidation and protein modification, disrupting cellular integrity and signaling pathways[23-25]. Crucially, oxidative stress is a known modulator of epigenetic landscapes and gene expression. Therefore, a key hypothesis is that AFB1-induced ROS could contribute to TIMP-3 downregulation by promoting its promoter hypermethylation or by altering the activity of transcription factors that regulate TIMP-3. Furthermore, ROS can influence the expression of microRNAs, some of which are known to target TIMP-3 mRNA for degradation, providing another potential link. The NFE2 Like BZIP transcription factor 2 (Nrf2) pathway plays a crucial role in cellular defense against oxidative stress induced by AFB1, by regulating antioxidant response genes that mitigate oxidative damage[26]. The interplay between Nrf2 activation by AFB1 and TIMP-3 expression is another unexplored facet; for example, does chronic Nrf2 activation in response to AFB1 inadvertently affect TIMP-3 Levels?
Furthermore, AFB1 impacts cell cycle regulation and apoptosis, mechanisms central to cancer development. It induces G1 phase arrest and can initiate apoptosis through pathways involving the tumor suppressor p53[27,28]. The compromised functionality of p53 due to specific mutations induced by AFB1 impedes its DNA binding capacity and transcriptional activity, leading to uncontrolled cell proliferation and survival[29,30]. As TIMP-3 itself can induce apoptosis and cell cycle arrest, its downregulation by AFB1 - whether through p53-dependent or -independent mechanisms - would synergize with these direct effects of AFB1 to promote tumorigenesis. Additionally, AFB1 has been shown to influence epigenetic patterns, notably DNA methylation, thus altering gene expression and further contributing to oncogenesis[31]. This capacity of AFB1 to induce widespread DNA methylation changes is perhaps the most direct and compelling hypothetical link to TIMP-3 suppression in AHCC. Given that TIMP-3 is frequently silenced by promoter hypermethylation in HCC, it is highly plausible that AFB1 exposure directly initiates or accelerates this specific epigenetic silencing event at the TIMP-3 Locus. Investigating the methylation status of the TIMP-3 promoter in response to AFB1 exposure is therefore a paramount research priority.
GENERAL MECHANISMS OF TIMP-3 IN CANCER SUPPRESSION
In the intricate landscape of cancer biology, the TIMP-3 emerges as a pivotal antagonist of matrix metalloproteinases (MMPs), enzymes integral to the ECM degradation[32,33]. Normally, MMPs facilitate tissue remodeling and repair; however, their dysregulation can lead to enhanced tumor invasion and metastasis[34,35]. TIMP-3 serves as an essential regulatory protein, distinguished by its unique characteristic of being tightly bound to the ECM, thereby localizing its inhibitory activity. It binds to and inhibits a broad spectrum of MMPs, including key players in cancer progression such as MMP-2 (gelatinase A) and MMP-9 (gelatinase B), primarily through a non-covalent, stoichiometric interaction where the N-terminal domain of TIMP-3 inserts into the active site cleft of the MMP, thus blocking substrate access and enzymatic activity. This action effectively curbs ECM breakdown and stymies cancer cell dissemination[36]. Beyond its role in inhibiting metalloproteinases, TIMP-3 exerts considerable influence in mitigating angiogenesis - the formation of new blood vessels - vital for tumor growth[37].
Furthermore, TIMP-3 enhances apoptosis, promoting programmed cell death which is crucial in preventing the unchecked proliferation of cancer cells. It achieves this by stabilizing cell surface death receptors such as tumor necrosis factor receptor-1, fas cell surface death receptor, and TNF-related apoptosis-inducing ligand receptor 1, thus amplifying apoptotic pathways within the tumor cells[33]. In addition to its roles in apoptosis and anti-angiogenesis, TIMP-3 also modulates the tumor microenvironment by controlling inflammatory cytokine levels, primarily through the inhibition of A disintegrin and metalloprotease 17[38]. This regulation helps diminish the chronic inflammation often associated with tumor progression. Given these general anti-cancer effects, it's crucial to examine TIMP-3’s specific role in HCC to better understand its regulation and therapeutic potential.
THE IMPACT OF TIMP-3 DOWNREGULATION IN HCC
TIMP-3 is an important protein in HCC because it helps control the breakdown of the ECM, which is crucial for liver health and cancer progression. Recent research has focused on how TIMP-3 is expressed in liver cancer cells, how it is controlled, and what its reduction means for cancer treatment.
The expression of TIMP-3 in HCC tissues significantly influences the severity of the disease and patient survival rates, with lower levels typically associated with increased tumor spread and poor cell differentiation[39]. Research has shown that TIMP-3 is frequently reduced in HCC due to the methylation of its promoter region, a type of genetic modification that suppresses its expression and leads to more aggressive tumor behavior[33]. This methylation is a reversible change, providing potential therapeutic opportunities to increase TIMP-3 Levels and curb tumor progression. Additionally, microRNAs, particularly miR-21 and the miR-221/222 cluster, play a role in TIMP-3 regulation by targeting its mRNA for degradation[33]. This interaction further diminishes TIMP-3's ability to inhibit MMPs, thereby enhancing tumor invasion and migration.
PROBING THE IMPLICATIONS OF TIMP-3 IN AHCC
While extensive research has been conducted on the role of TIMP-3 in HCC, its specific implications in AHCC remain unexplored. This gap highlights a critical need for focused studies that can clarify the role of TIMP-3 within the unique pathogenic context of AFB1 exposure.
The recent study published by Liang et al[32] provides critical insights into the prognostic significance of TIMP-3 in AHCC. The study employed enzyme-linked immunosorbent assay to measure AFB1-DNA adducts, a method known for its precision and reliability, which lends further credibility to the results. This rigorous approach supports the study’s conclusions that link aflatoxin exposure, TIMP-3 expression, and AHCC outcomes, highlighting the potential of TIMP-3 as a valuable biomarker for prognosis. These findings importantly bridge a knowledge gap in our understanding of HCC pathogenesis influenced by environmental carcinogens.
Beyond AFB1, aristolochic acid (AA), another potent liver carcinogen prevalent in herbal medicines, warrants consideration due to its mechanistic similarities with AFB1. Although direct evidence linking AA to TIMP-3 is lacking, AA’s induction of DNA adducts, oxidative stress, and epigenetic alterations could plausibly down-regulate TIMP-3, mirroring AFB1’s effects. For instance, studies indicate AA promotes HCC progression via pathways like the C3a/C3aR complement system[40], which may intersect with TIMP-3’s regulatory roles. Investigating whether AA induces TIMP-3 silencing, potentially via promoter methylation or ROS-mediated pathways, is essential to broaden the understanding of environmental carcinogens in HCC. Elucidating the AFB1-TIMP-3 and AA-TIMP-3 axes through targeted molecular studies, particularly in high-exposure regions, could validate TIMP-3 as a universal biomarker and therapeutic target, enhancing personalized management of HCC in diverse etiological contexts.
CHALLENGES AND FUTURE DIRECTIONS
The research surrounding the TIMP-3 in AHCC reveals significant gaps and prompts the need for further investigation. Although TIMP-3’s potential to inhibit HCC cell growth and promote cell death is recognized, the precise molecular mechanisms influenced by AFB1 exposure are not fully understood, necessitating detailed molecular and epigenetic studies. Additionally, establishing a robust link between TIMP-3 expression and clinical outcomes requires comprehensive in vivo studies and long-term clinical follow-ups. To advance the therapeutic potential of TIMP-3, well-designed clinical trials focusing on its efficacy as a biomarker for early detection and its role in therapeutic interventions are essential. Furthermore, integrating TIMP-3 analysis into liquid biopsy techniques could enhance early detection capabilities, while exploring its synergistic effects with existing treatments could lead to innovative therapeutic strategies. Research into the multi-generational impacts of AFB1 exposure on TIMP-3 expression will also provide deeper insights into preventative measures. This integrated approach combining molecular, clinical, and epidemiological research is key to translating these findings into substantial clinical benefits.
CONCLUSION
The exploration of TIMP-3 in the context of AHCC presents a pivotal opportunity to enhance our understanding of liver cancer pathogenesis and therapeutic strategies. The recent findings underscoring TIMP-3 as a potential prognostic biomarker in AHCC offer promising avenues for early detection and intervention, especially in populations at high risk due to aflatoxin exposure. To optimize the clinical utility of TIMP-3, further research is imperative to elucidate the detailed molecular mechanisms at play, as well as to validate its role through comprehensive clinical trials. The integration of such biomarkers into routine clinical practice could revolutionize the management of HCC, shifting the paradigm towards more personalized and proactive treatment approaches.
Provenance and peer review: Invited article; Externally peer reviewed.
Peer-review model: Single blind
Specialty type: Gastroenterology and hepatology
Country of origin: China
Peer-review report’s classification
Scientific Quality: Grade A
Novelty: Grade B
Creativity or Innovation: Grade B
Scientific Significance: Grade B
P-Reviewer: Su XB, PhD, Associate Professor, China S-Editor: Bai Y L-Editor: A P-Editor: Zhang YL
