Metformin and cancer hallmarks: shedding new lights on therapeutic repurposing

Metformin is one of the most commonly prescribed anti-diabetic drugs worldwide. Its history can be traced back to 1918 when guanidine, found in traditional herbal medicine in Europe known as Galega officinalis, was shown to lower glycemia [1]. A series of guanidine derivatives, including metformin, was subsequently synthesized [2]. Over time, the benefits associated with repurposing metformin for several challenging diseases, including obesity [3], cardiovascular diseases [4, 5], liver diseases [6], renal diseases [7], aging-related diseases [8], and cancers [9] have been shown. Epidemiological studies have revealed that metformin exerts protective effects on people with diabetes suffering from cancer [10‐12]. Intriguingly, several clinical studies have also reported encouraging outcomes in non-diabetic cancer patients [13‐15]. Given that metformin is safe, well-tolerated, and cost-effective, it is extremely appealing as a focus of antitumor research. Subsequent evidence has shown that metformin inhibits tumor growth, invasion, and metastasis both in vitro and in mouse tumor models for hepatocellular carcinoma [16], ocular melanoma [17], head and neck squamous cell carcinoma [18], and breast cancer [19], among others. Moreover, metformin has been used as a synergistic therapy for cancer, as it enhances sensitivity to radiotherapy [15, 20], chemotherapy [14, 21], and immunotherapy [22] and decreases side effects at lower therapeutic dosages of anticancer treatments.

Great interest has been attached to the basic and clinical study of metformin in cancer. The central mechanism by which metformin attenuates tumorigenesis and progression is through the regulation of energy metabolism. The master pathway of metformin anticancer activity is the activation of the adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway triggered by inhibition of complex I in the mitochondrial respiratory chain [23‐25]. However, the vague performance in a clinical study was in contrast with the excellent performance in a preclinical study. Metformin did not show any benefit in cancer treatment in some clinical trials. Therefore, there are great challenges in the clinical translation of metformin.

Abundant reviews have elaborated on the topic of metformin and cancer from different perspectives, such as specific cancer types [26, 27], diabetes [28, 29], pharmacology [30, 31], and molecular mechanisms [32, 33]. However, insight into the therapeutic repurposing of metformin is still insufficient [34]. Based on the literature review, we recognize that metformin exerts protective effects against multiple tumor types and an increasing number of subtypes [35, 36]. Hence, the mechanisms of action of metformin must be closely related to the hallmarks of cancer [37], which have been proposed as a common set of functional capabilities crucial to the transformation from normalcy to malignancy. This review focused on the effects of metformin on cancer cells in terms of the hallmarks of cancer and updated the clinical translation of metformin in cancer treatment. In this review, we aim to (1) update the readers on the molecular mechanisms through which metformin exhibits antitumor activities, (2) map the effects of metformin on cancer cells in terms of the hallmarks of cancer (Fig. 1), and (3) summarize seminal clinical trials and therapeutic prospects of metformin for cancer treatment.

The classic modes of metformin’s antitumor effects are the inhibition of respiratory complex I in the mitochondria and the activation of AMPK in succession. Recently, metformin was defined to inhibit complex I by binding in the quinone channel and exert an independent localized chaotropic effect by combining cryo-electron microscopy and enzyme kinetics [38]. Although the direct interaction between metformin and complex I is essential, metformin-induced complex I inhibition is not a consequence of the direct interaction but instead occurs through an indirect mechanism [39]. Ma et al. [40] conducted another novel study that focused on direct molecular targets of metformin and identified PEN2, a subunit of γ-secretase, as a direct molecular target of metformin. PEN2 binds to ATP6AP1, inhibits the activity of v-ATPase without increasing AMP or ADP, and then activates the lysosomal AMP-independent AMPK pathway.

Epidemiologic studies indicate that metformin decreased the risk of cancer incidence compared to other anti-diabetic medications. Hence, the anticarcinogenic effects of metformin were traditionally divided into direct (blood glucose- and insulin-independent) and indirect (blood glucose- and insulin-dependent) effects, being mindful that none of the effects are mutually exclusive.

Numerous clinical trials have been conducted in the pursuit of more convincing evidence of metformin’s anticancer effects and to determine the appropriate anticancer uses of metformin. Here, we have included 34 completed clinical trials with results available on the clinicaltrials.gov website among the 182 results searched until March 2023 (Table 1). These include brain tumors [149], HNSCC [15, 22, 136, 137], acute lymphoblastic leukemia [150], and breast [76, 77, 151‐157], lung [158], esophageal [136, 159], colorectal [160, 161], pancreatic [162‐164], endometrial [165‐169], ovarian [14, 170], and prostate [13, 171‐175] cancers. Among them, two trials are in phase 3, and 16 trials combine metformin with chemotherapy and/or radiotherapy and/or targeted therapy.

The results varied in biochemical indicators, PFS, OS, and adverse events. A double-blind, placebo-controlled crossover pilot study revealed that metformin was linked to better performance in the assessment of cognitive and neural recovery, with good safety and tolerance in survivors of pediatric brain tumors [149]. A multicenter phase 2 trial reported that metformin induced objective PSA responses and disease stabilization in patients with chemotherapy-naive castration-resistant prostate cancer without diabetes [13]. However, some clinical trial results did not support the application of metformin in cancer treatment. A triple-blinded, randomized, placebo-controlled phase 3 trial with 3649 participants reported that the addition of metformin did not significantly improve invasive disease-free survival among patients with high-risk operable breast cancer without diabetes [157]. A double-blind, randomized, placebo-controlled phase 2 trial reported that the addition of metformin to gemcitabine and erlotinib did not improve outcomes in patients with advanced pancreatic cancer [164]. Another phase 2 randomized clinical trial showed that the addition of metformin to chemoradiation was well tolerated but did not improve the OS or PFS of patients with stage III NSCLC [176]. A randomized, double-blind, phase 2 trial even discovered that the addition of metformin to gefitinib resulted in nonsignificantly worse outcomes and increased toxicity in NSCLC patients without diabetes harboring EGFR mutations [158]. Besides, there are no synergetic benefits of metformin and chemotherapy or antiandrogen therapy in prostate cancer patients [174, 175]. However, the clinical trials had some limitations, such as a lack of accurate molecular stratification of patients, insufficient compliance of patients and a limited study population. Hopefully, more well-designed clinical trials are being conducted, including phase 3 multicenter studies with participants with or without diabetes enrolled, and they may provide more convincing evidence regarding the anticancer efficacy and safety of metformin alone or in combination with chemoradiotherapy, targeted therapy or immunotherapy.

The repurposing of metformin has always been a research hotspot worldwide. Currently, the benefits of metformin to aging-related diseases have gained more importance since the speedup of aging in our society. Metformin was reported to mimic significant metabolic effects of caloric restriction, which was the only acknowledged strategy to robustly extend health and lifespan in mammals [177]. For cancer, an aging-related disease, metformin exhibited promising anticancer effects in preclinical studies. Thus, the anticancer mechanisms of metformin have received considerable attention. Metformin inhibits mitochondrial complex I and triggers energy depletion, which activates AMPK and inhibits mTOR, restraining cancer growth via the maintenance of energy homeostasis. Besides, metformin also exerts anticancer effects that are independent of AMPK but rather dependent on Rag GTPases or REDD1. Moreover, some additional anticancer pathways involve the IGF-1R/PI3K/AKT/mTOR pathway and the p53 pathway. Intriguingly, metformin can exert influences on the hallmarks of cancer, including regulation of the cell cycle, cell death, CSCs, cancer cell migration, invasion and metastasis, cancer metabolism, and cancer immunity. In any case, the specific mechanism remains to be clarified, and the explicit anticancer mechanisms of metformin should be further investigated.

For basic research on the molecular mechanisms of metformin in anticancer therapy, we consider that the main challenges lie in three aspects: (1) finding the direct cellular targets of metformin that mediate anticancer activities; (2) clarifying the key questions of the “direct effects” and the “indirect effects” as well as which one plays a greater role in anticancer actions; and (3) developing in vivo models that can mimic the indirect and direct effects of metformin. Many endeavors have been devoted to solving these problems. First, the latest studies have managed to explore the direct cellular targets of metformin. For example, Ma et al. [40] revealed that PEN2 is the direct molecular target of metformin using a photoactive metformin probe. Bridges et al. [38] recently defined the inhibitory drug-target interactions of metformin with mammalian respiratory complex I by combining cryo-electron microscopy and enzyme kinetics. The identification of the direct target of metformin’s anticancer effects may help to further investigation for drug development. Second, in in vitro research, the direct effects are emphasized and well-studied, while the indirect effects cannot be mimicked. Some in vivo and clinical studies suggest that indirect insulin-dependent effects may be of great significance in at least some cancers, such as breast cancer and lung cancer. The research directions varied for the two modes of anticancer effects (Table 2). In terms of “direct effects,” more effort should be put into markers such as LKB1, Rag GTPases and REDD1. In terms of “indirect effects,” more effort should be put into markers such as blood glucose and insulin levels, insulin resistance, expression of insulin receptors and insulin-like growth factor receptor 1, and targets in the liver. Understanding the markers helps to predict the therapeutic response of patients. For example, the synergistic effects between metformin and gefitinib were reported to rely on the presence of wild-type LKB1 in NSCLC cells [178]. Third, since indirect effects could not be simulated in vitro, more in vivo models are needed to reexamine the direct and indirect effects of metformin and the possible interactions. Some attempts have been made to develop related models. For instance, in research on the anticancer activity of metformin, hyperglycemic mice were reported to lose sensitivity to metformin compared with normoglycemic mice, probably through increased c-Myc expression, glycolytic enzymes hexokinase 2 and pyruvate dehydrogenase kinase 1 [179]. We expect more in vivo models with more complex designs that are currently used in most in vitro research.

There is a discrepancy in the antitumor effect of metformin between clinical research and preclinical studies, although metformin has shown notable benefits for cancer prevention and treatment in preclinical research, and the related molecular mechanisms have been extensively studied. The challenges mainly include (1) simulating pharmacokinetics consistent with clinical settings, including appropriate metformin concentrations and dosing time; (2) exploring suitable synergetic therapies and patients who are more sensitive to metformin; and (3) utilizing other forms of biguanides, such as phenformin or modified biguanides, which have better performances. Therefore, further research is required regarding the critical aspects mentioned above. First, many preclinical studies have employed metformin at concentrations that are considerably higher than what would be deemed safe in clinical settings [180]. The plasma concentrations of metformin were reported to be 5–30 μmol/L in patients taking clinical doses of 1.5–2 g per day, which were the most common doses for diabetes and were used in most clinical trials for cancer treatment [181]. However, the concentrations in most preclinical studies in vitro (300 μmol/L–10 mmol/L) were dozens or even a thousand times the clinical concentrations. The dosages of in vivo studies (200–1000 mg/kg per day) were 6–30 times the clinical dosages (approximately 30 mg/kg per day) with metformin diluted in the drinking water or intraperitoneally injected. Recent studies have noted this issue. Metformin can suppress cancer at a clinically safe concentration in vitro [40] and in vivo [16, 35, 65]. A recent report also proposed that AMPK activation by clinical concentrations of metformin was not completely the consequence of changes in the cellular AMP/ATP ratio or depletion of cellular energy charge [182]. For future research, it is possible to reasonably expand the metformin concentration and set more concentration gradients. Besides, metformin tends to be administered at the very beginning of preclinical models, which cannot be replicated in clinical settings. For example, a synergetic therapy of metformin and PD1 blockade for melanoma was reported to have a tumor size threshold. Once tumors were larger than 10 mm² before treatment in mice, metformin failed to exert synergistic effects with PD-1 blockade [183]. In the clinical setting, improved outcomes were observed only among patients with early-stage NSCLC or those who took metformin before the NSCLC diagnosis [184]. Therefore, it enlightens us to apply metformin once cancer is diagnosed or even in people with a high risk of cancer if possible. Second, based on our understanding of metformin’s effects on cancer hallmarks, metformin could be a useful adjuvant agent in combination therapy to combat cancer synergistically in certain patients with certain cancers [36]. It can be administered along with chemotherapy, radiotherapy, immunotherapy or targeted therapy. Taking immunotherapy as an example, a high level of lactate can lead to tumor immune tolerance, while metformin was reported to increase the level of lactate in the intra- and extracellular environment [31, 185]. However, from the current evidence, acidification of the TME made tumors more susceptible to metformin due to the loss of NAD+ regeneration capacity [78, 79, 186]. Therefore, whether metformin can cause tumor immune tolerance by increasing the acidification of the TME remains an interesting issue to explore. If indeed, the combination of metformin and immunotherapy might be a possible direction in further research. Besides, metformin was reported to show no more benefits in some cancers with certain mutations [158] or advanced stages [164, 175, 176] or in patients without diabetes [157, 158]. We call for further high-quality clinical trials on metformin combined with other therapies in different types of physiological conditions and cancers. Third, it was reported that phenformin may outperform metformin owing to its unique pharmacokinetic characteristics, which include better absorption and inhibition of the mitochondria [187]. Although the incidence of lactic acidosis associated with phenformin is higher than that associated with metformin, phenformin is in any case safer than other cancer treatments. Moreover, given the pharmacokinetic differences between metformin and phenformin, we can obtain more insights regarding drug modification. Once there is more evidence, we expect metformin or other forms of biguanides to exert a greater influence on anticancer therapy, at the appropriate dosage, on patients of appropriate metabolic state, and in combination with other therapies.

The review details the possible molecular mechanisms of metformin in cancer prevention and treatment, elucidates its role in terms of cancer hallmarks, and more importantly, analyses current challenges and future directions in clinical translation.