Major outcomes

Detailed Methodology

Metformin inhibits the development of L-DOPA-Induced Dyskinesia in a Murine Model of Parkinson’s Disease (6-hydroxydopamine-lesioned mouse model). having therapeutic potential for the suppression or management of L-DOPA-induced motor complications in patients with PD.

Metformin showed a long-lasting effect on axial, limb, and orofacial abnormal involuntary movement scores for up to 20 days after treatment initiation. Interestingly, persistent enhancement of the mammalian target of rapamycin, dopamine D1 receptor, and extracellular signaling-regulated kinase 1/2 signaling was maintained in the DA-denervated striatum during metformin treatment. Metformin globally normalized the increased glycogen synthase kinase 3β activity induced by chronic treatment of l-DOPA in a manner associated with Akt activation in unilaterally 6-OHDA-lesioned mice¹.

Metformin is a chemo-preventive agent against multiple cancers. There is also evidence in human studies that metformin is a cancer chemotherapeutic agent, and several clinical trials that use metformin alone or in combination with other drugs are ongoing. In vivo and in vitro cancer cell culture studies demonstrate that metformin induces both AMPK-dependent and AMPK-independent genes/pathways that result in inhibition of cancer cell growth and migration and induction of apoptosis.

The effects of metformin in cancer cells resemble the patterns observed after treatment with drugs that downregulate specificity protein 1 (Sp1), Sp3 and Sp4 or by knockdown of Sp1, Sp3 and Sp4 by RNA interference. Studies in pancreatic cancer cells clearly demonstrate that metformin decreases expression of Sp1, Sp3, Sp4 and pro-oncogenic Sp-regulated genes, demonstrating that one of the underlying mechanisms of action of metformin as an anticancer agent involves targeting of Sp transcription factors. These observations are consistent with metformin-mediated effects on genes/pathways in many other tumor types².

Metformin is a promising drug for cancer prevention and treatment, especially in the diabetic population. The authors aimed to test whether 14-3-3zeta affects the anticancer effect of metformin on colorectal carcinoma (CRC). The results suggest that 14-3-3zeta may be associated with carcinogenesis and poor prognosis of CRCs associated with diabetes, and metformin may reverse the AMPK inhibition caused by 14-3-3zeta in CRCs in the background of diabetes. The study should lead to a better understanding of the anticancer activity of metformin and points to possible application of metformin to the treatment of cancers overexpressing 14-3-3zeta.

The study confirmed that higher 14-3-3zeta expression was found in CRC tissues than in pericarcinoma tissues, and in CRC tissue of patients with diabetes than in those without diabetes. A knockdown of 14-3-3zeta inhibited CRC proliferation and promoted apoptosis in vitro and in vivo. Then, the study created stable cell lines with under-expressed 14-3-3zeta from SW480 and HCT15 cells after infection by a lentiviral vector carrying short hairpin RNA targeting 14-3-3zeta (named LV-sh14- 3-3zeta). Of note, metformin induced apoptosis and retarded tumor growth in the CRCs with overexpressed 14-3-3zeta, whereas this action was attenuated when 14-3-3zeta was knocked down. Moreover, either metformin or down-regulation of 14-3-3zeta noticeably activated AMP-dependent protein kinase (AMPK) signaling, whereas the effect of metformin was attenuated when the 14-3-3zeta expression was decreased³.

Repurposing Metformin for

Cardiovascular Disease

Its widespread use has largely been underpinned by the United Kingdom Prospective Diabetes Study that reported lower cardiovascular mortality and morbidity in patients treated with metformin in comparison with alternative glucose-lowering drugs, despite similar glycemic control. A recent meta-analysis suggests that the cardiovascular effects of metformin could be smaller than that reported by United Kingdom Prospective Diabetes Study; however, this should be interpreted with caution because there have only been a small number of randomized controlled trials. Although patients who have cardiovascular disease (CVD) with T2D comorbidity are likely to benefit most from metformin, indications of cardiovascular benefit over other diabetes treatments has driven interest in repurposing metformin to treat CVD, irrespective of diabetes status⁴.

Caffeine induces metformin anticancer effect on fibrosarcoma in hamsters. The combination of metformin and caffeine inhibited fibrosarcoma growth in hamsters without toxicity. Administration of metformin with caffeine might be an effective and safe approach in novel nontoxic adjuvant anticancer treatment.

Thirty two Syrian golden hamsters of both sexes, weighing approximately 100 g, were randomly allocated to 3 experimental and 2 control groups, with a minimum of 6 animals per group. 2 × 106 BHK-21/C13 cells in 1 ml were injected subcutaneously into the animals' back in 4 groups. The first experimental group started peroral treatment with metformin 500 mg/kg daily, the second with caffeine 100 mg/kg daily and the third with a combination of metformin 500 mg/kg and caffeine 100 mg/kg daily, via a gastric probe 3 days before tumor inoculation. After 2 weeks, when the tumors were approximately 2 cm in the control group, all animals were sacrificed. The blood was collected for glucose and other analyses. The tumors were excised and weighed and their diameters were measured. The tumor samples were pathohistologically (HE) and immunohistochemically (Ki-67, CD 31, COX IV, GLUT-1, iNOS) assessed and the main organs toxicologically analyzed, including the control animals that had received metformin and caffeine. Tumor volume was determined using the formula LxS2/2, where L was the longest and S the shortest diameter. Ki-67-positive cells in the tumor samples were quantified. Images were taken and processed by software UTHSCSA Image Tools for Windows Version 3.00. Statistical significances were determined by the Student's t-test⁵.

Metformin is a future therapy for neurodegenerative diseases. Apart from hypoglycaemic activity, it improves serum lipid profiles, positively influences the process of haemostasis, and possesses anti-inflammatory properties. Recently, scientists have put their efforts in establishing metformin’s role in the treatment of neurodegenerative diseases, such as AD, amnestic mild cognitive impairment and Parkinson’s disease. Results of several clinical studies confirm that long term use of metformin in diabetic patients contributes to better cognitive function, compared to participants using other anti-diabetic drugs.

The exact mechanism of metformin’s advantageous activity in AD is not fully understood, but scientists claim that activation of AMPK-dependent pathways in human neural stem cells might be responsible for the neuroprotective activity of metformin. Metformin was also found to markedly decease Beta-secretase 1 (BACE1) protein expression and activity in cell culture models and in vivo, thereby reducing BACE1 cleavage products and the production of Aβ (β-amyloid). Furthermore, there is also some evidence that metformin decreases the activity of acetylcholinesterase (AChE), which is responsible for the degradation of acetylcholine (Ach), a neurotransmitter involved in the process of learning and memory. In regard to the beneficial effects of metformin, its anti-inflammatory and anti-oxidative properties cannot be omitted. Numerous in vitro and in vivo studies have confirmed that metformin ameliorates oxidative damage⁶.

Modified Metformin as a More Potent Anticancer Drug: Mitochondrial Inhibition, Redox Signaling, Antiproliferative Effects and Future EPR Studies. Results indicate that the lead compound, mito-metformin10, was nearly 1000-fold more potent than metformin in inhibiting mitochondrial complex I activity, inducing reactive oxygen species (superoxide and hydrogen peroxide) that stimulate redox signaling mechanisms, including the activation of adenosinemonophosphate kinase and inhibition of proliferation of pancreatic cancer cells.

Epidemiological studies suggest that metformin exerts anticancer effects in diabetic patients with pancreatic cancer. However, at typical antidiabetic doses the bioavailability of metformin is presumably too low to exert antitumor effects. Thus, more potent analogs of metformin are needed in order to increase its anticancer efficacy. To this end, a new class of mitochondria-targeted metformin analogs (or mito-metformins) containing a positively-charged lipophilic triphenylphosphonium group was synthesized and tested for their antitumor efficacy in pancreatic cancer cells.

The potential use of the low-temperature electron paramagnetic resonance technique in assessing the role of mitochondrial complexes including complex I in tumor regression in response to metformin and mito-metformins in the in vivo setting is discussed⁷.

Metformin prevents dopaminergic neuron death in MPTP/P-induced mouse model of Parkinson's disease via autophagy and mitochondrial ROS clearance. Metformin may be a pluripotent and promising drug for dopaminergic neuron degeneration, which will give us insight into the potential of metformin in terms of opening up novel therapeutic avenues for Parkinson's disease.

In this study, a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine plus probenecid-induced mouse model of Parkinson's disease was established to explore the neuroprotective effect of metformin on dopaminergic neurons in substania nigra compacta. The study showed that treatment with metformin (5 mg/mL in drinking water) for 5 weeks significantly ameliorated the degeneration of substania nigra compacta dopaminergic neurons, increased striatal dopaminergic levels, and improved motor impairment induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine plus probenecid. Moreover, the study found that metformin inhibited microglia overactivation-induced neuroinflammation in substania nigra compacta of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine plus probenecid Parkinson's disease mice, which might contribute to the protective effect of metformin on neurodegeneration. Furthermore, metformin (2 mM) activated AMP-activated protein kinase in SH-SY5Y cells, in turn inducing microtubule-associated protein 1 light chain 3-II-mediated autophagy and eliminating mitochondrial reactive oxygen species. Consequently, metformin alleviated MPP+-induced cytotoxicity and attenuated neuronal apoptosis⁸.

Metformin lowers α-synuclein phosphorylation and upregulates neurotrophic factor in the MPTP mouse model of Parkinson's disease. The metformin regimen significantly increased the level of brain derived neurotrophic factor in the substantia nigra, and activated signaling pathways related to cell survival. Proof of concept study revealed that inhibition of PP2A or tropomyosin receptor kinase B reversed neuroprotective property of metformin in SH-SY5Y cells. Results indicate that metformin provides neuroprotection against MPTP neurotoxicity, which might be mediated by inhibition of α-synuclein phosphorylation and induction of neurotrophic factors.

The neuroprotective effects of metformin were assessed in the subchronic MPTP model of PD, and explored its feasible mechanisms for neuroprotection. Animals received saline or MPTP injection (30 mg/kg/day) for the first 7 days, and then saline or metformin (200 mg/kg/day) for the next 7 days. Immunohistochemical stainings showed that metformin rescued the tyrosine hydroxylase-positive neurons and attenuated astroglial activation in the nigrostriatal pathway. In parallel, metformin restored dopamine depletion and behavioral impairments exerted by MPTP. Metformin ameliorated MPTP-induced α-synuclein phosphorylation which was accompanied by increased methylation of protein phosphatase 2A (PP2A), a phosphatase related to α-synuclein dephosphorylation⁹.

Positive results of numerous preclinical studies on various types of cancer, many clinical trials are underway to study metformin's effect in chemoprevention and treatment of cancers in humans. Nowadays, applications of novel metformin analogues and nanotechnology based targeting have further enhanced the potential of metformin anticancer therapy.

Metformin targets many pathways that play an important role in cancer cell proliferation and angiogenesis; mTORC1 signaling is a crucial pathway among them. Metformin inhibits mTORC1 via AMPK dependent and AMPK independent pathways, thereby inhibiting cancer cell growth and development. Both AMPK dependent and AMPK independent mechanisms were involved in anticancer activity of metformin along with the outcome of preclinical and clinical studies¹⁰.

Repurposing metformin for the prevention of cancer and cancer recurrence was suggested. Multiple epidemiological studies have documented an association between metformin, used for treatment of type 2 diabetes, and reduced cancer incidence and mortality.

Cell line models may not accurately reflect the effects of metformin in the clinical setting. Moreover, findings from animal model studies have been inconsistent, whilst those from more recent epidemiological studies have tempered the overall effect size. The purpose of the study was to examine metformin’s chemopreventive potential by outlining relevant mechanisms of action, the most recent epidemiologic evidence, and recently completed and ongoing clinical trials¹¹.

Metformin reverses TRAP1 mutation-associated alterations in mitochondrial function in Parkinson's disease. Metformin reverses mutation-associated alterations on energy metabolism, mitochondrial biogenesis and restores mitochondrial membrane potential. TRAP1 acts downstream of PINK1 and HTRA2 for mitochondrial fine tuning, whereas TRAP1 loss of function leads to reduced control of energy metabolism, ultimately impacting mitochondrial membrane potential. These findings offer new insight into mitochondrial pathologies in Parkinson's disease and provide new prospects for targeted therapies.

Following genetic screening of Parkinson's disease patients and healthy controls, The study reported the first TRAP1 mutation leading to complete loss of functional protein in a patient with late onset Parkinson's disease. Analysis of fibroblasts derived from the patient reveal that oxygen consumption, ATP output and reactive oxygen species are increased compared to healthy individuals. This is coupled with an increased pool of free NADH, increased mitochondrial biogenesis, triggering of the mitochondrial unfolded protein response, loss of mitochondrial membrane potential and sensitivity to mitochondrial removal and apoptosis. These data highlight the role of TRAP1 in the regulation of energy metabolism and mitochondrial quality control¹².

Metformin and caffeic acid regulate metabolic reprogramming in human cervical carcinoma SiHa/HTB-35 cells and augment anticancer activity of Cisplatin via cell cycle regulation. The findings provide new insights into specific targeting of mitochondrial metabolism in neoplastic cells and into designing new cisplatin-based selective strategies for treating cervical cancer in humans with regard to the role of tumor microenvironment.

Normal human fibroblasts (FB) and metastatic cervical cancer cells (SiHa) were exposed to 10 mM Metformin (Met), 100 μM Caffeic Acid (trans-3,4-dihydroxycinnamic acid, CA) or combination of the compounds. Both drugs were selectively toxic towards cancer cells, but neither Met nor CA treatment suppressed growth of normal cells. Met and CA regulated metabolic reprogramming in SiHa tumor cells through different mechanisms: Met suppressed regulatory enzymes Glurtaminase (GLS) and Malic Enzyme 1 (ME1) and enhanced pyruvate oxidation via tricarboxylic acids (TCA) cycle, while CA acted as glycolytic inhibitor. Met/CA treatment impaired expression of Sterol Regulatory Element-Binding Protein 1 (SREBP1c) which resulted in alleviation of de novo synthesis of unsaturated fatty acid. The toxic action of CisPt was supported by Met and CA not only in tumor cells, but also during co-culture of SiHa GFP+ cells with fibroblasts. Furthermore, Met and CA augmented Cisplatin (CisPt) action against quiescent tumor cells involving reprogramming of cell cycle¹³.

Repurposing metformin in Fragile X syndrome (FXS) is caused by loss of the fragile X mental retardation 1 (FMR1) gene and is characterized by learning disabilities and cognitive impairment. Here, injection of the type 2 diabetes therapeutic metformin for 10 days corrected the social deficits, repetitive behaviour, cognitive impairment, aberrant dendritic spine morphology and exaggerated long-term depression of synaptic transmission in a mouse model of FXS.

Metformin reduced the elevated mRNA translation and normalized extracellular signal-related kinase signalling, eukaryotic translation initiation factor 4E phosphorylation and matrix metalloproteinase 9 expression¹⁴.

Repurposing metformin as a quorum sensing inhibitor in Pseudomonas aeruginosa. Inhibition of quorum sensing can disarm the virulence factors without exerting stress on bacterial growth that leads to emergence of antibiotic resistance. Finding a new quorum sensing inhibitor and determining its inhibitory activities against virulence factors of Pseudomonas aeruginosa PAO1 strain was performed. Metformin can act as a quorum sensing inhibitor and virulence inhibiting agent that may be useful in the treatment of Pseudomonas aeruginosa infection.

Quorum sensing was evaluated by estimation of violacein production by Chromobacterium violaceum CV026. Molecular docking was used to investigate the possible binding of metformin to LasR and rhlR receptors. The inhibition of pyocyanin, hemolysin, protease, elastase in addition to swimming and twitching motilities, biofilm formation and resistance to oxidative stress by metformin was also assessed. Metformin significantly reduced the production of violacein pigment. Significant inhibition of pyocyanin, hemolysin, protease and elastase was achieved. Metformin markedly decreased biofilm formation, swimming and twitching motilities and increased the sensitivity to oxidative stress. In the molecular docking study, metformin could bind to LasR by hydrogen bonding and electrostatic interaction and to rhlR by hydrogen bonding only¹⁵.

The cell-autonomous mechanisms underlying the activity of metformin as an anticancer drug.

Current understanding of the molecular mechanisms were discussed that are perturbed by metformin treatment and that might be relevant to understand its antitumour activities. The study focus on the cell-autonomous mechanisms modulating growth and death of cancer cells¹⁶.

Repurposing Metformin as Therapy for Prostate Cancer. Metformin is a safe, well-tolerated, inexpensive treatment that can be given in addition to current standard-of-care therapies for prostate cancer. Its use might mitigate the deleterious side effects of castration and exert an additional anticancer effect.

Repurposing Metformin as Therapy for Prostate Cancer will test its true utility as a repurposed treatment for men with high-risk locally advanced or metastatic prostate cancer at first presentation¹⁷.

Metformin potentiates the anticancer activities of gemcitabine and cisplatin against cholangiocarcinoma cells in vitro and in vivo.

Metformin inhibited the proliferation of human cholangiocarcinoma RBE and HCCC-9810 cells and induced cell cycle arrest at the G0/G1 phase by increasing the activation of AMP-activated protein kinase (AMPK) pathways. Metformin upregulated the expression of p21Waf1 and p27kip1, and downregulated the expression of cyclin D1, a key protein required for cell cycle progression. The combination of gemcitabine and cisplatin inhibited the proliferation and induced the apoptosis of human cholangiocarcinoma cells by inducing the phosphorylation of AMPK, downregulating cyclin D1, and activating caspase-3. Administration of metformin enhanced the efficacy of gemcitabine and cisplatin to suppress the growth of cholangiocarcinoma tumors established in experimental models by inhibiting cell proliferation and inducing cell apoptosis through their effects on AMPK, cyclin D1 and caspase-3¹⁸.

Metformin use and gynecological cancers: A novel treatment option emerging from drug repositioning. Recent in vitro and experimental data suggest that metformin electively targets cancer stem cells, and acts together with chemotherapy to block tumor growth in different cancers. Several epidemiological studies and meta-analysis have shown that metformin use is associated with decreased cancer risk and/or reduced cancer mortality for different malignancies. The present review analyzes the recent biological and clinical data suggesting a possible growth-static effect of metformin also in gynecological cancers. The large majority of available clinical data on the anti-cancer potential of metformin are based on observational studies. Therefore long-term phase II–III clinical trials are strongly warranted to further investigate metformin activity in gynecological cancers.

Metformin exerts antitumor effects mainly through AMP-activated protein kinase [AMPK] activation and phosphatidylinositol 3-kinase [PI3K]-Akt-mammalian target of rapamycin [mTOR] inhibition. This drug leads to activation of the cellular energy-sensing liver kinase B1 [LKB1]/AMPK pathway. LKB1 is implicated as a tumor suppressor gene in molecular pathogenesis of different malignancies. AMPK is a serine/threonine protein kinase that acts as an ultra-sensitive cellular energy sensor maintaining the energy balance within the cell. AMPK activation inhibits mRNA translation and proliferation in cancer cells via down-regulation of PI3K/Akt/mTOR pathway. Moreover, metformin decreases the production of insulin, insulin-like growth factor, inflammatory cytokines and vascular endothelial growth factor, and therefore it exerts anti-mitotic, anti-inflammatory and anti-angiogenetic effects¹⁹.

Anticancer effect of metformin on estrogen receptor-positive and tamoxifen-resistant breast cancer cell lines

one of the anticancer mechanisms of metformin could be attributable to the repression of expression and transcriptional activity of ERα. Metformin may be a good therapeutic agent for treating ERα-positive breast cancer by inhibiting the expression and function of ERα. In addition, metformin may be useful to treat tamoxifen-resistant breast cancer.

The objective of this study was to investigate the anticancer activity of metformin in relation to ERα expression and its signaling pathway in ERα-positive MCF-7 and MDA-MB-361 breast cancer cells as well as TR MCF-7 breast cancer cells. Metformin inhibited both protein and mRNA levels of ERα in the presence or absence of estrogen (E2) in the MCF-7, TR MCF-7 and MDA-MB-361 cells. Metformin repressed E2-inducible estrogen response element (ERE) luciferase activity, protein levels and mRNA levels of E2/ERα-regulated genes [including c-Myc, cyclin D1, progesterone receptor (PR) and pS2] to a greater degree than tamoxifen, resulting in inhibition of cell proliferation of MCF-7, TR MCF-7 and MDA-MB-361 cells.²⁰

Repurposing of metformin in liver injury. It describes a protective role of metformin in acetaminophen mediated liver injury.

It describes a protective role of metformin by down-regulating MKK4-JNK activity in a Gaad45ß-dependent and AMPK-independent manner. The study shown that metformin has protective and therapeutic effects against acetaminophen (APAP) overdose-evoked hepatotoxicity via Gadd45β-dependent MKK4-JNK signaling regulation^21-22.

Toward Repurposing Metformin as a Precision Anti-Cancer Therapy Using Structural Systems Pharmacology as safe, effective, personalized therapies.

To repurpose metformin as a precision anti-cancer therapy, the authors have developed a novel structural systems pharmacology approach to elucidate metformin's molecular basis and genetic biomarkers of action. The authors integrated structural proteome-scale drug target identification with network biology analysis by combining structural genomic, functional genomic, and interactomic data. Through searching the human structural proteome, the authors identified twenty putative metformin binding targets and their interaction models and experimentally verified the interactions between metformin and our top-ranked kinase targets. Notably, kinases, particularly SGK1 and EGFR were identified as key molecular targets of metformin. Subsequently, they linked these putative binding targets to genes that do not directly bind to metformin but whose expressions are altered by metformin through protein-protein interactions, and identified network biomarkers of phenotypic response of metformin. The molecular targets and the key nodes in genetic networks are largely consistent with the existing experimental evidence. Their interactions can be affected by the observed cancer mutations²³.

Repurposing metformin to chemoprevention

The purpose of this paper is to examine metformin's chemopreventive potential by reviewing relevant mechanisms of action, preclinical evidence of efficacy, updated epidemiologic evidence after correction for potential biases and confounders, and recently completed and ongoing clinical trials. Although repurposing drugs with well described mechanisms of action and safety profiles is an appealing strategy for cancer prevention, there is no substitute for well executed late phase clinical trials to define efficacy and populations that are most likely to benefit from an intervention²⁴.

Metformin is a candidate for the treatment of gynecological tumors based on drug repositioning. The use of metformin for the treatment of gynecological cancer may become a successful example of drug repositioning, following establishment of the drug's antitumor effects, risk evaluation, screening and validation of efficacy.

The anticancer action of metformin involves the enhancement of phosphorylation of liver kinase B1, activation of adenosine monophosphate-activated protein kinase and inhibition of mammalian target of rapamycin, which reduces cell growth. Metformin is anticipated to exert antitumor effects in gynecological cancer and its efficacy for the treatment of endometrial, breast and ovarian cancer has been suggested in preclinical studies and clinical trials. Although the effect of metformin on cervical cancer remains to be examined in clinical trials, its antitumor effects have been reported in preclinical studies²⁵.

Repurposing metformin for cancer treatment

Preclinical studies have demonstrated several anticancer molecular mechanisms of metformin including mTOR inhibition, cytotoxic effects, and immunomodulation. Epidemiologic data have demonstrated decreased cancer incidence and mortality in patients taking metformin. Several clinical trials, focused on evaluation of metformin as an anti-cancer agent are presently underway. Data published from a small number of completed trials has put forth intriguing results. Clinical trials in pre-surgical endometrial cancer patients exhibited a significant decrease in Ki67 with metformin monotherapy. Another interesting observation was made in patients with breast cancer, wherein a trend towards improvement in cancer proliferation markers was noted in patients without insulin resistance. Data on survival outcomes with the use of metformin as an anti-cancer agent is awaited²⁶.

Metformin pharmacogenomics: A genome-wide association study to identify genetic and epigenetic biomarkers involved in metformin anticancer response using human lymphoblastoid cell lines was suggested. Mechanistic studies revealed that the E3 ubiquitin ligase, STUB1, could influence metformin response by facilitating proteasome-mediated degradation of cyclin A. GWAS using a genomic data-enriched LCL model system, together with functional and mechanistic studies using cancer cell lines, help us to identify novel genetic and epigenetic biomarkers involved in metformin anticancer response.

The authors conducted a pharmacogenomic study using 266 lymphoblastoid cell lines (LCLs). Metformin cytotoxicity assay was performed using the MTS assay. Genomewide association (GWA) analyses were performed in LCLs using 1.3 million SNPs, 485k DNA methylation probes, 54k mRNA expression probe sets, and metformin cytotoxicity (IC50s). Top candidate genes were functionally validated using siRNA screening, followed by MTS assay in breast cancer cell lines. Further study of one top candidate, STUB1, was performed to elucidate the mechanisms by which STUB1 might contribute to metformin action. GWA analyses in LCLs identified 198 mRNA expression probe sets, 12 SNP loci, and 5 DNA methylation loci associated with metformin IC50 with P-values < 10-4 or < 10-5. Integrated SNP/methylation loci-expression-IC50 analyses found 3 SNP loci or 5 DNA methylation loci associated with metformin IC50 through trans-regulation of expression of 11 or 26 genes with P-value < 10-4 Functional validation of top 61 candidate genes in 4 IPA networks indicated down regulation of 14 genes significantly altered metformin sensitivity in two breast cancer cell lines²⁷.