Antitumor Effects Of Metformin Research Articles

Metformin as an adjuvant option for systemic treatment of breast cancer

The modification of the used and development of new treatment regimens significantly improved the overall, recurrence-free survival and quality of life of patients with malignant oncological diseases. Recently, drugs used in non-oncological pathology have been introduced into cancer treatment regimens. This phenomenon is associated with a better understanding of the biology of tumor cells and the mutations that can change this biology. Metformin is actively researched in terms of the treatment of various oncological diseases. For the most part, the modification of the neoadjuvant treatment regimen for local or locally advanced stages of breast cancer results in a less traumatic variant of surgical procedure, thereby increasing recurrence-free survival. The aim is to systematize the data on the possibilities of the antitumor metformin usage for neoadjuvant treatment of breast cancer and to study the results of clinical and morphological effectiveness of the treatment by reviewing the literature. A search of PubMed from February 2023 showed 258 results on the antitumor effects of metformin, of which only 159 were published in the last 5 years. On this subject only four clinical studies were carried out, and only one of them pertained to the implementation of metformin in the systemic treatment of breast cancer. For this review, 55 sources of general and specialized information on anticancer effects of metformin were analyzed. It should be noted that approximately 60% of the study results were published more than 5 years ago and primarily focused on the biological, not clinical aspects of metformin usage. Only one study regarding the implementation of metformin for systemic treatment of breast cancer was carried out by Ukrainian scientists. Currently, there are 2 main hypotheses regarding the antitumor effect of metformin. First one is driven by its impact on the metabolic function of cells and energy deficit. Second - the method of regulating the proliferation of tumor cells involves the PIK3 branch of the regulatory cascade of biological reactions in cancer cells. In addition, metformin reduces the development of resistance in breast cancer cells, thus allowing active chemotherapy agents to act in synergism. However, further studies on the effect of metformin used alone or in combination with standard chemotherapy regimens require a more adequate definition of proto-oncogenic mutations and somatic mutation load. However, it should be considered that more aggressive therapy of oncological diseases can be a nosocomial selector of more aggressive clones of the pool of tumor cells. The main questions are whether metformin can be a targeted drug for the treatment of tumor, whether it is appropriate to use it at the time when the manifestation of evolution disturbances of tumor cell is minimal and homogeneity is maximal.

Metformin may improve the outcome of patients with colorectal cancer and type 2 diabetes mellitus partly through effects on neutrophil extracellular traps

BackgroundAlthough metformin reduces the risk of cancer-related mortality in patents with type 2 diabetes, the mechanism of its anti-cancer effects has not been fully understood.MethodImpact of metformin on survival was examined in patients who underwent curative colectomy for colorectal cancer (CRC). The effects of metformin in neutrophil extracellular traps (NETs) were examined with in-vitro experiments and multiplex immunohistochemistry of surgically resected CRC specimens.ResultsPrior intake of metformin prolonged relapse-free (P = 0.036) and overall survival (P = 0.041) in 289 patients with T2DM to the comparable levels to those of 1576 non-diabetic patients. Metformin reduced the production of NETs stimulated with lipopolysaccharide or HT-29 colon cancer cells to 60% of control. Neutrophils markedly suppressed the chemotactic migration of activated T cells in an NET-dependent manner, which was reversed by metformin treatment up to approximately half of the migration without neutrophils. Immunohistochemical analysis revealed a significant association between metformin intake and a reduction in the numbers of tumor-associated neutrophils (TANs) and NETs. Simultaneously, metformin intake was found to increase the presence of CD3(+) and CD8(+) tumor-infiltrating T cells (TILs), particularly at the tumor-invasion front, especially in areas with fewer TANs and NETs.ConclusionMetformin suppresses the diabetes-associated enhancement of NET formation, which can augment the infiltration of TILs in CRC tissues. The anti-tumor effect of metformin in patients with T2DM may be, at least partly, attributable to the inhibition of NETs.

Integration of metabolomics and transcriptomics reveals metformin suppresses thyroid cancer progression via inhibiting glycolysis and restraining DNA replication

The anti-tumoral effects of metformin have been widely studied in several types of cancer, including thyroid cancer; however, the underlying molecular mechanisms remain poorly understood. As an oral hypoglycemic drug, metformin facilitates glucose catabolism and disrupts metabolic homeostasis. Metabolic reprogramming, particularly cellular glucose metabolism, is an important characteristic of malignant tumors. This study aimed to explore the therapeutic effects of metformin in thyroid cancer and the underlying metabolic mechanism. In the present study, it was shown that metformin reduced cell viability, invasion, migration, and EMT, and induced apoptosis and cell cycle G1 phase arrest in thyroid cancer. Transcriptome analysis demonstrated that the differentially expressed genes induced by metformin were involved in several signaling pathways including apoptosis singling pathways, TGF-β signaling, and cell cycle regulation in human thyroid cancer cell lines. In addition, the helicase activity of the CDC45-MCM2–7-GINS complex and DNA replication related genes such as RPA2, RAD51, and PCNA were downregulated in metformin-treated thyroid cancer cells. Moreover, metabolomics analysis showed that metformin-induced significant alterations in metabolic pathways such as glutathione metabolism and polyamine synthesis. Integrative analysis of transcriptomes and metabolomics revealed that metformin suppressed glycolysis by downregulating the key glycolytic enzymes LDHA and PKM2 and upregulating IDH1 expression in thyroid cancer. Furthermore, the anti-tumor role of metformin in thyroid cancer in vivo was shown. Together these results show that metformin plays an anti-tumor role by inhibiting glycolysis and restraining DNA replication in thyroid cancer.

Metformin: A New Inhibitor of the Wnt Signaling Pathway in Cancer.

The biguanide drug metformin is widely used in type 2 diabetes mellitus therapy, due to its ability to decrease serum glucose levels, mainly by reducing hepatic gluconeogenesis and glycogenolysis. A considerable number of studies have shown that metformin, besides its antidiabetic action, can improve other disease states, such as polycystic ovary disease, acute kidney injury, neurological disorders, cognitive impairment and renal damage. In addition, metformin is well known to suppress the growth and progression of different types of cancer cells both in vitro and in vivo. Accordingly, several epidemiological studies suggest that metformin is capable of lowering cancer risk and reducing the rate of cancer deaths among diabetic patients. The antitumoral effects of metformin have been proposed to be mainly mediated by the activation of the AMP-activated protein kinase (AMPK). However, a number of signaling pathways, both dependent and independent of AMPK activation, have been reported to be involved in metformin antitumoral action. Among these, the Wingless and Int signaling pathway have recently been included. Here, we will focus our attention on the main molecular mechanisms involved.

Abstract 510: Targeting metabolic vulnerabilities to reduce triple negative breast cancer health disparities

Abstract Triple negative breast cancer (TNBC) occurs in 10-15% of all breast cancer (BC) patients, yet it accounts for almost half of all BC deaths. Women of African ancestry (WAA) are twice as likely as women of European Ancestry (WEA) to be diagnosed with advanced TNBC with worse prognosis. Emerging data shows that insulin resistance and high circulating levels of insulin are more prevalent in WAA with invasive BCs than in WEA, and activation of the AKT/mTOR pathway by insulin may occur in aggressive TNBC. Reports show diabetic patients treated with metformin, a biguanide drug used to treat diabetes type 2, have reduced incidence of BC and improved survival. However, anticancer actions of metformin require use of high drug doses with limiting side effects in vivo. To address this challenge, we used a structure-activity strategy to develop biguanide analogues with more potent anticancer action and safety at lower doses in vivo. Using TNBC cell proliferation in vitro to screen analogues, promising candidates were identified that exerted dose-dependent inhibition of cell proliferation at significantly lower doses than that of parental metformin (P<0.01). As antitumor effects of metformin are attributed in part to activation of LKB1-AMPK pathways, we find that biguanide analogues also strongly induce AMPK phosphorylation on Western immunoblots and significantly reduce phosphorylation of downstream mTOR signaling pathway components including p70S6K, S6 ribosomal protein and 4E-BP1. Further, analogues induce TNBC cell apoptosis in vitro at lower doses than metformin. In vivo, analogues were more effective than metformin in stopping human TNBC xenograft progression in nude mouse models (P<0.001). Notably, analogues were also more effective than metformin at blocking lung metastases in syngeneic murine 4T1 TNBC models (P<0.05). Transcriptome analyses comparing mammary tumors and lung metastases revealed that analogue JD006 down-regulated genes related to oxidative phosphorylation in lung metastases treated with JD006 and increased expression of genes related to T-cell activation. Further, gene expression in tumors treated with JD006 showed significant down-regulation of long non-coding RNAs that associate with the up-regulation of malignant transformation and activation of M1 macrophages. Importantly, our data indicate that analogue JD006 modulates the activity/trafficking of myeloid-derived suppressor cells (MDSC) and tumor infiltrating lymphocytes (TIL) that may significantly impact TNBC responses to immune checkpoint inhibitors. Further understating of potential biologic differences between TNBC of WAA and WEA is needed to design more effective therapeutic strategies to reduce TNBC health disparities. New targeted treatments could be beneficial for patients afflicted with this deadly disease. (Funding: CBCRP B27IB3869, 4IB-0058; NCI U54 CA143930; JCCC BC Award, Team Research Grant; UCLA TDG). Citation Format: Diana C. Márquez-Garban, Gang Deng, Lorena P. Burton, Gaoyuan Ma, Vishaka Muhunthan, Begonya Comin-Anduix, Gabriela Llarena, Neda Moatamed, Jennifer Murphy, Nalo Hamilton, Daivd Shackelford, Michael E. Jung, Richard J. Pietras. Targeting metabolic vulnerabilities to reduce triple negative breast cancer health disparities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 510.

Antitumor activity of Metformin through p53 and Cyclin D1 in the Urothelial Cell Carcinoma

Bladder cancer is considered as one of the main drivers of cancer related mortality in adult men. Data from Global Cancer Statistics 2018 (GLOBOCAN) showed that the bladder cancer was included among the Top 10 cancer incidence in worldwide. Meanwhile, metformin, an antidiabetic agent, is believed to be able to impede the varying cancer cells expansion. Many examinations had displayed that metformin interferes via the AMPK/mTOR axis pathway, thereby suppressing tumor growth. AMPK activation can also increase stromal cell survival through p53 activation. Metformin also disrupts the cell cycle by decreasing the cyclin D1 protein in cancer cells. The human cell line 5637 was treated with metformin 15 mM, examined for cyclin D1 and p53 by immunohistochemical staining and assessed for the viability of cancer cells. The Statistic test was utilized to make a comparison of tumor viabilities and other variables. No significant differences were found in the expression of wild type p53 and cyclin D1 but significant differences were observed in the viability between the control and metformin groups. We have proven in our study that the anti-tumor effect of metformin in reducing the viability of urothelial carcinoma tumor cells not only through p53 and cyclin D1.

Abstract 898: Metformin and ICG-001 act synergistically to abrogate cancer stem cell-mediated chemoresistance in colorectal cancer by promoting apoptosis and autophagy

Abstract Background: Colorectal cancer (CRC) remains the third most frequently diagnosed cancers in the United States. Current treatment modalities for patients with CRC include surgery, which is often followed with 5-fluorouracil (5FU) based adjuvant chemotherapy. While such treatment regimens are effective at improving disease outcomes, their clinical usefulness is often hampered due to emergence of chemotherapeutic resistance. Presence of cancer stem cells (CSCs) have been attributed towards 5FU-mediated drug resistance which subsequently leads to poor prognosis in CRC patients. In this study we investigated a novel therapeutic strategy by using metformin along with ICG-001, a Wnt signaling inhibitor, as an approach to abrogate CSC mediated chemoresistance in 5FU resistant (5FUR) and parental CRC cells, as well as CRC patient derived tumor organoids. Methods: Two 5FU resistant (5FUR)-CRC cell lines previously generated and characterized in our lab were used in this study. First, we performed genome-wide transcriptomic profiling followed by pathway enrichment analysis to identify differentially expressed genes and its associated pathways between parental and 5FUR CRC cells. Next, we determine the anti-tumor effects of metformin and ICG-001 by performing cell viability, colony formation, migration, and invasion analysis. Next, we explore the possible mechanism of action of these two drugs by performing gene and protein expression analysis of CSC, autophagy and apoptotic markers. We also evaluated the anti-tumor activity of metformin and ICG-001 in CRC patient derived tumor organoids. Results: We observed that 5FUR-CRC cells exhibited increased expression of CSC markers and increased spheroid forming ability. Genome-wide transcriptomic analysis followed by pathway analysis revealed that Wnt signaling pathway, pathways regulating pluripotency of stem cells etc. were enriched between parental and 5FUR CRC cells. These findings prompted us to use ICG-001 which selectively targets Wnt signaling along with metformin in abrogating CSC mediated chemoresistance in CRC. We observed that a combined treatment of metformin and ICG-001 exert anti-tumor activity in by decreasing cell viability (p<0.01), colony formation (p<0.001), migration (p<0.01), and invasion ability (p<0.01) in synergistic manner. We also observed that combined treatment decreases the expression of CSC markers and promote autophagy and apoptosis. In accordance with our in vitro studies, we observed that metformin and ICG-001 exhibited anti-tumor activity in patient derived CRC organoid models in combination (p<0.001). Conclusion: We conclude that metformin and ICG-001 act synergistically, which can be used as a therapeutic strategy to overcome 5FU mediated therapeutic resistance in CRC. Citation Format: Souvick Roy, Yinghui Zhao, Yate-Ching Yuan, Ajay Goel. Metformin and ICG-001 act synergistically to abrogate cancer stem cell-mediated chemoresistance in colorectal cancer by promoting apoptosis and autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 898.

Metformin has no Significant Anticancer Effect on Patients with Advanced or Unresectable Cancer: A Systematic Review and Meta-analysis

At present, the antitumor effect of metformin is controversial. Previous meta-analyses included observational studies, of which the results can be influenced by many confounders, affecting the result of meta-analyses and weakening the strength of evidence. Therefore, we conducted a meta-analysis to confirm the effect of metformin use on patients with advanced or unresectable cancers, including randomized clinical trials (RCTs). We searched for RCTs in accordance with the inclusion and exclusion criteria. A meta-analysis was conducted to combine hazard ratios (HRs) or risk ratios (RRs) and their 95% confidence intervals (CIs) using a random-effects model. Finally, 7 eligible RCTs were included in the meta-analysis. Overall, the combined results revealed that treatment with metformin did not improve the overall survival (OS) of patients (HR, 1.12; 95% CI, 0.91-1.37, p>0.05), and there was no clear evidence that metformin use was related to improved progression-free survival (PFS) (HR, 1.17; 95% CI, 0.97-1.40; p>0.05). The pooled RR for grade III or IV adverse events was 0.92 (95% CI, 0.52- 1.60; p>0.05), indicating that the use of metformin was not significantly related to increased toxicity. Metformin does not significantly improve the survival of patients with advanced or unresectable cancer, regardless of cancer type and region.

Transcriptome Analysis of Liver Cancer Cell Huh-7 Treated With Metformin.

Metformin is a kind of widely used antidiabetic drug that regulates glucose homeostasis by inhibiting liver glucose production and increasing muscle glucose uptake. Recently, some studies showed that metformin exhibits anticancer properties in a variety of cancers. Although several antitumor mechanisms have been proposed for metformin action, its mode of action in human liver cancer remains not elucidated. In our study, we investigated the underlying molecular mechanisms of metformin's antitumor effect on Huh-7 cells of hepatocellular carcinoma (HCC) in vitro. RNA sequencing was performed to explore the effect of metformin on the transcriptome of Huh-7 cells. The results revealed that 4,518 genes (with log2 fold change > 1 or < −1, adjusted p-value < 0.05) were differentially expressed in Huh-7 cells with treatment of 25-mM metformin compared with 0-mM metformin, including 1,812 upregulated and 2,706 downregulated genes. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses identified 54 classical pathways that were significantly enriched, and 16 pathways are closely associated with cancer, such as cell cycle, DNA replication, extracellular matrix–receptor interaction, and so on. We selected 11 differentially expressed genes, which are closely associated with HCC, to validate their differential expressions through a quantitative real-time reverse transcription-polymerase chain reaction. The result exhibited that the genes of fatty acid synthase, mini-chromosome maintenance complex components 6 and 5, myristoylated alanine-rich C-kinase substrate, fatty acid desaturase 2, C-X-C motif chemokine ligand 1, bone morphogenetic protein 4, S-phase kinase-associated protein 2, kininogen 1, and proliferating cell nuclear antigen were downregulated, and Dual-specificity phosphatase-1 is significantly upregulated in Huh-7 cells with treatment of 25-mM metformin. These differentially expressed genes and pathways might play a crucial part in the antitumor effect of metformin and might be potential targets of metformin treating HCC. Further investigations are required to evaluate the metformin mechanisms of anticancer action in vivo.

Genome-wide CRISPR screen identifies synthetic lethality between DOCK1 inhibition and metformin in liver cancer

Metformin is currently a strong candidate anti-tumor agent in multiple cancers. However, its anti-tumor effectiveness varies among different cancers or subpopulations, potentially due to tumor heterogeneity. It thus remains unclear which hepatocellular carcinoma (HCC) patient subpopulation(s) can benefit from metformin treatment. Here, through a genome-wide CRISPR-Cas9-based knockout screen, we find that DOCK1 levels determine the anti-tumor effects of metformin and that DOCK1 is a synthetic lethal target of metformin in HCC. Mechanistically, metformin promotes DOCK1 phosphorylation, which activates RAC1 to facilitate cell survival, leading to metformin resistance. The DOCK1-selective inhibitor, TBOPP, potentiates anti-tumor activity by metformin in vitro in liver cancer cell lines and patient-derived HCC organoids, and in vivo in xenografted liver cancer cells and immunocompetent mouse liver cancer models. Notably, metformin improves overall survival of HCC patients with low DOCK1 levels but not among patients with high DOCK1 expression. This study shows that metformin effectiveness depends on DOCK1 levels and that combining metformin with DOCK1 inhibition may provide a promising personalized therapeutic strategy for metformin-resistant HCC patients.

Metformin-Induced Heat Shock Protein Family A Member 6 Is a Promising Biomarker of Esophageal Squamous Cell Carcinoma

Introduction: Antidiabetic drug metformin exerts various antitumor effects on different cancers. Esophageal squamous cell carcinoma (ESCC) is an intractable digestive organ cancer and new treatment strategy is required. In this study, we performed a comprehensive gene expression analysis of ESCC cell lines treated with metformin, which provided helpful information on the antitumor effects of metformin in ESCC. Next, we selected a promising gene among them and examined its effects on ESCC properties. Methods: We examined metformin-induced mRNA expression changes in two human ESCC cell lines by performing next-generation sequencing (NGS) and pathway analysis. Heat shock protein family A (Hsp70) member 6 (HSPA6) expression in surgical specimens obtained from 83 ESCC patients who underwent curative operations was evaluated immunohistochemically and analyzed. Results: Metformin upregulated mRNA expression of the many genes, including HSPA6, a cancer immune-related gene, and inhibited mRNA expression of the other many genes. Pathway analysis indicated major canonical pathways and upstream regulators related to metformin. The result indicated HSPA6 as a promising biomarker. HSPA6 expression correlated with disease-free survival (DFS) of the patients with all stage ESCC (p = 0.021), especially with stage I/II ESCC (p < 0.001). With stage III, low HSPA6 expression was not associated with poor DFS (p = 0.918). Multivariate analysis indicated that independent low HSPA6 expression was an independent poor prognostic factor of stage I/II ESCC (p < 0.001). However, HSPA6 expression did not correlate with the clinicopathological characteristics, including age, sex, tumor depth, lymph node metastasis, tumor stage, and tumor markers of the patients with stage I/II ESCC. Conclusions: This NGS analysis detected prospective candidate genes, including HSPA6. Our results indicate that HSPA6 is a promising biomarker of the recurrence risk of stage I/II ESCC. Further studies on HSPA6 would lead to better treatment.

The use of metformin in the treatment of gastrointestinal cancer

Type 2 diabetes mellitus and malignancies have several common risk factors, such as obesity, metabolic syndrome, hyperinsulinemia, and chronic inflammation. Currently these factors are considered as possible mechanisms that increase the risk of the development of malignanttumors, including gastrointestinal cancer. Epidemiological studies and meta-analyses indicate that patients with diabetes have a higher incidence and mortality from malignancies. Metformin is well-known oral hypoglycemic drug that belongs to the biguanide class and is being used to treat diabetes mellitus for almost a century. It has been established that in patients with diabetes mellitus, long-term use of metformin reduces the incidence of tumors and mortality from various cancers. The results of recent studies suggest that metformin may have direct pleiotropic anticancer activity against many tumor cells and their microenvironment. Its potential mechanisms include both insulin-dependent and insulin-independent. Moreover, metformin promotes antitumor immunosuppressive metabolic control of T lymphocytes and cancer cells, it is able to modulate the intestinal microbiota and renders a systemic effect on a body metabolism. The article presents data that reveal possible mechanisms of anticancer action of metformin and justify the use of metformin as a drug that may be useful for the treatment of gastrointestinal cancer. Although most of the clinical trials have retrospective design, they are often limited in time and patient bias, recent data from preclinical studies on the antitumor effects of metformin suggest that its effects on carcinogenesis and gastrointestinal cancer progression need to be further investigated. Future studies should consider the diabetic status, prognostic biomarkers, disease stage, and treatment regimens. Given that metformin is a safe and widely available drug with low cost, the feasibility of further study of its anti-cancer effects is beyond doubt.

Additive antitumor effect of metformin and simvastatin combination in glioblastoma: evidence for a potential drug repurposing

Additive antitumor effect of metformin and simvastatin combination in glioblastoma: evidence for a potential drug repurposing

Dynamic switch of immunity and antitumor effects of metformin in rat spontaneous esophageal carcinogenesis

Chronic inflammation contributes to tumor development by creating a local microenvironment that facilitates neoplastic transformation and potentiates the progression of cancer. Esophageal cancer (EC) is an inflammation-associated malignancy with a poor prognosis. The nature of the switch between chronic inflammation of the esophagus and EC-related immunological changes remains unclear. Here, we examined the dynamic alterations of immune cells at different stages of chronic esophagitis, Barrett’s esophagus (BE) and EC using an esophageal spontaneous carcinogenesis rat model. We also investigated the anticancer effects of metformin. To stimulate EC carcinogenesis, chronic gastroduodenal reflux esophagitis via esophagojejunostomy was induced in 120 rats in metformin-treated and non-treated (control) groups. After 40 weeks, BE and EC developed in 96.7% and 63.3% of the control group, and in 66.7% and 23.3% of the metformin-treated group, respectively. Flow cytometric analysis demonstrated that the balance of M1/M2-polarized or phospho-Stat3-positive macrophages, regulatory T, cytotoxic T, natural killer (NK), NK T cells, and Th17 T cells was dynamically changed at each stage of the disease and were resolved by metformin treatment. These findings clarify the immunity in esophageal carcinogenesis and suggest that metformin could suppress this disease by improving the immunosuppressive tumor microenvironment and immune evasion.

Antitumor Effect of Metformin in Combination with Binimetinib on Melanoma Cells.

Cutaneous melanoma is a fatal disease for patients with distant metastasis. Metformin is the most widely used anti-diabetic drug, and proved to suppress cell proliferation and metastasis in diverse cancers including melanoma. We previously reported that MEK inhibitor trametinib increases the expression of epithelial-mesenchymal transition (EMT) regulators and melanoma cell motility, which are suppressed by addition of metformin in A375 melanoma cells. To confirm our findings further, we first evaluated the effect of metformin in combination with another MEK inhibitor binimetinib on cell viability in G361 melanoma cells. We then investigated whether binimetinib affects the expression of EMT regulators and cell motility. We finally monitored the effect of metformin on binimetinib-induced cell migration. Cell viability assay showed that combination index (CI) value at ED50 is 0.80, suggesting synergy for the combination of metformin with binimetinib. Our results also revealed that binimetinib increased the expression of EMT regulators such as integrin αV, fibronectin and slug, which correlate well with the enhanced cell migration in wound healing assay. Metformin, on the contrary, suppressed the expression of sparc, integrin αV, fibronectin and N-cadherin with the reduced cell motility. The combination treatment showed that metformin counteracts the binimetinib-induced increase of cell motility. Overall, these results suggest that metformin with binimetinib might be useful as a potential therapeutic adjuvant against cell survival and metastatic activity in melanoma patients.

Novel Metformin Analogues for Treatment of Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDCA) is a leading cause of cancer death in the US. Patients diagnosed with PDCA generally present with advanced disease with poor prognosis and limited treatment options. African American patients have higher incidence and mortality of PDCA than Caucasian American or any other ethnic group. Different factors have been attributed to contribute to this health disparity, among them higher incidence of Diabetes Mellitus type 2. To address the need for new therapeutic approaches, we note epidemiologic reports that patients with diabetes mellitus-type 2 treated with the biguanide drug metformin, but not other antidiabetic drugs, have a reduced risk of PDCA and an increased survival rate among those with PDCA. The main physiologic effect of metformin is to lower blood glucose and reduce hyperinsulinemia associated with insulin resistance. In the cell, metformin stimulates AMP-activated protein kinase (AMPK) that in turn inhibits mTORC1 which integrates signals from an array of intracellular pathways to regulate cell growth. Recent clinical trials describe modest antiproliferative effects from use of neoadjuvant metformin, but no significant clinical benefit occurred when metformin was dosed at glycemic control levels in patients with advanced cancers. These findings suggest that development of more potent anticancer analogues of metformin may help to boost clinical benefit and patient survival. Hence, we have designed new biguanide analogues of metformin, and screening of these compounds in preclinical PDCA models show that selected analogues are more efficacious in blocking tumor progression than parental metformin at lower doses. Using proliferation assays in vitro, PDCA cells (Panc 1, MIA Paca-2) were treated 72-hrs with metformin or analogues, and greater dose-dependent inhibition of PDCA cell proliferation was found with analogues as compared to metformin (P<0.001). Further, apoptosis was also markedly induced by metformin analogues as compared to parental metformin (P<0.01). Antitumor effects of metformin are attributed in part to activation LKB1-AMPK pathways and downstream blockade of mTOR signaling, which is often increased in PDCA cells. Using PDCA cells treated in vitro with analogues for 24-hrs, we find that analogues induce AMPK phosphorylation and suppression of mTOR signaling, thus blocking protein synthesis and tumor proliferation. With an in vivo PANC 1 xenograft model in nude mice, lead metformin analogues given by oral gavage daily significantly inhibited tumor progression over 28-days as compared to appropriate controls (P<0.0001). Our findings show that selected metformin analogues have potent anticancer activity in preclinical PDCA models and may have promise as new targeted therapeutics for patients afflicted with this deadly disease. [Funded by NIH/NCI R21CA176337 and NIH/NCI U54 CA143930]

Abstract 692: Discovery of novel metformin derivatives with potent antitumor activity in combination with immunotherapy for treatment of triple-negative breast cancer

Abstract Triple negative breast cancer (TNBC) occurs in about 15% of breast cancer (BC) patients, yet it accounts for almost 50% of all BC deaths. TNBC is characterized by lack of expression of estrogen, progesterone and HER2 receptors, and it cannot be treated with current targeted therapies. TNBCs are heterogeneous and occur often in younger and African American women. Although initially responsive to some chemotherapies, TNBCs tend to relapse early and metastasize, leading to poor survival. Development of new therapeutic approaches for clinical translation is a high priority. Emerging evidence from epidemiologic and preclinical work suggests that metformin, the most widely used drug to treat type 2 diabetes mellitus, exerts anticancer activity in BC. Diabetic patients treated with metformin have a reduced incidence and better survival from BC. Moreover, TNBC cells are reported to be uniquely sensitive to metformin in vitro, but strong antitumor effects require relatively high metformin doses in vivo. To address this challenge, we used a structure-activity strategy to develop metformin analogues with more potent anticancer action and safety at lower doses in vivo. Using TNBC cell proliferation in vitro to screen drug candidates, selected new metformin analogues were identified that exerted dose-dependent inhibition of cell proliferation at significantly lower concentrations than that of parental metformin (P&amp;lt;0.01). As antitumor effects of metformin are attributed in part to activation of the LKB1-AMPK pathway, we find that metformin analogues also strongly induced phosphorylation of AMPK by Western blot assays and significantly reduced phosphorylation of mTOR downstream signaling pathway components (p70S6K, S6 ribosomal protein, 4E-BP1). Analogues also induced TNBC cell apoptosis at lower doses than metformin. In vivo, metformin analogues were more effective at lower doses given by oral gavage than metformin in suppressing TNBC human tumor xenograft progression in nude mouse models (P&amp;lt;0.001). It is notable that data from clinical trials with immune checkpoint inhibitors (ICI) indicate that TNBCs are susceptible to immunotherapy, but only a minority of patients to date have had clinical benefit. Importantly, our studies indicate that metformin analogues modulate the activity and trafficking of myeloid-derived suppressor cells (MDSC) and tumor infiltrating lymphocytes (TIL) that may significantly impact TNBC responses to ICIs. Thus, combination therapy with metformin analogues and anti-PD-L1 antibodies was more effective at blocking tumor growth in vivo than either treatment given alone using murine 4T1 TNBC implants in syngeneic, immune-competent BALB/C mouse models. In the TNBC microenvironment, analogue treatment with ICIs elicited significant infiltration of predominantly effector T-cells, increased populations of tissue-resident memory T-cells, and a reduction of exhausted T-cells as compared to controls (P&amp;lt;0.01). Moreover, MDSC were significantly decreased after combination therapy as compared to appropriate controls (P&amp;lt;0.05). Since there are currently no specific treatments for TNBC, identification of a new targeted therapeutic approach could ultimately be beneficial for managing patients afflicted with this deadly disease. Funding by California BC RP, NCI U54 CA143930. Citation Format: Diana C. Márquez-Garban, Gang Deng, Begonya Comin-Anduix, Vishaka Muhunthan, Gaoyuan Ma, Jennifer Murphy, Alejandro J. Garcia, Cristian D. Yanes, Lorena R. Burton, Nalo Hamilton, David Shackelford, Michael E. Jung, Richard J. Pietras. Discovery of novel metformin derivatives with potent antitumor activity in combination with immunotherapy for treatment of triple-negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 692.

Low glucose enhanced metformin’s inhibitory effect on pancreatic cancer cells by suppressing glycolysis and inducing energy stress via up-regulation of miR-210-5p

ABSTRACT To explore mechanisms underlying the discrepancy in anti-tumor effects of metformin on pancreatic cancer cells PANC-1 under different glucose conditions. We cultured PANC-1 cells in 25 mM and 5 mM glucose media, then treated with or without metformin. It showed that metformin significantly inhibited proliferation and viability, induced apoptosis of PANC-1 cells, which was more pronounced in low-glucose than in high-glucose group. Metformin up-regulated the expression of miR-210-5p in low glucose, but not in high glucose. miR-210-5p mimic inhibited the viability of PANC-1 cells and further enhanced the inhibitory effect of metformin. miR-210-5p down-regulated the expression of PFKFB2, a predicted target gene of miR-210-5p, reduced the activity of PFK1 and LDH. Metformin significantly inhibited the expression of phosphorylation-PFKFB2(p-PFKFB2) in the low-glucose group and inhibited the LDH activity both in the low and high glucose groups, thus inhibiting anaerobic glycolysis and inducing energy stress. Cells in the high glucose group could make a compensatory adaptation to the energy stress induced by metformin through increasing glucose consumption. However, due to the limited glucose supply and high dependence on anaerobic glycolysis of cells in the low glucose group, they couldn’t make effective adaptive compensation. Therefore, cells in the low-glucose group were more vulnerable to the toxicity of metformin. In conclusion, the enhanced inhibitory effect of metformin on PANC-1 cells cultured in low glucose may be due to the up-regulation of the expression of miR-210-5p, then inhibiting anaerobic glycolytic flux and inducing energy stress via repressing the expression of p-PFKFB2 and activity of LDH. Abbreviations PC: pancreatic cancer; DM: diabetes mellitus; PFKFB2: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase2; PFK1: phosphofructokinases; LDH: lactate dehydrogenase; F-2,6-BP: fructose 2,6-bisphosphate

Abstract A16: Metformin is a potential nontoxic adjuvant to enhance the efficacy of non-PDL1/PD-1 targeting immune therapies

Abstract Investigations into various immunotherapies combined with conventional anticancer drugs are ongoing to increase therapeutic efficacy. However, combination therapy generally increases the risk of side effects. To achieve high efficacy with minimal side effects, nontoxic adjuvants should be identified and appropriate combinations should be designed based on the functional mechanism. In this regard, metformin can be an attractive candidate for immunotherapeutic adjuvants. Metformin is a widely used oral medication for type 2 diabetes (T2D) and has been recognized as a safe and well-tolerated drug through several decades of clinical experience. Interestingly, metformin also exhibits antitumor effects as several case-control studies for T2D patients indicated that metformin reduces the incidence of various cancer types. However, the functions and the detailed mechanism of metformin related to cancer immunity are not fully understood. In this study, we investigated the antitumor effects of metformin in relation to cancer immunity in the tumor microenvironment. Our data showed that AMPK activated by metformin decreases the expression of PD-L1 in the cancer cells, blocking PD-L1’s ability to aid cancer cells in escaping immune surveillance. This is caused by the mechanism in which phosphorylation of PD-L1 at S195 induces an abnormal glycan structure that leads to endoplasmic reticulum-associated degradation. In addition, we have obtained human breast tumor tissues from a previous clinical trial investigating metformin as treatment for breast cancer patients. The data from human tumor tissues also provided strong support to our current conclusion, namely AMPK activated by metformin reduces the level of PD-L1. On the basis of these results, we validated the possibility of metformin as an adjuvant to boost the efficacy of previous immunotherapy without toxicity. Our findings suggest that metformin has strong potential to be used as an adjuvant for immunotherapy. Metformin is expected to have synergistic effect with various non-PDL1/PD-1 targeting immune therapies without additional toxicity. Citation Format: Jong-Ho Cha, Wen-Hao Yang, Weiya Xia, Yongkun Wei, Li-Chuan Chan, Seung-Oe Lim, Chia-Wei Li, Jennifer Hsu, Hung-Ling Wang, Chu-Wei Kuo, Wei-Chao Chang, Sirwan Hadad, Colin Purdie, Aaron McCoy, Jennifer Litton, Elizabeth Mittendorf, Stacy Moulder, William Symmans, Alastair M Thompson, Helen Piwnica-Worms, Chung-Hsuan Chen, Kay-Hooi Khoo, Mien-Chie Hung. Metformin is a potential nontoxic adjuvant to enhance the efficacy of non-PDL1/PD-1 targeting immune therapies [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A16.

Anti-diabetic drug metformin inhibits cell proliferation and tumor growth in gallbladder cancer via G0/G1 cell cycle arrest.

Gallbladder cancer is the most common biliary tract cancer with poor prognosis and wide variation in incidence rates worldwide, being very high in some countries in Latin America and Asia. Treatment of type 2 diabetes with metformin causes a reduction in the incidence of cancer. Till date, there are no reports on the anti-tumor effects of metformin in gall bladder cancer. Therefore, this study evaluated the effects of metformin on the proliferation of human gallbladder adenocarcinoma cells in vitro and in vivo, as well as explored the microRNAs associated with the anti-tumor effects of metformin. Metformin inhibited the proliferation in gallbladder adenocarcinoma cell lines NOZ, TGBC14TKB, and TGBC24TKB, and blocked the G0 to G1 transition in the cell cycle. This was accompanied by strong reduction in the expression of G1 cyclins, especially cyclin D1 and its catalytic subunits including cyclin-dependent kinase 4, and in retinoblastoma protein phosphorylation. In addition, metformin reduced the phosphorylation of receptor tyrosine kinases, especially Tie-2, ALK, PYK, EphA4, and EphA10, as well as angiogenesis-related proteins, including RANTES, TGF-β, and TIMP-1. Moreover, metformin also markedly altered microRNA expression profile leading to an anti-tumor effect. Treatment of athymic nude mice bearing xenograft tumors with metformin inhibited tumor growth. These results suggest that metformin may be used clinically for the treatment of gallbladder adenocarcinoma.