1. Increased cancer-related mortality for patients with type 2 diabetes who use sulfonylureas or insulin.Bowker SL, Majumdar SR, Veugelers P, Johnson JA – Diabetes Care 2006 Feb
2. Common variants near ATM are associated with glycemic response to metformin in type 2 diabetes. Kaixin Zhou, Celine Bellenguez-Nat Genet 2011 Feb
3. Cell cycle arrest in Metformin treated breast cancer cells involves activation of AMPK, downregulation of cyclin D1, and requires p27Kip1 or p21Cip1-Yongxian Zhuang and W Keith Miskimins- J Mol signals 2008
4. Metformin activates an ataxia telangiectasia mutated (ATM)/Chk2-regulated DNA damage-like response. Vazquez-Martin A, Oliveras-Ferraros C, Cufí S, Martin-Castillo B, Menendez JA-Cell cycle, 2011 May(ATM)%2FChk2-regulated%20DNA%20damage-like%20response.
5. Metformin: multi-faceted protection against cancer Del Barco S. – Oncotarget December 2011
6. Metformin blocks the stimulative effect of a high-energy diet on colon carcinoma growth in vivo and is associated with reduced expression of fatty acid synthase. Algire C, Amrein L, Zakikhani M, Panasci L, Pollak M.- Endocr Relat Cancer 2010 Jun
7. Promoter-specific effects of metformin on aromatase transcript expression. Samarajeewa NU, Ham S, Yang F, Simpson ER, Brown KA – Steroids 2011 July
Metformin, the well-known hypoglycemic drug used as a first-line therapy for NIDDM, is emerging in the last years as a possible antitumor drug, due to his effects preventing carcinorigenesis in healthy people and reducing mortality in those who have already developed a tumor form.
FIRST EPIDEMIOLOGICAL EVIDENCES
The first evidence of this collateral action of metformin were obtained from some epidemiological studies, such as that conducted by Bowker et al. (1). This was a cohort study in which was demonstrated a possible link between assumption of metformin and reduced cancer-mortality, particularly if compared with the mortality in those patient which assume other classes of drugs, such as sulfuylureas and synthetic insulin. This results, however, if partially demonstrate the theoretical postulate that a substance which makes increase the circulating levels of a proliferative and antiapoptotic hormone such as insulin is also potentially a carcinogenic, were not sufficient at all to support the thesis of metformin as an anticancer. The absolute reduction of cancer-mortality in metformin-treated patient compared with generic population in fact could be due to selection-bias or to other type of statistical errors, event which is very common in a complex pathology like type II diabetes. These studies, like almost all statistical studies, weren’t solving; however helped to shed light to the problem and gave rise in the scientific community to that interest which finally brought to the determination, in bio-molecular terms, of the mechanism of action of this drug as an anticancer.
MOLECULAR MECHANISMS
1. Direct effects on ATM and on the DDR
Metformin is known to be an activator of AMPK, enzyme sensible to the AMP/ATP ratio and whose activity increases proportionally to this ratio. When AMP levels increase in the cell (situation which occurs when the cell is in a stress condition), activated AMPK enhances all catabolic processes, disadvantaging anabolic ones. Activation of AMPK is mediated by many other kinases, the most relevant being: CaMK (calcium modulated kinase), TAK1 (TGF β dependent protein 1) and LKB1 (liver kinase B1). The latter seems to be very important for metformin to exert its antitumor action: in fact LKB1 is phosphorylated by ATM (ataxia telangiectasia mutated), in which was recently discovered a possible medium of metformin action. This protein, so called after a rare pathology, ataxia telangiectasia, present in subject with a particular mutation of its gene, is a 370 kD member of the PIKK family (Kinases related to the PI-3K), well-known as a major component of the DDR (DNA damage response) (2). In fact ATM is a serine-threonine kinase sensible to DNA double-strand breaks and to DNA alkylation, working in a paraller and complementary way with the other “stress-sensor” ATR (Ataxia teleangiectasia and Rad3 related protein) . His activation brings to the phosphorylation of many downstream protein (MRE11, NSB1) and finally the signals converge on the activation of Chk2 (Checkpoint homologue 2) and on p53, both characterized by an oncosuppressor role. The activation of p53 activates the transcription of p21 and the arrest in the cell’s cycle. Moreover metformin is able to induce cycle arrest independently of p53 status, as was observed in one of the first studies conducted about this item (3). However, should be clear that if the enhancement of catabolic pathway, that is known from a long time as the best-characterized effect of metformin, depends on activation of AMPK ATM-mediated, this could also partially justify the antitumor effect of the drug, considering this position of “molecular crossroad” of ATM.
Extremely important to confirm this particular role of ATM were the studies conducted by Zhou et al. In these studies, in order to highlight a causal link between activation of ATM and downstream molecular effects of metformin, was used a selective inhibitor of ATM, KU-55933. Using the cell line of rat hepatoma, was confirmed the original hypothesis: the addition of metformin to the cell culture medium elicited an increased AMPK activity. If it was added KU-55933, the slope of the AMPK in function of metformin dramatically decreased, clearly indicating that ATM is a cellular mediator of metformin effects.
Furthermore, confirming this hypothesis, were recently published by Vasquez-Martin et al. some interesting results according to which also the kinase Chk2 is activated after the exposition of the cell to metformin: the activation of ATM not only enhances the pathway ATM/LKB1/AMPK but also the apparently more relevant (at least in oncologic terms) DDR pathway (4).
Moreover has been demonstrated that a chronic activation of AMPK, independently from the phosphorylation status of ATM causes an activation of p53 and consequently altering the mitosis’ processes and enhancing apoptosis.
2. Inhibition of mTOR
mTOR (mammalian Target of Rapamycin) is a PI3KK involved in the pathogenesis of many tumor, whose activation in the cancer cell is considered an index of bad prognosis and of resistance to chemotherapy. Has been demonstrated that AMPK, activated by metformin, phosphorylates and stabilizes the tumor suppressor TCS2 (tuberous sclerosis 2) which is able to inhibit mTOR (5).
3. How metabolic effects directly elicit anticancer activity
Besides its effect on stimulating the DDR pathway, a great contribute to metformin anticancer activity seems to be given by the strictly metabolic effects of the drug. This effects too are mediated by the activation of ATM and, as exposed in the previous paragraph, by ATM-mediated phosphorylation of LKB1 and, downstream, of AMPK. In fact, is known from a long time that cancer cell express a singular metabolic pattern (the so called “cancer metabotype”), characterized by a high rate of glycolysis independent of the oxygen content of the cell - the Warburg Effect – and by a copious lipogenesis. Some recent researches have highlighted how metformin is responsible for an inhibition of both these pathways, dramatically changing the metabotype of the tumor cell. If it’s certainly easy to see how a hypoglycemic drug like metformin, stimulating the assumption of glucose by adipose and muscular tissue, can deprive the hypothetical tumor mass of his supply of glucose, are at first sight not so evident all the reasons why this drug should inhibit the de novo synthesis of fatty acids and the lipogenesis. The shortage of glucose and the consequent lack of ATP only partially justify the inefficiency of this pathways, the reason of which indeed is to be found in an inhibition of ACC, FAS, HMGcoAR and many other lipogenic enzymes mediated by AMPK (6).
4. Effects of metformin on estradiol secretion
Another interesting demonstration of how metformin can lead to cancer prevention (or better prognosis) is emerged from some studies on breast cancer in which was retrospectively analyzed the link between the onset (and the prognosis) of this pathology in the post-menopausal period and the production of estrogens in this period of woman’s life. It is known that many post-menopausal breast cancer are hormone-dependent and that their growth depends especially on the stimulation of the tumor mass by estrogens: in fact, also during menopause, small amounts of estrogens continue to be produced, from adrenal androgens, by aromatization in hepatic, muscular and adipose tissue. A correlation so can be obtained between the total fat mass and the amount of estrogens. Considering this phenomenon of enhancement of tumor cell’s growth, have been synthesized many drugs which acts as antagonists in the estrogens signaling pathway (SERM) or as inhibitors of aromatase and consequently of estrogen synthesis (Als); however, this latter class of drugs are responsible for many side effect, due to the whole-body inhibition of aromatase. A possible candidate as a inhibitor of estrogen production, selectively for the mammary district, is metformin. The aromatase gene, CYP19A1, has a very complex structure composed by many exons which are expressed in a highly differentiated and tissue specific way. In the adipose tissue surrounding many breast cancer has been found to be very active a particular promoter for an exon of this enzyme, the PII/PI.3 promoter. In such sites this promoter is active because of the PGE2-rich environment: this prostaglandin brings to an increase in intracellular cAMP in the cancer cell and to over-expression of the exon which is controlled by this promoter, finally conducing to an increase of aromatase in this area. Has been shown that metformin is able directly to inhibit the transcription of this exon, without altering the expression of PI.4 (the classical aromatase promoter sequence) and so behaving as a breast selective Als. To prove this phenomenon was used a hASCs line (breast adipose stromal cell) (7), in which was induced activation of both PII/PI.3 and PI.4 promoters; the first activation was achieved through exposure of the cells with forskolin/phorbol ester, the second through exposure with dexametasone/TNFα. In presence of this activators large amount of aromatase were transcribed by this cells. However metformin, already at relatively low concentrations, has been shown to diminish the aromatase production; this was a consequence only of the inhibition of forskolin/phorbol ester-induced transcription of aromatase, since, via PCR, was highlighted a decrease only in the specific-PII/PI.3 transcripts, announcing a possible future use of this drug also as a targeted therapy for post-menopausal breast cancers.
