Research Question

The goal of this project is to investigate the anti-diabetic drug metformin which has been shown in various studies to prevent the progression of precancerous pancreatic cells to pancreatic ductal adenocarcinoma (PDAC) (Mohammed, et al. 2013). The main target of this drug is the activation of the AMPKinase pathway and the inhibition of the mTOR complex (Mohammed, et al. 2013).


Why is Studying Pancreatic Cancer Important?


Pancreatic cancer is one of the most lethal forms of cancer with a less than 6% estimated five year survival. In 2013 there were about 42,220 cases of pancreatic cancer in the United States. About 38,460 of these cases resulted in patient death making the disease the fourth leading cause of cancer mortality (Wang, et al. 2014). The mortality rate is so high because of a lack of early diagnosis techniques and effective treatments. Pancreatic intraepithelial neoplasia (PanIN) is the precursor to PDAC. The form that PanIN takes in our pancreas is a lesion. More information on PanIN lesions can be found on the pancreatic cancer page. Epidemiological studies have shown that more than 80% of patients that have pancreatic cancer have diabetes mellitus (Wang, et al. 2014). Whether diabetes is a precursor to or a result of pancreatic cancer is highly controversial but there is evidence to support both arguments. A chemopreventative is in high demand for those that might be at a greater risk of developing the disease because of its poor prognosis due largely in part to late detection. Researchers have found that the anti-diabetic drug metformin might be the chemopreventative they have been looking for.

Researchers from the Department of Radiation Oncology and Department of Statistics at the Fudan University Shanghai Cancer Center in Shanghai, China conducted a meta-analysis of observational studies analyzing metformin's potential ability to decrease the risk of various cancers among patients with type 2 diabetes mellitus (T2DM) was conducted (Wang, et al. 2014). Included in this analysis was 11 articles (13 studies) made up of 10 cohort studies and 3 case-control studies looking specifically at pancreatic cancer. The summarized results concluded that metformin was in fact associated with a significantly lower risk of pancreatic cancer (Wang, et al. 2014).

BIO179 Figure from Metformin&T2DM.png
Figure 1. The risk ratio as determined by each of the studies involved in the meta-analysis and the subsequent position of the study plotted on a scale of favoring vs. not favoring metformin use as a cancer preventive (Wang, et al. 2014).

This figure is known as a Forest plot and demonstrates the drug's odds ratio, with 1 being the median value. Forest plots are crucial to analyzing results of studies used in meta-analyses (Islas 2014). The more participants that were tested in each of the studies used in this meta-analysis decreased the associated standard error and therefore made the plotted risk ratio more accurate for the results (Islas 2014). Studies were weighted based on their reliability, sample sizes, and study type. It is certainly favorable that this meta-analysis used 10 cohort studies and only 3 case-control studies because cohort studies are better types of trials. This strengthens the validity of this meta-analysis. 5/13 studies were significantly significant, favoring metformin as a chemopreventative, while 8/13 showed no statistical significance (none were statistically significant favoring no metformin). We can see at the bottom of the plot the total of all of the studies contained in this analysis, and with 95% confidence interval, metformin is a favored intervention in the prevention of cancer progression. The risk ratio therefore determines that metformin is effective in humans (Figure 1).

Because of the results in this plot, we wanted to look deeper into the effects of metformin, and how it affects the cells on a molecular level, as in signaling pathways. We have included in our report the results from a multi-part research project that aims to demonstrate how metformin works directly on the prevention of PanIN lesion progression to PDAC, the decrease of mTOR signaling, and the increase of activated AMP kinase in precancerous cells (Mohammed, et al. 2013).



Understanding the Anti-Diabetic Drug Metformin

What is Metformin?
Metformin is an anti-diabetic drug sold under the prescription names Glumetza, Fortamet, Riomet, and Glucophage. It is prescribed to patients with type 2 diabetes to help lower blood glucose levels as a main form of treatment along with diet and exercise. Metformin helps the body become more sensitive to its own insulin, decreases the amount of sugar the liver produces, and decreases the amount of sugar the intestine absorbs (Learn More... 2014).

Metformin Structure.png
Figure 2. Molecular structure of metformin (Ref. 6)

How Metformin Treats Patients with Type 2 Diabetes
The liver is the main site of action for Metformin where is has been shown to reduce hepatic glucose output by 75% (Pernicova, et al. 2014).

  • Reduces glucose output by inhibiting liver gluconeogenesis which is the formation of glucose by the liver from noncarbohydrate sources, such as amino acids (Boitard 2014). In addition the uptake of gluconeogenic substrates (Alanine and Lactate) is reduced by metformin (Pernicova, et al. 2014).
  • Increased Glucose Uptake in Skeletal Muscle: Insulin sensitivity is increased in skeletal muscles because of an increase in the tyrosine kinase activity of the insulin receptor along with increased GLUT1 (glucose transporter) transport activity by increasing translocation to the plasma membrane (Pernicova, et al. 2014).
  • Altered Endocrine Function in the Pancreas: Metformin stimulates the expression of the GLP-1 (glucagon-like peptide 1) receptor and GLP-1 protein in the pancreas. GLP-1 is responsible for increasing the secretion of insulin and lowering the secretion levels of glucagon (a hormone that raises blood glucose levels) (Pernicova, et al. 2014).
  • Slightly delays the absorption of glucose through the gastrointestinal (GI) tract (Pernicova, et al. 2014).

Molecular Targets of Metformin in Diabetics
nrendo.2013.256-f3.jpg

Figure 3. Mechanistic action of Metformin in the hepatocytes of the liver in diabetic patients (Pernicova, et al. 2014).

  • Metformin is a polar molecule that relies on membrane transporters such as OCT1 and hENT3 for cellular uptake and secretion (Pernicova, et al. 2014). Metformin acts selectively on the liver by binding to the Organic Cation Transporter (OCT) of hepatocytes (liver specific cells) (Boitard 2104).

  • Mitochondria: The mitochondria is the primary target of Metformin where it inhibits the complex 1 of the mitochondrial transport chain where the NADH is reduced so the production of ATP is blocked (Boitard 2014). The result of this is a drop in ATP production and rise in AMP levels. These changes in energy levels will result in the activation of the AMP activated protein Kinase (AMPK) (Pernicova, et al. 2014).
    When activated the AMPK turns on metabolic pathways that make ATP and turns off anabolic pathways that consume ATP such as hepatic gluconeogenesis (Boitard 2014).

  • Glucagon Signaling and Metformin: Individuals with diabetes have higher levels of circulating glucagon because hyperglycemia prevents the suppression of the alpha cells that secrete the glucagon. Glucagon acts by activating adenylate cyclase which allows for an increase in cAMP levels that then activate the protein Kinase A (PKA). Eventually through a signal cascade glycolysis is inhibited and gluconeogenesis is activated by alteration of several enzymes in these pathways. Metformin interrupts this pathway by inhibiting glucagon signalling. This is thought to occur by way of increasing AMP levels through inhibition of mitochondrial complex 1 (Pernicova, et al. 2014).

  • Metformin improves insulin secretion and sensitivity by lowering the levels of glucose and free fatty acids as well as preventing lipid deposition in insulin sensitive tissues. High levels of free fatty acids can reduce glucose transport by dampening the insulin signaling (Pernicova, et al. 2014).

Understanding the Connection Between Metformin and Cancer

Indirect Systemic Effects on Tumorigenesis
Under insulin resistant conditions metformin lowers systemic glucose levels and improves secondary hyperinsulemia in the liver which prevents tumor growth and progression. This means that it can affect insulin sensitive neoplastic tissues indirectly without the need to accumulate in cancer cells (Pernicova, et al. 2014).


AMPK-dependent Metformin Effects in Cancer
LKB-1 activation of the AMPK pathway and its suppression of mTOR pathway is the most potent antineoplastic effect of metformin because it disrupts protein synthesis and therefore tumor cell proliferation. The mTOR complexes are critical in tumor growth and are responsible for integrating signals received from hormones and energy sensing pathways (Pernicova, et al. 2014).


MetforminCancerMechanism.png

Figure 4. Mechanisms of action of Metformin in cancer cells (Pernicova, et al. 2014).


Activating the AMPKinase has many downstream effects on the cell. AMPK activates tumor suppressor genes (TSC1 and TSC2) that inhibit mTOR. AMPK also directly inhibits RAPTOR which is an activating member of the mTOR complex. Activating AMPK will phosphorylate the IRS-1 protein which results in the inhibition of the transmittance of the signal from the insulin receptor to the PI3K-AKT pathway. Inhibition of PIK3 allows activation of TSC1/TSC2 tumor suppressors that inhibit the mTOR pathway. The ability for AMPK to activate the crucial p53 tumor suppressor that frequently undergoes a loss of function mutation in cancer is still controversial but some studies have found this to be the case which would then inhibit the AKT and mTOR pathways. Through AMPK activation, metformin inhibits the proto-oncogene cMYC as well as HIF-1alpha (Pernicova, et al. 2014).
cMYC is a transcription factor that regulates many genes responsible in rapid cellular proliferation (Miller, et al. 2012). It is very commonly mutated in many forms of cancers but has yet to be successfully inhibited by pharmaceuticals. HIF-1alpha is a transcription factor that promotes the expression of GLUT1. HIF1alpha is found in mammalian cells growing at low oxygen concentrations which is a characteristic of cancer cells (Miller, et al. 2012). A decrease in HIF-1alpha signaling helps in the altering of metabolic processes in tumor cells allowing them to favor glycolysis as an energy source even under aerobic conditions. This is also known as the Warburg effect which is one of the two emerging hallmarks of cancer. Cancer cells undergo a metabolic transformation promoting fatty acid synthesis and ensuring the availability of intermediates as building blocks for proliferating cells (Pernicova, et al. 2014). In preneoplastic cells with a functioning AMPK pathway, metformin can counteract the Warburg effect (Pernicova, et al. 2014). Specifically in LBK1-AMPK mutation driven pancreatic cancers the cells are selectively more vulnerable to the decrease in ATP caused by metformin because their ability to restore their energy is already impaired (Pernicova, et al. 2014).


Mouse Model Studies of Metformin as a Chemopreventative

Various studies have been done on the mechanisms of metformin and its potential as a chemopreventative because a preventive for pancreatic cancer is in such high demand. Due to the highly aggressive nature and the treatment-resistant properties of the cancer, "the overall 5 year survival rate has not exceeded 5% and the median survival period remains less than 6 months" (Wang, et al. 2014). The incidence rate of PC is high and has decreased very little in the past years, despite the development and optimization of treatments. A multi-part study conducted by researchers from the University of Oklahoma Health Sciences Center in Oklahoma City, OK and the Chemopreventative Agent Development Research Group from Bethesda, MD looked at the effects of metformin on PanIN and their progression to PDAC in p48Cre/+.LSL-KrasG12D/+ mice. We have focused our project around this study because we feel that it best represents the potential of metformin but also critically analyzes the results. It gives the most realistic picture of metformin's abilities as a potential chemopreventative.

Looking at Metformin's Effects on Gene Expression
Within this study they analyzed, among other things, the expression of mTOR mRNA in mice fed diets of varying dosages of metformin and metformin-free diets (Mohammed, et al. 2013). Because mTOR is vital to cell proliferation and the reduction of apoptosis, it becomes a key player in the growth of tumors. In the case of pancreatic cancer, we see the over-activation of the PI3K/Akt/mTOR pathway which boosts a cancer cell's ability to growth and proliferate within the pancreatic tissues (Mohammed, et al. 2013). The Materials and Methods page explains more details of how the study was carried out.

In this study, mTOR expression and its related signaling molecules were analyzed using quantitative, real-time PCR. Frozen samples of pancreatic tissue from the mice were also analyzed for fold change increases in mRNA expression for a variety of proteins and transcription factors including mTOR, Rheb, cyclin D1, among others (Mohammed, et al. 2013). Rheb is a GTP-binding protein involved in growth and cell cycle progression while Cyclin D1 is a CDK 4/6 regulatory protein. If mutations, amplifications, or over-expression of cyclin D1 occurs tumorigenesis can occur because of the proteins direct link with cell cycle regulation. The following image shows results from this experiment, analyzing the mRNA expression of mTOR in p48Cre/+.LSL-KrasG12D/+ mice fed metformin-supplemented diets of varying amounts in comparison with mice fed an AIN-76A diet alone (the control)(Mohammed, et al. 2013). The mice fed diets with metformin showed nearly two fold decreases in mTOR, Rheb, and Cyclin D1 expression (Figure 3. A, B, E). It is important to note that according to the researchers conducting the study, "None of the animals fed the metformin diets exhibited any observable toxicity or any gross morphologic changes to liver, spleen, kidney, or lung" (Mohammed, et al. 2013). This is vital for metformin ever having a chance to progress forward and potentially be a standard of care for preventing the progression of PDAC.

BIO179 Wikipage Figure 4A.jpg BIO179 Wikipage Figure 4B.jpg BIO179 Wikipage Figure 4E.jpg
Figure 3. Effect of metformin on expression of mRNA for A) mTOR, B) Rheb, and E) Cyclin D1 as determined by real-time quantitative PCR (Ref. 1).

This drug appears to be effective at decreasing the expression of certain proteins and transcription factors contributing to the continued growth of pancreatic cancer cells, however, as the primary researchers of this study pointed out, "It is important to elucidate whether this anti-diabetic drug can exert direct effects on in vivo transgenic animal models of PC that recapitulate the stepwise progression of precursor lesions to carcinoma as seen in humans" (Mohammed, et al. 2013). We have seen test results that suggest a mechanism that should work in humans, but cancer in human pancreatic tissue is much more complex than in these simple mice models. It would be interesting to see the results of a similar test in humans, or at least in human cells grown in culture. These type of experiments would be telling if the mechanism of metformin affecting the expression of mTOR mRNA, and other related proteins, holds true in human cells.

Looking at Metformin's Effect on mTOR and pAMPK Pathways
Results from Immunohistochemical (IHC) analyses and fluorescence further solidified the argument in favor of metformin as a chemopreventative. Samples of mice pancreata (of the same study as the results above) were stained and photographed to examine active mTOR signaling and phosphorylated AMP kinase (pAMPK) activity.The photos taken below are from the pacreata of the control, lower dose, and higher dose of metformin. Again, the Materials and Methods page will have all information regarding the mice type and protocol for analysis (Mohammed, et al. 2013).


BIO179 Wikipage Figure 3.jpg

Figure 5. Effect of metformin on mTOR and pAMPK in pancreatic tumors. IHC and immunofluorescence analyses were performed with paraffin-embedded and microsectioned pancreatic tissues as described in the Materials and Methods section. A significantly decreased expression of mTOR and an increased pAMPK expression were seen with metformin treatment (Mohammed, et al. 2013).

It is clear in the photos that mTOR activity in the pancreatic cancer cells was significantly decreased in mice treated with metformin, particularly in the lower (1000ppm) dose in comparison to the control. This illustrates that metformin is in fact acting as a chemopreventative by inhibiting mTOR (via the activation of AMPK) to inhibit cell growth and proliferation, processes vital to cancer cell development and progression (Figure 5). Both the IHC analysis and the fluorescence support these conclusions for mTOR activity. As for the phosphorlyated (activated) AMPK, the mice treated with metformin show significantly greater expression than the control mice, again in higher amounts in the 1000ppm dose (Figure 5). This too supports metformin as a possibly effective chemopreventative. As pAMPK activity increases, as it does in the two experimental mice, mTOR is inhibited along with the activation of certain cancer suppressor genes, such as TSC1 and 2 as was mentioned above (Mohammed, et al. 2013).


Looking at Metformin's Effect on PanIN Lesion Progression
This part of the research study looked at the number of PainIN lesions observed in the four conditions (wild type mice, control diet-fed mice, 1000ppm metformin-fed mice, and 2000ppm metformin-fed mice). The type of mice used for this analysis is consistent with the type used above, but the methods will of course vary and can be found of the Materials and Methods page (Mohammed, et al. 2013). There were no lesions found in the pancreata of the wild type mice (mice not induced to develop pancreatic cancer), however the number of lesions were counted in the control and experimental mice pancreata and the results summarized below.


BIO179 Wikipage Figure 2.jpg
Figure 4. (A and B) Effect of metformin on the PanIN multiplicity (means ± SE; A—male, B—female) (Ref. 1).

According to these results, there is a significantly decrease number of PanIN 3 lesions (carcinoma in situ) in the experimental mice pancreata (at both dosages, but particularly in the 1000ppm dosage of metformin, in comparison with the control, non-metformin treated mice pancreata. This shows that the number of lesions that could potentially progress into PDAC (full, invasive pancreatic cancer), is decreased in those mice given metformin. Interestingly enough, the number of PanIN 2 and especially PanIN 1 lesions are much greater in the experimental mice than in the control mice. This observation suggests that meformin is acting as a blockade to these lesions' further progression to PanIN 3 grade or further to PDAC (Figure 4). All of these findings are consistent across both sexes of mice. This is consistent with and supports the idea of metformin acting as an effective pancreatic cancer chemopreventative.


Future Potential

In non-diabetic patients showed a short term treatment with metformin suppressed the formation of colorectal abberant crypt focci (Pernicova, et al. 2014). Metformin also reduced cellular marker proliferation, Ki67, in biopsy samples obtained from nondiabetic women with breast cancer (Pernicova, et al. 2014). There are currently clinical trials looking into metformin as a potential cancer chemopreventative in breast cancer, colorectal cancer, lung cancer, and prostate cancer. The next step will be to determine how to use this drug, for what types of cancer, and at what point in the treatment.


Resources


  1. Mohammed, Altaf, Naveena B. Janakiram, and Misty Brewer, et al. "Antidiabetic Drug Metformin Prevents Progression of Pancreatic Cancer by Targeting in Part Cancer Stem Cells and mTOR Signaling." Translation Oncology 6.6 (2013): 649-659. Print.
  2. "mTOR Signaling Pathway." Cell Signaling Technology (CST): Antibodies, Reagents, Proteomics, Kits and Consumables. Cell Signaling Technology, Nov 2012. Web. 12 May 2014. <http://www.cellsignal.com/contents/science-pathway-research-pi3k-akt-signaling-resources/mtor-signaling-pathway/pathways-mtor-signaling>.
  3. American Diabetes Association. "Diagnosis and Classification of Diabetes Mellitus," “Insulin Basics,” “Statistics about Diabetes.” Diabetes Care 3.1 (2008): 533. Print.
  4. "Learn More About the TREATMENT of Type 2 Diabetes With GLUMETZA®." Treatment for Type 2 Diabetes. Salix Pharmaceuticals Inc. Web. 13 May 2014.
  5. Boitard, Celia. "Metformin: What Cellular Target?" International Group on Insulin Secretion. Web. 13 May 2014.
  6. Harahap, Yahdiana-, Santi Purnasari, Hayun Hayun, Krisnasari Dianpratami, and Mahi Wulandari. "Bioequivalence Study of Metformin HCl XR Caplet Formulations in Healthy Indonesian Volunteers." Journal of Bioequivalence & Bioavailability 03.01 (2011) Print.
  7. "AMPK Signaling Pathway." Cell Signaling Technology (CST): Antibodies, Reagents, Proteomics, Kits and Consumables. Web. 13 May 2014.
  8. "MTOR Signaling Pathway." Cell Signaling Technology (CST): Antibodies, Reagents, Proteomics, Kits and Consumables. Web. 12 May 2014.
  9. Pernicova, Ida, and Márta Korbonits. "Metformin—mode of Action and Clinical Implications for Diabetes and Cancer." Nature Reviews Endocrinology (2014) Print.
  10. Wang, Zheng, Song-tao Lai, Jian-dong Zhao, et al. "Metformin is associated with reduced risk of pancreatic cancer in patients with type 2 diabetes mellitus: A systematic review and meta-analysis." Science Direct (2014) Science Direct. Web. 21 May 2014.
  11. "Diabetes Mellitus: Types, Symptoms, Causes, Treatments." WebMD. WebMD, Web. 17 May 2014. <http://www.webmd.com/diabetes/types-of-diabetes-mellitus>.
  12. "Cellular Classification of Pancreatic Cancer." Pancreatic Cancer Treatment. National Cancer Institute, Web. 21 May 2014. <http://www.cancer.gov/cancertopics/pdq/treatment/pancreatic/HealthProfessional/page2>.
  13. Karolinska Institutet - Stockholm, Sweden. "New research redraws pancreas anatomy." Medical Xpress. Karolinska Institutet of Stockholm, 6 July 2011. Web. 31 May 2014. <http://medicalxpress.com/news/2011-07-redraws-pancreas-anatomy.html>
  14. Green, Shon. "Genetically Engineered Mouse Models." Cancer Research Intro Lecture. UCSF Helen Diller Family Comprehensive Research Center. UCSF, San Francisco. 30 June 2013. Lecture.
  15. Islas, Angel. "Clinical Trials and Human-Subject Studies." Cancer Biology. Santa Clara University. SCU Biology Dept, Santa Clara. 5 May 2014. Class lecture.
  16. Miller, D. M., S. D. Thomas, A. Islam, D. Muench, and K. Sedoris. "C-Myc and Cancer Metabolism." Clinical Cancer Research 18.20 (2012): 5546-553. Web
  17. Hezel, A. F. "Genetics and Biology of Pancreatic Ductal Adenocarcinoma."Genes & Development 20.10 (2006): 1218-249. Web.
  18. Eng, Charis, Maija Kiuru, Magali J. Fernandez, and Lauri A. Aaltonen. "A Role for Mitochondrial Enzymes in Inherited Neoplasia and beyond."Nature Reviews Cancer 3.3 (2003): 193-202. Web.
  19. "Pancreatic intraepithelial neoplasia (PanIN-3)." World Health Organization: Classification of Tumors. International Agency for Research on Cancer, Web. 3 June 2014. <http://www.pubcan.org/icdotopo.php?id=5875>.
  20. Costello, Eithne, and Anthony Evans. "The role of inflammatory cells in fostering pancreatic cancer cell growth and invasion." Frontiers in Physiology 3 (2012): NA. Print.
  21. "Pancreatic Cancer Treatment (PDQ®)." The University of Chicago Medicine. University of Chicago Medicine, n.d. Web. 4 June 2014. <http://www.uchospitals.edu/online-library/content=CDR62957>.