Tuesday, May 29, 2012

Transcription Factor KLF11, Diabetes, and Pancreatic Cancer

Image Above: Six insulin molecules in a hexamer (6).
KLF11 also known as, TIEG2, is a pancreas transcription factor that has started to gain attention due to its discovered role of being a negative regulator of exocrine cellular growth both found in vitro and in vivo. This is a very interesting case because for the first time KLF11 has been characterized as a glucose regulated transcription factor specific to the insulin gene. In addition, type II diabetes has been linked too two rare variants of the KLF11 gene and has been found to impair transcriptional activity. KLF11 works by altering the TGF-β signaling pathway  binding pathway that can impair the insulin promoter and cause lower levels of insulin release in beta cells.  Two specific diseases that can result in a mutation to the exocrine pathway (which KLF11 is a part of) are pancreatic cancer and type 2 diabetes.   


In the case of pancreatic cancer, KLF11 has been cited to regulate exocrine cell growth and behave as a tumor suppressor (however, this has yet to been fully documented and proven) (2).  In diabetes, KLF11 has been named a candidate gene that leads to a predisposition to diabetes or may even cause diabetes.  One study presented today by (Bernadette and his team) will prove KLF11 is a glucose- induced regulator of insulin and that variations of the KLF11 gene have shown a significant association with diabetes. The other paper (Fernandez- Zapico and team) has found that KLF11 is down regulated in human cancers, one of which is pancreatic cancer.


Using a combination of these two studies, I am going to attempt to assimilate and prove that  KLF11 causing pancreatic cancer can lead to type 2 diabetes.  And even further does a rare form of the KLF11 gene, predispose an individual to pancreatic cancer, and not just type 2 diabetes.       

Study #1 (Neve and team)  (Quick link to study here: KLF11 and Diabetes)

- Methods: 
First in this experiment the promoter sequence was analyzed.  To analyze the promoter sequence, fusion proteins of KLF11 were incubated in a buffer and allowed to incubate.  From here the reactions were analyzed using gel electrophoresis.  For the portion of the chromatin immunoprecipitation (ChIP), transfections of the KLF11 plasmids were fragmented.  These samples were then immunoprecipitated using an antibody and a combination of agarose beads and a ChIP kit.  Next, crosslinks were removed and immunoprecipitated DNA was purified by a phenol/ chloroform extraction and an ethanol precipitation.  The last step of the chromatin immunoprecipitation was to amplify a 200 base pair region of the insulin promoter and to use primers to visualize the DNA on agarose gel.  After that was completed, the final portion of the experiment involved the genetic analysis of KLF11 in the study population.  There were two studies done that consisted of 313 cases of type 2 diabetes and 313 control subjects and 1,383 type 2 diabetes patients and 1,463 control subjects, respectively. (4)  

KLF11 is involved in glucose signaling within pancreatic beta cells.  KLF11 is responsible for the binding and activation of the human insulin promoter inside beta cells, when exposed to high- circulating levels of glucose.  In addition, KLF11 is induced by glucose and leads to the up regulation of insulin expression.  (Figure 1)

Variations of the KLF11 gene have been proven to be associated with type 2 diabetes. Three distinct families genes were sequenced and through this process researchers discovered two rare KLF11 variants.  Using a peptide identification system tool, the analysis vaticinated both variants were involved in the modification of the secondary protein structure of KLF11.
To associate the link between type 2 diabetes and KLF11 variants, researchers analyzed the transcriptional regulatory activity in the two rare variants.  From here it was found that KLF11's impairment on the function of diabetes, could have come from KLF11's altered transcriptional activity.  (Figure 3)  

Study #2 (Fernandez- Zapico and team) (Quick link to study here: KLF11 and Growth Regulation)

For this experiment, transgenic mice were used.  Plasmid/ vector genetic engineering (an interest note is the restriction enzymes used were HindIII/ BamHI [sound familiar to Biology 25] were done and injected into the mice and a founder population was conceived.  Once the offspring were born, the founder population was killed and their organs were harvested for evidence and observations.  In addition to using transgenic mice, many different assays were performed using transfected mice cells from the pancreas.  Chromatin immunoprecipitation was done using transfected plasmids, fragmented, and then visualized using PCR on agarose gel.  

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Image Above: An over expression of KLF11 leads to an
increase in apoptosis both in vivo and in vitro (2).
KLF11 has been shown to be down regulated in epithelial cell tumors.  In addition, it can lead to the inhibition of neoplastic transformation and cell growth.  To combat these effects KLF11, likely, decreases cell proliferation and increases apoptosis.  These cellular mechanisms have been viewed both in cultured cells and in vivo.  In addition, KLF11 being expressed in the pancreas leads to a vast reduction in the weight and size of the organ.  Lastly, using chromatin immunoprecipitation assays researchers have seen KLF11 directly targets oxidative stress genes superoxide dismutase2 and catalase1.  Expression profiling experiments and reporter assays have shown that KLF11 decreases the levels of these genes.  Combining all these results together, researchers have found that KLF11 works as a surpressor of cell growth and neoplastic transformation and effects growth regulation by down regulating genes that are involved in oxidative stress. (2)  


1) Evidence to target the hypothesis.  
In the second paper Fernandez- Zapico and his team of researchers proved that KLF11 is involved in a variety of cancers, one of which is pancreatic cancer (2).  They found that KLF11 directly targets oxidative stress genes (2). Two that were particularly affected were superoxide dismutase2 and catalase1 (2).  The down regulation of these genes that KLF11 causes leads me to believe this could induce pancreatic cancer and diabetes.  Since altering oxidative stress genes could affect glucose and glycogen synthesis I believe this could lead to diabetes.  Diabetes (type 2 diabetes) is caused by a problem in the way the body makes insulin or the way the body uses insulin.  An important note with type 2 diabetes is people can produce insulin, where as, in type 1 diabetes people can not make insulin (5).  The body uses insulin to transport glucose into cells.  Because people with type 2 diabetes' insulin does not function properly, glucose can not get into the cells and leads to a build up of glucose in the blood, which is termed hyperglycemia (1).  As proven by Neve and his team, KLF11 has been shown to be directly involved in the binding and activating of the insulin promoter (4).  The combination of a mutated KLF11 gene causing an alteration in insulin and affecting oxidative stress genes, likely, could lead to type 2 diabetes.  

A backwards link of pancreatic cancer causing diabetes could be possible with KLF11. Thinking back to Fernandez- Zapico and his team's results, if KLF11 causes pancreatic cancer and KLF11 affects oxidative stress genes, the affected oxidative stress genes, may not, be digesting the left over glucose or in some way leading to an altered glucose metabolism. This leftover glucose or alteration in glucose metabolism could lead to higher levels of glucose in the blood stream or all over the body. A high level of glucose in the blood is hyperglycemia. If hyperglycemia is caused by KLF11 this could directly result in a person developing type 2 diabetes.  Using this information it would suggests that pancreatic cancer that is caused by or allowed to proliferate by an altered KLF11 gene could lead to diabetes.  

Thus, in the future patients that have been diagnosed with type 2 diabetes, upon diagnosis, the doctor would be able to test for an altered form of KLF11 and if an altered form of KLF11 is seen the doctor can then resort to an invasive test to make sure the individual doesn't have pancreatic cancer (because in the last paragraph it was discussed how pancreatic cancer could cause diabetes as a result of KLF11).  In the near future, an altered KLF11 gene could become a known target for testing whether someone has pancreatic cancer and/or diabetes.  

The other aspect that I have not yet discussed is if a rare form of KLF11 can cause a predisposition to pancreatic cancer.  After reading both of these studies, I would say it could.  Because of the already present mutation and the affect it has on the insulin pathway, I would think it could lead to a greater chance of getting pancreatic cancer.  Mainly, due to the fact that KLF11 has already caused a vital mutation it would seem it could only be a matter of time or luck before another mutation occurs that could cause enough of a mutation to lead to some form of cancer, particularly, pancreatic cancer.  

2) A possible follow- up.
Since the researchers in Neve and his team proved that rare mutations of KLF11 can cause diabetes.  A very interesting follow- up they could perform is taking patients from their previous study (discussed in this post) and following up on these patients until death and see if at any point they are suspected to have any form of pancreatic cancer.  In addition, it would be interesting if they could harvest the pancreas' of the deceased test subjects and see if there are any signs of potentially cancerous tumors.  It would also be beneficial to see if they develop any other types of cancer because KLF11 has been proven to be down regulated in certain cancers.  This could confirm  Fernandez- Zapico's experiment's results. Ultimately, it would be great if they were able to follow- up with these patients and find out if they had pancreatic cancer and use that information to a triumphant breakthrough in the link of KLF11, diabetes, and pancreatic cancer.  

3) Statistics for both papers and sample size.
In the first study performed, by Neve and team, I would say the sample size was definitely large enough to give an accurate representation.  There were over 2,000 test subjects (4).  However, when the researchers actually looked at specific family lines of KLF11, there were only 3 families chosen (4).  It would have been nice to have seen more families just to see more variants of the gene, but 3 families is definitely a start. On a statistical note, the researchers in the Neve experiment were safe.  They had statistical tests, where necessary, including X^2 analysis and ANOVA tests for the association of type 2 diabetes and KLF11 (4).
For the Fernandez- Zapico paper, they did not discuss any statistical tests that were performed (2) (Note: I do not discredit their findings because of other tests they ran and the high number of test subjects (n) involved in their experiments).  In addition, Fernandez- Zapico's team compared their results that said KLF11 was down regulated in certain cancers to the multitumor array data base provided by Stanford (2) (http://source.stanford.edu/cgi-bin/sourceSearch).  This also leads to their credit, as the data has already been tested prior to their experiment.  

4) A follow- up that could be done for this blog post. 
A very interesting potential next blog post could be on if a mutation in KLF11 is caused by obesity or a mutated KLF11 causes obesity.  Obesity has already been a proven risk factor for both diabetes (1) and pancreatic cancer (3).    


The first study in this blog post links KLF11 to diabetes, while the second paper links KLF11 to pancreatic cancer.  Using their observations and data, I believe it is reasonable to conclude that if an individual has diabetes, in the future, a test for a mutation in KLF11 should be done, and a test for pancreatic cancer should not be far behind due to the connection of KLF11, pancreatic cancer, and diabetes.  

Thanks for reading
-- Quincy


1) A.D.A.M. Medical Encyclopedia. "Type 2 Diabetes." PubMed Health. 28 June 2011. Web. 29 May 2012. <http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001356/>.

2) Fernandez- Zapico, Martin E. "An MSin3A Interaction Domain Links the Transcriptional Activity of KLF11 with Its Role in Growth Regulation." EMBO Journal 22.18 (2003): 4748-758. NCBI. US National Library of Medicine. Web. 29 May 2012. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC212736/?tool=pubmed>.

3) Mayo Clinic Staff. "Pancreatic Cancer: Risk Factors." Mayo Clinic. 10 Apr. 2012. Web. 29 May 2012. <http://www.mayoclinic.com/health/pancreatic-cancer/ds00357/dsection=risk-factors>.

4) Neve, Bernadette. "Role of Transcription Factor KLF11 and Its Diabetes-associated Gene Variants in Pancreatic Beta Cell Function." Proceedings of the National Academy of Sciences of the United States of America 102.12 (2005): 4807-812. NCBI. US National Library of Medicine. Web. 29 May 2012. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC554843/>.

5) WebMD Staff. "Type 2 Diabetes Overview." WebMD. Web. 29 May 2012. <http://diabetes.webmd.com/guide/type-2-diabetes>.

6) Yonemoto, Isaac. Insulin Hexamer. Digital image. Wikipedia. Web. 29 May 2012.<http://en.wikipedia.org/wiki/File:InsulinHexamer.jpg>.