PROJECT
Motivation
World Health Organization Statement
The World Health Organization (WHO) has recently published a statement (October 2015) where it was announced that red meat and processed meat increased the probability of suffering from colorectal cancer. In fact, processed meat, which includes bacon, sausages and ham, was classified as carcinogenic for human. “For an individual, the risk of developing colorectal (bowel) cancer because of their consumption of processed meat remains small, but this risk increases with the amount of meat consumed,” said Dr Kurt Straif from the WHO. The organisation has come to this conclusion on the advice of its International Agency for Research on Cancer, which assesses the best available scientific evidence. Even though this statement may be overlooked with scepticism by many people, meat actually contains a component which has been correlated with colorectal cancer: the polyamines.
Colorectal Cancer
Colorectal cancer statistics
Colorectal cancer [7] is the third most common cancer in the world, with nearly 1.4 million new cases diagnosed in 2012.
Approximately 95 per cent of colorectal cancers are adenocarcinomas. Other types of cancer that can occur here include mucinous carinomas and adenosquamous carcinomas.
The Continuous Update Project Panel judged that there was convincing evidence that consuming red meat, processed meat and alcoholic drinks (men); body fatness, abdominal fatness and adult attained height increase the risk of colorectal cancer. There was also convincing evidence that physical activity and consuming foods high in dietary fibre protect against this cancer. Garlic, milk and calcium probably protect and consuming alcoholic drinks (women) probably increase the risk of this cancer.
Preventability estimates using the new findings from the Continuous Update Project show that about 47% of cases of colorectal cancer in the UK can be prevented by eating and drinking healthily, being physically active and maintaining a healthy weight.
Both sexes + Republic of Korea had the highest rate of colorectal cancer, followed by Slovakia and Hungary. + About 54 per cent of colorectal cancer cases occurred in more developed countries. + The highest incidence of colorectal cancer was in Oceania and Europe and the lowest incidence in Africa and Asia.
A snapshot of CRC
Colorectal cancer is the third most common non-skin cancer in both men and women. It is the second leading cause of cancer-related mortality in the United States. Over the past decade, colorectal cancer incidence and mortality rates have decreased in all racial/ethnic populations except American Indians/Alaska Natives. Men and women have similar incidence rates through age 39; at and above age 40, rates are higher in men.
Differences exist between racial/ethnic groups in both incidence and mortality. African Americans have higher mortality rates than all other racial/ethnic groups and higher incidence rates than all except American Indians/Alaska Natives. Incidence and mortality rates are lowest among Hispanics and Asians/Pacific Islanders. Overall colorectal cancer incidence and mortality rates have been declining over the past two decades; these declines have been attributed largely to increased use of screening tests.
Risk factors for colorectal cancer include increasing age, colorectal polyps, a family history of colorectal cancer, certain genetic mutations, excessive alcohol use, obesity, being physically inactive, cigarette smoking, and a history of inflammatory bowel disease. Effective colorectal cancer screening tests include the fecal occult blood test, sigmoidoscopy, and colonoscopy. Standard treatments for colorectal cancer include surgery, chemotherapy, radiation therapy, cryosurgery, radiofrequency ablation, and targeted therapy.
Assuming that incidence and survival rates follow recent trends, it is estimated that $13.8 billion will be spent on colorectal cancer care in the United States in 2014.
Selected Advances in Colorectal Cancer Research
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A preclinical study has shown that gut microflora can influence the treatment response of tumors formed by colon carcinoma cells. Published November 2013.
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In a study comparing the gut microflora of colorectal cancer patients and noncancer controls, colorectal cancer risk was associated with decreased bacterial diversity. Published December 2013.
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Overexpression of PLAC8, a protein whose levels are elevated in colon cancer, shifts the normal cells lining the colon into a state that encourages metastasis. Published April 2014.
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The integration of proteomic and genomic data from colorectal cancer studies identified five colon cancer subtypes (three of which were unique to this combined analysis) and demonstrated the value of using proteomics to understand the mutations that drive cancer. Published July 2014.
Preventive treatment
Even if the statement from the WHO gave us the main idea for our project, our true motivation for dedicating one whole year to that idea lied elsewhere. There are many studies performed by public healthcare entities stating that the morbidity of harsh diseases such as cancer, and the money required to treat them, is astronomically larger than what it takes to prevent them. This happens because too often, in order to diagnose these pathologies, they must be developed enough for our limited diagnostic tests to detect them, just when it is too late for treatment or when its effectiveness is already reduced. For our project, we wanted to design a handy preventive treatment - rather than a therapy - for colorectal cancer using synthetic biology.
Polyamines
Molecules
Polyamines are polycations and thus one of their main features is to interact with negatively charged molecules, such as DNA, RNA or proteins. Given their promiscuity in binding other molecules, they are involved in many functions, mostly linked with cell growth, survival and proliferation.
Polyamines are important players in plant growth, stress and disease resistance, but they are also involved in diseases, for example Alzheimer’s or infectious diseases. The main research area for the involvement of polyamines in diseases is cancer, as high levels of polyamines are observed in cancer cells. [1]
There exist 4 different polyamine molecules: Putrescine, Spermidine and Spermine (from low to high molecular weight), which are found in animal cells. Bacteria also include Cadaverine, which has a separate pathway from the three mentioned before, which are synthesized from L-methionine, L-ornithine and L-arginine (if bacteria) in contrast with this polyamine which is synthesized from L-Lysine.
Figure 1: Putrescine molecule
Figure 2: Spermidine molecule
Figure 3: Spermine molecule
Functions
Polyamines are involved in diverse functions involved in cell growth and differentiation, such as DNA synthesis and stability, regulation of transcription, ion channel regulation, and protein phosphorylation. [3]
Polyamines are essential for promoting cell growth. They are also implicated in apoptosis but as often with polyamines, with contradictory results. Polyamines increase Ca2+ accumulation in mitochondria, modulating the mitochondrial permeability transition, and triggering apoptosis. Polyamines can also directly promote cytochrome c release, a prelude to apoptosis.They can also regulate the other type of cell death, necrosis.
Finally, polyamine levels generally increase with inflammation. However, whether they are pro- or anti- inflammatory is still unclear. [1]
High polyamine levels alter histone acetylation and histone acetylases and deacetylases activities in proliferative cells.
Stark et al. [4] investigated whether CK2 was a target of polyamines by which they would modulate the MAPK pathway (Ras, Raf, MERK, Erk pathway). The authors suggested that CK2 could sense relative polyamine levels and translate the information to the MAPK pathway to trigger the appropriate cellular response. [1]
Figure 4: Polyamines functions
Sources
Three main sources for polyamines exist in organisms: Food intake, cellular synthesis, microbial synthesis in the gut. [1]
Food is an important source of polyamines. Polyamines in the intestinal lumen are absorbed quickly and distributed to all organs and tissues. Moreover, continuous intake of polyamine-rich food gradually increases blood polyamine levels. Therefore, the restricted intake of food polyamine and inhibition of polyamine synthesis by microbiota in the intestine with or without inhibitor-induced inhibition of polyamine synthesis is reported to have favorable effects on cancer therapy. [3]
In blood circulation, the majority of polyamines are contained in blood cells, especially in red and white blood cells, and therefore increases in blood polyamine concentration indicate concurrent increases in polyamine levels in blood cells.
Orally administered radiolabeled polyamines have been shown to be immediately distributed to almost all organs and tissues. [3]
Pathway
Polyamines are synthesized from the amino acids ornithine and methionine in animal cells, and lysine and arginine in the case of bacteria cells. The first step in the pathway is the production of ornithine from arginine by the mitochondrial enzyme arginase. Ornithine is then decarboxylated by ornithine decarboxylase (ODC) to produce putrescine. In bacteria, another putrescine synthesis pathway exist from L-Arginine, which is converted into agmatine and then hydrolyzed into putrescine by the agmatine ureohydrolase (speB). In parallel to putrescine production (in both bacteria and animal cells), L-methionine is converted into S-adenosyl-L-methionine (AdoMet), which is then decarboxylated by AdoMet decarboxylase (AdoMetDC) to produce decarboxylated AdoMet (DcAdoMet). DcAdoMet is then used as an aminopropyl group donor either to putrescine by spermidine synthase to produce spermidine, or to spermidine to produce spermine by spermine synthase. [1]
E. coli has two polyamine uptake systems belonging to the ABC transporters family. One system is a spermidine-preferential system and the second one a putrescine-specific system. Each system consists of 4 transporters: PotA to D for spermidine transport and PotF to I for putrescine transport. There are also two exporters (PotE and CadB), uptaking polyamines at neutral pH and excreting them at acidic pH. Finally, a spermidine excretion protein, MdtII was recently identified. [1]
Figure 5: Polyamines Pathway
Polyamines and cancer
Cancer and proliferative cells exhibit high levels of polyamines and this is thought to be a feature by which cancer cells maintain their proliferative capacity. However, the exact role of polyamines in cancer is still unclear.
A strategy to study the involvement of polyamines in cancer has been to use polyamine biosynthesis pathway inhibitors or polyamine analogues.
Although the ability of polyamines to induce cancer is still a matter of debate, it is likely that their high levels in cancer cells can help these cells maintain their proliferative capacity. This could happen by the interaction of polyamines with oncogenes. Polyamines have thus been the target for chemotherapeutic agents for a relatively long time. Such potential chemotherapeutic drugs developed are polyamine synthesis inhibitors, polyamine analogues or polyamine-conjugated compounds, as mentioned before. Many of these compounds show no effect during clinical trials, and are currently being studied as a chemopreventive agents rather than a chemotherapeutic ones. [1]
Polyamine concentrations are often increased in the blood and urine of cancer patients, and these increased levels have been shown to correlate with poor prognosis. The increased blood and urinary polyamine levels are attributable to increased polyamine synthesis by cancer cells, since these increases can be abolished by complete eradication of tumors by surgery or radio-chemotherapy. The capacity of cancer tissue to produce abundant polyamines likely contributes to cancer cells’ enhanced growth rates because polyamines are indispensable for cellular growth, which may at least partially explain why cancer patients with increased polyamine levels have a poorer prognosis. [3]
Polyamines synthesized by cancer tissues are transferred to the blood circulation and kidney, where they are excreted into the urine.
Polyamines are also produced in other parts of the body and can be transported to various organs and tissues such as the intestinal lumen where polyamines are absorbed quickly to increase portal vein polyamine concentrations. The majority of spermine and spermidine in the intestinal lumen is absorbed in their original forms because there is no apparent enzymatic activity present to catalyze their degradation. [3]
Patients with increased polyamine levels either in the blood or urine are reported to have more advanced disease and worse prognosis compared to those with low levels, regardless of the type of malignancy. [3]
Polyamines and colorectal cancer
Polyamines are involved in almost all steps of colonic tumorigenesis, their roles in hyperproliferation.
Regarding all described roles of polyamines in colonic tumorigenesis, it seems likely that polyamine deprivation, modulations of the polyamine metabolic pathway, as well as impairment of polyamine uptake into neoplastic cells in the colon, can be a logical though not specific way of chemoprevention and chemotherapy of colorectal cancer. [2]
Polyamines and their metabolizing enzymes could be, above all, reliable intermediate markers of neoplastic proliferation in the colon, and clinically relevant parameters of follow-up in colon cancer chemoprevention trials.
Specific inhibition of polyamine-synthesizing enzymes, and ODC in particular, has been long considered as a desired goal in chemoprevention and chemotherapy for hyperproliferative diseases, including cancer. [2]
The results of these trials are eagerly awaited, and will be most helpful in estimating a true value of the concept that depletion of colonic mucosal polyamines can prevent the development of colorectal cancer. [2]
Polyamines as tumor markers
N1,N12-diacetylspermine (DiAcSpm) and N1,N8-diacetylspermidine (DiAcSpd) are minor components of human urinary polyamine that have been associated to tumor markers in urine. [5]
Diacetylated polyamine derivatives, N1,N8-diacetylspermidine (DiAcSpd) and N1,N12- diacetylspermine (DiAcSpm), are derivatives of spermidine and spermine, respectively, in which both of the primary amino groups are acetylated.
Russell [6] reported in 1971 that the amount of polyamines excreted in urine was higher in patients with cancer than in healthy persons. This evoked a surge of studies on polyamine analysis that were intended to answer the question of whether the level of polyamines in urine could serve as indicators of malignant diseases.
Polyamines are excreted in human urine mainly as mono- acetylpolyamines. Acetylputrescine (AcPut) is the most abundant urinary polyamine, constituting 43% of total polyamines, followed by acetylcadaverine, N1-AcSpd and N8-AcSpd in decreasing order. Diacetylpolyamines represent minor polyamine species in human urine. The average amounts of DiAcSpd and DiAcSpm are only approximately 1.4 and 0.46%, respectively, of total polyamines, but their CV values are very small, taking their low content into account. This indicates that variation of these diacetylpolyamines in urine is very small from one individual to another, and may imply that they are secreted in a highly controlled manner.
DiAcSpm was sensitive for detecting cancer patients in that it increased markedly in about 80% of patients with cancer, reaching as high as 150 times the normal limits, while DiAcSpd was highly specific for cancer in that it was elevated only marginally in cases of benign diseases. This strongly suggested that diacetylpolyamines but not monoacetylpolyamines would serve as novel tumor markers, and these early observations prompted us to perform further studies of diacetylpolyamines. [5]
Project
As stated, in the Polybiome Project we believe that prevention is the best way to fight cancer. For this reason, we wanted to focus our research in two main working lines. The first one, is a probiotic proof-of-concept which would act as a preventive treatment for colorectal cancer. Our aim is to modify Escherichia coli so that they are able to absorb the excess of polyamines that we ingest through our diet as well as the ones that are produced by the intestinal microbiota. This way, we will decrease their concentration to healthy levels and reduce the probability of developing colorectal cancer.
The second idea was inspired by the fact that polyamines are found in high concentrations in urine in cancer patients with a very low variation between patients, according to some studies. Despite this good correlation, measuring the amount of polyamines in a liquid sample would take days - even weeks - to get to the lab and return. Moreover, polyamines are measured usually through High Performance Liquid Cromatography, which is an expensive test considering the bad properties of the polyamines. Following this idea, we decided to develop a tumor risk marker using yet once more synthetic biology. Engineered bacteria dried frozen in a strip would, that through a urine sample, alert the user whether he/she has or hasn’t a high probability of suffering from cancer. Bacteria would be provided with a sensing genetic system so that when high levels of polyamines are detected in their environment, they would change color and thus the strip too. Depending on the purple saturation of the device, the cancer risk level could be deduced.
Methods
The Probiotic
To accomplish our first objective, the creation of polyamine-degrading bacteria, first of all we needed to make Escherichia Coli auxotrophic for polyamines so that they needed to uptake the polyamines from the medium to survive. This way, they would diminish their levels in the colon and at the same time reduce the independent living capabilities of the cell, to avoid any possible invasion of our own microbiome. In order to get this, we initially decided to make speC (consitutive onrnithine decarboxylase), speF (biodegradative ornithine decarbozylase) and speB (agmatine ureohydrolase) knock-outs, so that they were deprived of the enzymes involved in the production of putrescine, which is the precursor of the polyamine synthesis. However, we saw that speF was not necessary since it was an enzyme that only worked at very acid media which was not the case of this study. So, we decided to create our PACO (Escherichia Coli Auxotrophic Bcteria which we made this way because it sounds cool). We used Crispr-Cas9 (pCas9 from Addgene) to generate these knock-outs, for which purpose we designed pairs of primers that annealed with the first bases of these genes. However, we realized that this were not easy knock-outs since they are compromising the survival of the cell because polyamines are completely necessary for living. For this reason, we decided to buy polyamine auxotrophic bacteria, in concrete the Escherichia Coli strain MA255 from Yale, which is the most used strain to analyze polyamines behaviour in bacteria. Since we really liked the name PACO, our new Yale bacteria was baptized with this same name few days from their arrival to our lab.
With the chromosomic knock-outs, the bacteria would be quite polyamine hungry, so we needed to implement a system to degrade the concentration excess of the own bacteria or otherwise they could end up killing themselves. For this, we reengineered E. Coli again by introducing some specific genes, codifying for enzymes capable of destroying all polyamines (from putrescine, which is the smallest with molecular weight, up to spermine, which bacterias do not usually degrade). Those enzymes in particular were the putrescine aminotransferase (PatA) from E. Coli K-12 and a polyamine oxidase (FMS1) from Yeast.
PatA catalytic activity:
.
FMS1 catalytic activity (reactions with more relevance according to our project):
Using E. Coli is a proof of concept since the initial idea was to create a probiotic that could consist of 8 different bacterias, all with different beneficials to our body and that in addition, could act as a colon cancer preventive treatment. These bacterias were selected from the VSL3 probiotic, which is already commercialized and that gives many good results with the users. Having so much troubles to clone just one bacteria, we are very happy we took the decision to reduce the number and complexity of the bacterias we wanted to modify. Still, this is a proof of concept of how our bacterias could be able to degrade polyamines, and thus reduce the probability of having colorectal cancer. The main objective is that our future bacteria (ones that are usually used in probiotics) are capable of modifying the human microbiome.
Polyamines can be found all around our body, however, they are higher in the colon. One challenge of all the probiotics is the delivery of the bacteria, since they must go through the entire digestive tract. In order to sort out this issue, ideally, they would be ingested in a lipid capsule, so that they do not die in the stomach due to the extreme acid medium to which they are exposed. Thus, we aim at creating a capsule that could be uptaken as a dietary complement, in order to help regulate the levels of polyamines taken up in the meals.
Cancer Risk Marker
The second objective of the Polybiome project was to be able to accomplish a fine device that could be able to detect cancer risk at home. This method is thought to be an easy friendly-user device that could be used at home as a simple urine test. Many cancers do not cause any symptons and when they are clinically detected, it is because they have achieved a considerable size and cause more harm. Our idea is to solve this problem by a simple method to detect if you are in risk or not of suffering from cancer.
The concept behind is to create a reactive strip with freeze dried modified bacteria that when in contact with urine, will produce a color that will warn the user of its risk. These modified bacteria, as the ones from the probiotic, will be polyamine auxotrphic, with the same Knock-ins but with the difference that will have a polyamine sensible promoter that will express a specific color depending on the amounts of polyamines.
The problem we found when iniating this project was that in urine there are 11 different types of polyamine-like molecules that would interfere in our analysis. We also saw that until now, the only methodes used to identify Diacetylspermine was by HPLC or Mass spectroscopy, tedious processes that need an expert hand to be performed. This is why we decided to create a way in which we could have accurate results in a much easier manner. For this reason, we will treat urine with our Reaction Mix, this mix will contain four different enzymes (mentioned above) that will degrade all the polyamine-like molecules except Diacetylspermine. This way, when our strip gets in contact with the pre-treated urine, the only molecule the auxotrophic bacteria will sense is the one we have left.
Enzymes from the reaction mix: - Polyamine Oxidase - Putrescine Oxidase - Acetylputrescine Deacetylase - Acetylspermidine Deacetylase (sent by Dr. Nicolas Porter with a pET-21b plasmid)
We first thought on purifying these enzymes, but since it was very expensive, we thought of a way to solve this problem: lyse bacteria via sonication. We would like to thank Dr. Sergi Aranda, who kindly gave us some BL21 Competent E. coli so that we could transform the genes’ enzymes into a recombinant bacteria. Once we could lyze the cells, the idea was to perform some analysis via HPLC or Mas Spectroscopy. Unfortunately, we did not have time to perform this last assay but we will some day continue with this idea, since we truly believe that a it can provide the world an accurate and easy tumor risk marker.
REFERENCES
[1] Nadège Minois, Didac Carmona-Gutierrez, Frank Madeo. Polyamines in aging and disease. AGING, August 2011 Vol. 3. No 8
[2] V. Milovic and L. Turchanowa. Polyamines and colon cancer. Biochemical Society Transactions (2003) Volume 31, part 2
[3] Kuniyasu Soda. The mechanisms by which polyamines accelerate tumor spread. Soda Journal of Experimental & Clinical Cancer Research 2011, 30:95
[4] Stark F, Pfannstiel J, Klaiber I, Raabe T. Protein kinase CK2 links polyamine metabolism to MAPK signaling in Drosophila. Cell Signal. 2011; DOI: 10.1016/j.cellsig.2011.01.013
[5] Masao Kawakita1, and Kyoko Hiramatsu. Diacetylated Derivatives of Spermine and Spermidine as Novel Promising Tumor Markers. The Japanese Biochemical Society. Vol. 139, No. 3, 2006
[6] Russell, D.H. (1971) Increased polyamine concentrations in the urine of human cancer patients. Nature New Biol. 233, 144–145
[7]Source: Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray, F. GLOBOCAN 2012 v1.1, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11 [Internet]. Lyon, France: International Agency for Research on Cancer; 2014.
