For UC (ulcerative colitis) PATIENTS, chronic inflammatory responses often accompany periodic tissue damage and recovery and finally lead to intestinal cancer.

However, during the cyclic damage and recovery process, what changes happen to the cells in tissues? How do the changes lead to the occurrence of cancer? Currently, there isn’t a clear answer. 

Two online articles published on Nature today analyze intestinal tissue cell mutations and tissue remodeling characteristics of UC patients at different stages from chronic inflammation to cancer and further disclose genes having a key influence on development of enteritis and intestinal cancer.

Two online articles published on Nature today analyze intestinal tissue cell mutations and tissue remodeling characteristics of UC patients at different stages from chronic inflammation to cancer and further disclose genes having a key influence on development of enteritis and intestinal cancer.

Research Background

Somatic evolution refers to the process that the somatic mutations increase constantly and the degree of mutation accumulates during occurrence and development of cancer 1. 

We know that most tissues inside human bodies maintain homeostasis through constant self-renewal. During self-renewal, somatic cells will have gene mutations inevitably 2. 

In recent years, the technical breakthrough in sequencing enables us to identify gene mutation types in normal tissues at the single cell level. 

It's worth noting that the mutant genes are quite similar to cancer driver genes.

Genetic research also proves that cells develop the ability of spontaneous self-renewal because of somatic evolution, which facilitates occurrence of cancer. The results emphasize the importance of somatic evolution during occurrence of cancer. 

According to the natural selection theory of Charles Darwin, somatic mutation may also be influenced by surroundings. However, the hypothesis now has been proved by experimental evidences.

Main Content

1. The Influence of the Inflammatory Environment on Directed Evolution of Intestinal Epithelial Cells

In the research paper with the name Somatic Inflammatory Gene Mutations in Human Ulcerative Colitis Epithelium, Professor Toshiro Sato’s team from Keio University School of Medicine analyzed the influence of the chronic inflammatory environment on intestinal epithelial cell mutation 3.

Firstly, the author collected intestinal tissues from HCs, UC patients without cancer and UC patients with cancer. 

The picture below describes the thinking of sample collection.

Specifically, all tissues from HCs are regarded as the normal control; the tissues from UC patients without cancer can be further divided into noninflammatory tissues (green) and inflammatory tissues (red); the tissues from UC patients with cancer can be divided into pulmonary inflammatory tissues (green), inflammatory tissues (black) and cancer tissues (purple).

Thinking of Sample Collection

In order to obtain the information of somatic mutation related to the inflammatory environment at the single cell level, the author built a single-cell cloning-based in vitro organoid culturing system by utilizing the collected samples and conducted exome sequencing. 

As shown in the picture below, in the normal control, with age, the SNVs will occur gradually and accumulate (0.019 time/ Mb /year). The result conforms to the previous research. 

Furthermore, the author analyzed the mutation frequency of different tissue parts during the progression of disease.

The result shows that, compared to the normal control (grey) and the healthy tissue of patients (blue), the mutation frequency of the diseased tissues increases significantly (red and black).

The result reflects that the dependence of the inflammatory environment and cell mutations exists. 

In order to avoid the influence on results caused by the individual differences of patients, the author selected samples from some patients for internal pair analysis. In other words, samples for pairwise comparison all come from one patient but are collected from different parts of the patient. 

The result shows that, different from the HC, the inflammatory tissue of patients has a higher mutation rate than the inflammatory tissue. The result further affirms the connection between the intestinal inflammatory environment and cell mutations. 

Through the further analysis of the sequencing result, the author discovered that the accumulative mutations of immune genes such as NFKBIZ, PIGR and TRAF3IP2 exist in inflammatory tissue-derived organoids, including SNVs and deletion mutations. 

For SNVs, the above two kinds of genes have a much higher missense mutation rate than the silent mutation rate.

In addition, since the above mutations happen in two copies of chromosomes, it means the genome stability declines. 

Later, the author studied the biological effect generated by the above mutations. 

The previous research shows that NFKBIZ is responsible for coding IκBζ, which is regulated upwards because of the influence of the NF-KB signal, and further promotes continuous activation of NF-KB to form positive feedback regulation.

PIGR codes a cell expressed on the cell surface and regulates transcytosis and IgA secretion.

TRAF3IP2 (also called ACT1) is the adapter protein of the IL-17 receptor and plays a very important role in activating IL-17-mediated NF-KB. Therefore, the above mutation may influence the IL-17-mediated NF-KB activation. 

In order to verify the hypothesis, the author compared the expressions of the above gene of the healthy tissue-derived organoids under the stimulation of IL-17A. 

The result shows that, under the healthy condition, the above genes will significantly increase the expression amount after the simulation of IL-17A. 

The above result shows that the IL-17 signal pathway-related somatic evolution of intestinal cells may be related to the selective pressure caused by the intestinal inflammatory environment. 

In order to verify the guess, the author utilized the CRISPR-CAS9 technology to artificially knock out the activity of specific genes in the healthy tissue-derived organoids.

Through the IL-17 stimulation, the author discovered that the organoid multiplies significantly under the IL-17 simulation after NF-KBIZ or IL17RA is knocked out. It indicates that the directed evolution of organoids is good for them to survive in an inflammatory environment. 

However, the PIGR doesn’t have corresponding effect. It indicates that the function of PIGR may involve the complex downstream mechanism including IgA activity, which is subject to further research. 

The above result shows that the IL-17 signal pathway-related somatic evolution of intestinal cells may be related to the selective pressure caused by the intestinal inflammatory environment. 

In order to verify the guess, the author utilized the CRISPR-CAS9 technology to artificially knock out the activity of specific genes in the healthy tissue-derived organoids.

Through the IL-17 stimulation, the author discovered that the organoid multiplies significantly under the IL-17 simulation after NF-KBIZ or IL17RA is knocked out. It indicates that the directed evolution of organoids is good for them to survive in an inflammatory environment.

However, the PIGR doesn’t have corresponding effect. It indicates that the function of PIGR may involve the complex downstream mechanism including IgA activity, which is subject to further research. 

2. The key gene mutation of intestinal epithelial cells regulates the transformation from enteritis into intestinal cancer.

In the research paper with the name Frequent Mutations that Converge on the NFKBIZ Pathway in Ulcerative Colitis, Seishi Ogawa’s team from Kyoto University also studied enteritis and revealed how the mutation of key genes influences the transformation from enteritis into intestinal cancer.

Firstly, the author analyzed the healthy person-derived and enteritis patient-derived crypt tissues by exome sequencing, and discovered that, compared to the normal control, enteritis patients obviously have a higher rate of gene mutation within the same time. 

Similarly, the author discovered that the mutation degrees of three genes (NFKBIZ, PIGR and ARID1A) in the crypt tissues of enteritis patients are significantly different from each by analyzing sequencing results. 

Then, the author tracked and analyzed the expansion course of crypt tissues.

The author conducted the crypt sequence similarity comparison of different parts by exome sequencing and described their inheritance and space relationships. 

The results indicate that the differentiation and expansion of crypt tissues of a normal person have completed at the age of 20 and then stay at a relatively stable status for a long time. 

In contract, the crypt tissues of enteritis patients experience the specific gene mutation-led repeated destruction and reconstruction process. Therefore, most mutant crypt tissues have the same previous-generation cells featuring a very short interval. 

In order to reveal the mechanism behind the directed evolution of intestinal epithelial cells, the author analyzed over 30,000 inspected from 399 non-cancer enteritis samples and verified 14 driver genes playing an important role in directed evolution.

As for the frequency of occurrence, they are NFKBIZ, ARID1A, PIGR, KRAS and ZC3H12A from high to low.

In addition, the very important point is that most of these mutant genes are related to NFKBIZ and IL-17 signals. Therefore, the author believed that the above gene mutation-driven directed evolution of intestinal epithelial cells may have an internal connection with IL-17 signals.

In order to verify the guess, the author adopted the organoid culture as well. 

By comparing the intestinal organoid of a wild mouse with that of a mouse with NFKBIZ deletion mutation, the author discovered that wild mice have a significantly higher expression level than mutants in terms of intestinal tissue NF-KBIZ and PIGR genes. In addition, under the IL-17A simulation, the mutant mouse-derived organoid grows faster than that of a wild mouse significantly.

The result indicates that NF-KBIZ, namely relevant gene mutation, can make intestinal epithelial cells grow better in the IL-17A environment. 

At last, the author studied the influence of NFKBIZ mutation on the occurrence of intestinal cancer.

The result indicates that NFKBIZ and relevant gene mutations are more in enteritis samples but the mutation rate is very low in intestinal cancer tissues. In addition, among mice with NFKBIZ deletion mutation, the occurrence rate and severity of cancer decline significantly. 

The above result indicates that the NFKBIZ mutation is good for inhibiting the transformation from enteritis into intestinal cancer.

Further Reading

Seishi Ogawa has devoted himself to research of cancer genetics and has a very important influence in the field. As early as in 2014, Professor Ogawa published a paper on Science to disclose the somatic mutation mechanism behind Cushing’s syndrome and provided a new thinking for treating endocrine disorders.

Summary and Discussion

The two studies reveal the complicated relation between enteritis and the directed mutation of intestinal epithelial cells.

Professor Toshiro Sato revealed the influence of the intestinal inflammatory environment on intestinal epithelial cells by in vitro culture of patient derived organoids and high throughput exome sequencing, and explained why such directed mutation is good for the survival of intestinal epithelial cells from the prospective of natural selection.

People like Professor Seishi Ogawa revealed the internal causes of the high-frequency mutation of intestinal epithelial cells of enteritis patients and the homogenization of mutant cells by analyzing human-derived samples. 

In addition, the author determined the key genes participating in the directed evolution of intestinal epithelial cells by exome sequencing and genetic models of mice and proved the function of the genes in inhibiting occurrence of cancer.

Cancerization of cells is always caused by constantly accumulative gene mutations. And inflammatory stimulation is one of the many reasons causing gene mutations. 

Through the two latest studies, we understand the damage of chronic inflammation to intestinal tissues and the internal mechanism for gene mutation of cells, and will study some specific genes precisely. 

However, now there isn’t a clear answer on how the above gene mutations inhibit the occurrence of cancer. It still needs further exploration. 

The culture of patient derived organoids is a new research method in the basic and translational medical field. Compared to the traditional animal models, this method has obvious advantages in terms of clinical samples for research. 

The above studies provide excellent cases of application of POD culture technologies. With technical development, we hope that we can further increase the use efficiency and application scope of PODs.