Cancer Stem Cells: their roles in tumour progression and their potential as therapeutic targets in colorectal cancer.
Adenomatous polyposis coli...................................APC
Colorectal cancer................................................. CRC
Cancer stem cells..................................................CSCs
Familial adenomatous polyposis............................FAP
Hereditary nonpolyposis colon cancer...................HNPCC
Hormone replacement therapy..............................HRT
Non steroidal anti inflammatory drugs...................NSAIDs
Colorectal cancer (CRC) is, now a day leading cause of death from cancer. It is the fourth and third common cause of death from cancer in females and males respectively. CRC is more common in Western Europe, North America and Australia but rare in Asian and African. This project is on to review surface markers of cancer stem cells (CSCs) in CRC.
The treatments that are available for CRCs are surgery, chemotherapy, radiotherapy and biological therapy. In most cases after therapies relapses do occur, the main cause has revealed by recent studies is the presence of CSCs in those tumours. CD44 is an important marker in colorectal CSC to initiate cancer, have hundred times grater tumorigenicity than CD44- cells whereas CD133 did not increase tumour growth in xenograft.
A lot of work has been done on CSCs and studies found a couple of surface markers including CD133, CD44 and CD166 but which marker in case of prognosis has greater impact in not known yet. In case of CSCs markers if exact markers are known for CRC then novel therapies can help to cure CRC permanently and also stop relapses. The therapies those are available to cure CRC to date not only cause tumour cell to die but also damage normal cell of the body. One best advantage of therapy against CSCs is it will only target CSCs so there is fewer chances for normal cells get affected.
Key words: Colorectal Cancer, Stem Cells, Cancer Stem Cells, Surface markers of CSCs, CD133, CD44, CD166, Tumour relapses and Novel therapies.
Colorectal cancer (CRC) is now a day leading cause of death from cancer, and incidence varies for CRC all over the world. The cases of CRC have been decreased since 1980's because of better screening and preventions. It is the third common cancer and also the second most common cause of death from cancer in UK and 90% of patients who are suffering from colorectal cancer are over the age of 50 (http://www.medicinenet.com).
CRC is more common in Western Europe, North America and Australia while rare in Asian and African. Also there has been an increase in rate in those countries that have started eating western diet (Ajani et al 2004). CRC is fourth and third common cause of death from cancer in females and males respectively (http://www.medicinenet.com).
CRC generally thought to arises from adenomatous polyposis coli (APC) that is mainly forms hyperproliferation in epithelial cells and crypt dysplasia (Ajani et al 2004). A national polyp study by Winawer (1999) reported that 2/3 of removed polyps were APC and were able to form malignant transformation, if the size of polyp is larger than 1cm it took five and a half years and in case of smaller polyps it took ten years. Winawer, (1999) also found that removal of polyps decrease the incidence of colorectal cancer. Mettlin et al, (1997) studies shows most colorectal cancer arises in proximal colon.
In case of colon tumour the inner wall of the large intestine being affected and tumour in colon formed, it can be benign or malignant. Benign tumours are called polyps and malignant tumours are called cancerous. Colon polyps develop when chromosome damage occurs within the cells in the inner site of the large intestine, affects the cell growth which then result into an extra massive tissue called polyps (http://www.medicinenet.com).
The risk factors are mainly aging, high fat and low fibre diet, more consumption of alcohol, smoking, obesity, hereditary colon cancer syndromes mainly FAP and HNPCC and also person with family history of CRC, as are explained in table 1. Also patient with crohn's disease or ulcerative colitis are 20 to 30 times more at risk than the person without those diseases (Ajani et al 2004). There are some other therapies that can reduce the risk of CRC as explained in appendix in table two.
In case of colorectal cancer patient might be suffering from rectal bleeding, fatigue because of anaemia, changes in bowl habits and also abdominal pain. On the other hand patient could be asymptomatic but results for hemoccult test shows positive on stool specimens (Ajani et al 2004).
Stages of colorectal cancer:
The treatments that are available for CRCs are surgery, chemotherapy, radiotherapy and biological therapy. Chemotherapy given after surgery is called adjuvant chemotherapy that causes microscopic metastatic cells to die. The results for adjuvant chemotherapy demonstrated by recent studies shows very positive results for therapy within 5 weeks of surgery. Whereas only chemotherapy used to treat tumour shows very poor results. On the other hand results for reoccurrence after radiotherapy either before or after surgery are decreased; chances for reoccurrence without radiotherapy are 50% while with radiotherapy chances are less than 7% (http://www.medicinenet.com). In most cases after therapies relapses do occur, the main cause has been suggested to be due to the presence of CSCs in those tumours, which are resistant to therapies.
The aim of the project is to critically evaluate the existence of cancer stem cells and their importance in the progression of colorectal cancers. In addition, i will search for specific markers, for CSCs and whether they could be targeted specifically using various approaches.
Risk factors that can cause colorectal cancer.
Age and Diet.
Age is very important factor in case of colorectal cancer and people with higher age are at higher risk. In case of diet food with higher saturated fat and red meat and also low fibre food increased the risk of colorectal cancer. On the other hand more intakes of fruits and vegetables showed a very positive effect in most studies (Ajani et al. 2004).
Smoking and Alcohol consumption.
Those people who start smoking at early age and also who smoke a higher number of cigarettes before age 30 are at higher risk of colorectal cancer (Ajani et al. 2004). Longnecker et al. (1990) studies demonstrated that people who used to drink at least 2 drinks a day are at 10% of higher risk of colorectal cancer than those who do not drink.
Family history of colorectal cancer.
Johns and Houlston. (2001) studies showed that colorectal cancer risk increases if a very close relative of individual developed colorectal cancer. The result of studies showed for 1st degree relative of colorectal cancer patients were 2.25 for 27 studies, the further increased in risk was shown if 2nd degree relative had colorectal cancer that was 4.25. Hereditary condition increased the risk that includes FAP, HNPCC and Peutz-jeghers syndrome.
Person with HNPCC (hereditary nonpolyposis colon cancer).
Whereas in case of HNPCC colon polyps also developed and cause cancer if left untreated, but usually in the right colon, in the 30s to 40s years of age. Person with HNPCC can also develop uterine cancer, stomach cancer, ovarian cancer and the biliary tract (website). Peltomaki. (2003) study revealed HNPCC is more common than other hereditary syndromes and most of the cases based on mutation in DNA mismatch repair gene that were identified as Hmsh2, Hmlh1, Hpms2 and Hmsh6. According to Vasen et al. (2002) two most common form of cancer in case of HNPCC family member are colorectal and endometrial cancer.
Person with FAP (familial adenomatous polyposis).
It is a hereditary colon cancer syndrome. In case of FAP mutation in APC gene on chromosome 5q occurs and cause polyps to develop. If those polyps are left untreated they will then convert into cancers. A person can usually develop cancer in 40s, but there are greater risk of developing cancer of thyroid gland, stomach and ampulla. Burn et al. (1991) studies shows FAP rate is 2 cases per 10000, whereas for peutz-jeghers syndrome is four time rarer.
Table 3: TNM classification of colon and rectum carcinoma.
Histologic features of the Neoplasm
At that stage carcinoma in situ or intramucosal carcinoma occurred.
At this stage tumour invade submucosa.
Tumour extends into the muscularis propria but not penetrate through it.
Tumour penetrates through the muscularis propria into subserosa.
Tumour penetrates through serosa and may directly invade other organs.
At this stage regional lymph node cannot be assessed.
It shows no metastasis of regional lymph nodes.
At this stage metastasis occur in one to three lymph nodes.
At this stage metastasis occur in four or more lymph nodes.
At this stage distant metastasis cannot be assessed.
Tumour shows no distant metastasis.
Tumour shows distant metastasis.
From: Kumar, et al. (2004).
Stem cells are special type of cells that has the ability to self-renewal and differentiate into specialized cell types. They are rare in most tissues and found mainly in nerves, muscles and the cells lining of digestive system. In most parts of the body they are not very active apart from the gastrointestinal tract where they proliferate and differentiate constantly to replace dead cells (Li and Neaves 2006).
In SCs development niche play a very important role, controls asymmetrical division of SCs and niche loss causes SCs loss which shows SCs depend on niche. It controls proliferation and differentiation by primarily inhibitory signals to prevent tumorigenesis. On the other hand also make transient signals available to SCs division for tissue renewal. In case of genetic mutation SCs will be independent or resistant to growth signal and anti-growth signal respectively, causes SCs uncontrolled proliferation and then may cause tumorigenesis as shown in figure 1 (Li and Neaves 2006).
Cancer stem cells:
CSCs are distinctive cells in tumours that have the ability to initiate tumour growth and prolonged tumour self-renewal. They thought to arise from normal stem cells or progenitor cells, because they have the ability of self-renewal and proliferation. The transformation of SCs into CSCs is because of variations in genes. CSCs are resistant to drugs, also express typical surface markers of stem cells (Soltysova et al. 2005). In case of CRC recent studies found some surface markers for CSCs that are CD44, CD133, CD166, and CD24. There is still more work going related to markers mainly which marker exactly causes CRC and how to use those markers to target CSCs directly to treat CRC permanently.
Colorectal cancer stem cells
Normal adult SCs are important for the production of colonic epithelium, which exist at the base of the crypt and mutation in stem cells causes neoplastic changes because of that SCs dissociate from epithelium and move towards mesenchyme and as a result invasive cancer forms (Salama and Platell 2009).
Salama and Platell (2009) also mentioned the presence of <1% cells those actually initiate tumours which are resistant to chemotherapies and radiotherapies. So the resistance of CSCs to therapies causes tumour relapses. In colorectal cancer the identification of normal stem cells and cancer stem cells is quite difficult because of presence in lower number. The most frequently used methods to identify surface markers of CSCs are immunohistochemistry, identification of DNA methylation pattern and RNA binding protein method.
The Wnt pathway plays an important role in crypt cells growth, transcription factors and adhesion molecules controls SCs proliferation and migration. In case of CRC the normal function of Wnt pathway get disturbed and β-catenin accumulation take place, because of that cell receive continuous signal for proliferation and eventually APC formed in intestine (Li and Neaves 2006).
Markers for cancer stem cells in colorectal cancer.
It is a transmembrane glycoprotein and play important role in cellular process mainly growth, differentiation, survival and motility. It acts as an adhesion molecule and because of cellular microenvironment it migrate cancer cells and matrix adhesion.
Cancer stem cells markers:
Recent studies have found some markers for isolating colorectal CSCs including CD133, CD44, CD24 and CD166 as are explained in table 4.
Ricci-Vitiani et al. (2007) studies reported CD133 as the only colorectal CSCs to initiate cancer. Their results showed 105 CD133- cells were not able to form tumour while 106 CD133+ cells formed tumour within 4 to 5 weeks after transplant, so they concluded CD133+ cells were able to form original tumour whereas CD133- cells could not and suggested CD133+ cells should be targeted for further therapies. Also demonstrated that CD133+ cells were in higher cellular density area whereas for normal tissues CD133 expression was rare, only six patient's samples were used and results were same for all the samples.
Whereas Sergey et al. (2008) studies showed that expression of CD133 expression not only restricted to intestinal stem cells or cancer initiating cells but a universal marker of organ specific stem cells and tumour initiating stem cells. The study used knockin lacZ model and immunostaining and found, both CD133+ and CD133- metastatic cells were able to form colonospheres in vitro and tumorigenesis in vivo. Also found CD133+ cells can give rise to metastatic CD133- cells, form more aggressive tumours and expressed CD44 (CD44+CD24-) and CD44lowCD24+ markers respectively.
Sergey et al. (2008) study used tumour from 19 patients with colon cancer metastasis to the liver, 11 of them had CD133+ while in 8 tumours CD133+ was not detected. Also CD133- population showed larger tumour with earlier and faster growth rate, which shows involvement of other factors. In primary colon cancer EPCAM+CD133- were not detected but CD133+ was detected and for metastatic tumour 40% results were CD133- which suggested CD133+ not responsible for tumour growth.
Horest, et al. (2009) studies revealed that colorectal cancer cells with higher expression of CD133 were more likely to cause liver metastasis. In their study results CD133 not detected in normal mucosa but in tumour over expression of CD133 detected. Because of higher expression of CD133 they proposed that could be clinically a significant marker to use for CRC patient. On the other hand study did mention this marker did not contribute towards disease fierceness because in colon cancer cell lines, knockdown of CD133 results did not affect colonel formation.
Li, et al (2009) studies proposed that cancer cells with higher expression of CD133+ cells shows poor prognosis in patients with advanced CRC and causes overpopulation of CSCs. Their finding suggested that cancer cells with less than 5% and greater than 55% CD133+ shows higher and lower five year survival rate respectively. Also no correlation was suggested between CD133+ expression and clinicopathologic factors.
Du et al. (2008) studies revealed that CD44 is an important marker in colorectal CSC to initiate cancer. They used sixty sample tissues and found clustered growth of CD44+ cells but not with CD133+ cells in the same tumour tissue of colorectal cancer and also demonstrated that CD44+ cells have greater capacities in both in vivo and in vitro tumours.
Also Du et al. (2008) study found that knockdown of only CD44, not CD133 inhibit tumour growth. It also down regulate stemness genes like Oct3/4, Bmi and β-catenin but less affect Nanog gene which propose CD44 control them differentially and study also suggested further study is required to see CD44 affects. Antibodies only against CD44 not against CD133 were found that stop colonel formation in cancer cells. Different isoforms of CD44 were found by western blotting and RT-PCR and also concluded CD44+ cancer cells have hundred times grater tumorigenicity than CD44- cells whereas CD133 did not increase tumour growth in xenograft.
Huh, et al, (2009) studies also investigate the expression of CD44 in CRC and their results for CD44 expression were 100% and 37.9% in primary tumour and normal mucosa. Their results suggested CD44 expression was linked to invasive strength and involvement of lymph node, though further study was suggested for biological role of CD44 and its importance as new therapeutic target in CRC. Also study found no association between CD44 expression and clinicopathological characteristics.
Weichert, et al. (2005) studies for the first time describe CD24 expression in CRC and revealed CD24 unregulated in CRC, their finding suggested cytoplasmic CD24 over expression relate to short patient survival. CD24 Immunostaining shows membranous and cytoplasmic staining in colon tissue. Study results found CD24 negative and CD24 over expression for normal colon and for colorectal cancer respectively. In case of tumour CD24 membranous staining were 68.7% positive and 17% strong positive whereas cytoplasmic staining were 84.4% positive and 10.2% strong positive. Study concluded that membranous and cytoplasmin CD24 staining were associated with systemic metastasis whereas not correlate with clinicopathologic factors for membranous, but do correlate with clinicopathologic factors for cytoplasmic. However study suggested further investigation required for biological role of CD24.
Whereas Ahmed, et al. (2009) studies proposed that, CD24 not a good prognostic marker in case of CRC. Their studies for first time revealed lower expression of CD24 was associated with poor outcome in CRC. Results showed central and strong expression of CD24 in normal mucosa and 90% of adenomas respectively. Study demonstrated that CD24 expression upregulated in early stage of CRC and in cancerous cells can be expressed anywhere in cell, as result shows it was expressed in nucleus apart from cytoplasmic and membranous expression.
Colorectal cancer is one of the leading causes of death from cancer and the therapies available nowadays are not that useful to cure it in most of the cases as in other diseases. Work done by different groups and end results demonstrate that the reason therapies are not able to cure CRC from the root is the presence of very small number of cells that are resistance to therapies and so called CSCs. Because of their resistance they are able to grow tumour again, that cells have similar properties to SCs such as self-renewal and proliferation.
CD133 not only restricted to stem cells, also expressed on differentiated epithelium in colon and CD133- population showed larger tumour with earlier and faster growth rate (Sergey, et al. 2008). Whereas for CD44, is an important marker in colorectal CSC to initiate cancer. CD44+ cancer cells have hundred times grater tumorigenicity than CD44- cells whereas CD133 did not increase tumour growth in xenograft (Du, et al. 2008).
Even though a lot of work has been done on CSCs and studies found couple of surface markers including CD133, CD44 and CD166 but which marker in case of prognosis has greater impact in not known yet. So to treat CRC by using CSCs markers more work is needed to be done to know exactly which marker has more prognostic effect towards patient treatment.
In case of CSCs markers if exact markers are known for CRC then future therapies can help to cure CRC permanently and also stop relapses. The therapies that are available to cure CRC now days not only damage tumour cells but also damage normal cells of the body. One best advantage of therapy against CSCs is it will only target CSCs so very fewer chances for normal cells get affected.
Table 2: Therapies used to decrease the risk of colorectal cancer.
According to Thun et al. (2002) studies the results for 5 cohort and 6 control case studies shows protection against CRC by using no steroidal anti inflammatory drugs (NSAIDs). Also the study of Steinbach et al. (2000) demonstrated that in CRC the reoccurrence was successfully prevented in FAP patient by using NSAIDs.
The use of HRT shows positive results against CRC protection. The control trial was done by women's health initiation and the report shows 38% decrease in CRC after HRT used for five years (Ajani et al. 2004).
The incidence of CRC can be decreased by doing screening. For example by doing fecal occult test following with endoscopy at 5 to 10 years period. This will help people by decreasing the risk to develop CRC and then dying of it afterwards (Ajani et al. 2004).
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