Chapter 6 Recent Concepts in the Pathogenesis and Management of Colorectal Cancer

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1 Chapter 6 Recent Concepts in the Pathogenesis and Management of Colorectal Cancer Amal H Hamza 1, Hadeer A Aglan 2 and Hanaa H Ahmed 2 * 1 Biochemistry and Nutrition Department, Faculty of Women, Ain Shams University, Egypt 2 Hormones Department, Medical Research Division, National Research Centre, Egypt * Corresponding Author: Hanaa H Ahmed, Hormones Department, National Research Centre, Giza, Egypt, Tel: 202( ); Fax: 202( ); hanaaomr@ yahoo.com First Published January 05, 2017 Copyright: 2017 Amal H Hamza, Hadeer A Aglan and Hanaa H Ahmed. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source. Abstract Colorectal cancer (CRC) is a major cause of morbidity and mortality throughout the world. It accounts for over 9% of all cancer incidence. It is the third most common cancer worldwide and the fourth most common cause of death. CRC develops slowly, over years, even decades. Most CRC starts with the polyps occurring on the epithelial lining of the colon or rectum. These polys may be benign (e.g., hyperplastic polyp), pre-malignant (e.g., tubular adenoma) or malignant (e.g., colorectal adenocarcinoma). It is estimated that about 20% cases of CRC have a family history of CRC. Some genetic syndromes are associated with greater risks of CRC, for example, hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome) accounts for about 3% of people with CRC, and Gardner syndrome and familial adenomatous polyposis (FAP) are nearly always associated with CRC and are the causes of about 1% of all CRC cases. However, the majority of CRC cases are linked to environmental factors rather than heritable genetic changes. Occurrence of CRC may be dependent on various factors, namely diet, life style and other environmental parameters, including environmental and food-borne mutagens. In addition, CRC risk factors also include intestinal microbiota and chronic intestinal inflammation that precede tumor development. It is estimated that chronic inflammation, likely to be caused by a dysregulated intestinal microbiota, contributes to approximately 20% of all CRC cases. Chemotherapeutic regimens based on 5-fluorouracil (5-FU) are commonly used for treatment of patients with advanced colon can- 2 3

2 cer. Although the combinations of 5-FU with irinotecan and oxaliplatin have improved the response rate, chemotherapeutic regimens are usually not very effective against disseminated colon cancer due to multiple drug resistance. Thus, the development of new therapeutic agents can overcome this resistance has become one of the most important goals in the management of this malignancy. Several useful nanotechnological applications have been identified in cancer biology, including early detection of tumors and the development of treatment approaches that cannot be achieved using the existing conventional technologies. In fact, in certain cancers, nanometer-sized particles of diverse shapes and compositions have come out as promising and important new tools for CRC diagnosis, staging, and therapeutics. Early detection of CRC is the key for prevention, and it has the ability to impact the long-term survival of patients with CRC. Morphology of Colon Colon is the first and longest part of the large intestine, a muscular tube about 5 feet long. The first section of the colon is the ascending colon; it begins with a pouch called the cecum, where undigested food is received from the small intestine, and extends upward on the right side of the abdomen. The second section is called the transverse colon because it crosses the body from the right to the left side. The third section is called the descending colon because it descends on the left side. The fourth section is called the sigmoid colon because of its S shape; the sigmoid colon joins the rectum, which connects to the anus. The ascending and transverse sections are collectively referred to as the proximal colon, while the descending and sigmoid colon are referred to as the distal colon [1]. Colorectal Cancer Overview Colorectal cancer is one of the most common malignancies in many regions of the world (Figure 1). It is the third most common cancer in both men and women, also it is the second most common cause of cancer death in the United States [2]. CRC arises from the accumulation of mutations in a single epithelial cell of the colon and rectum [3]. It has different characteristics based on their location within the colon or rectum; tumors in the proximal, or right, colon are more common among women and older patients whereas distal, or left-sided, tumors are more common among men and younger patients [4]. The incidence of CRC is increasing due to smoking, lack of physical activities, overweight and obesity, red and processed meat consumption, and excessive alcohol consumption [5]. Colorectal cancer usually develops slowly, over a period of 10 to 20 years. Most begin as a noncancerous growth called a polyp that develops on the inner lining of the colon or rectum [6]. The most common kind of polyp is called an adenomatous polyp or adenoma. Adenomas arise from glandular cells, which produce mucus to lubricate the colorectum. An estimated one-third to one-half 4 5

3 of all individuals will eventually develop one or more adenomas [7]. Although all adenomas have the capacity to become cancerous, fewer than 10% are estimated to progress to invasive cancer [8]. The likelihood that an adenoma will evolve into cancer increases as it becomes larger. Cancer that develops in glandular cells is called adenocarcinoma. Most colorectal cancers (approximately 96%) are adenocarcinomas [9]. The complex sequence of events occurring during initiation, development and propagation of tumours is likely the result of lifelong accumulation of a series of mutations [10]. Figure 1: Incidences of colon cancer worldwide. Prevalence of Colorectal Cancer In the United States, CRC is the third most common cancer diagnosis among men and women. There are similar incidence rates for cancer of the colon in both sexes, and a slight male predominance for rectal cancer [11]. In 2005, 108,100 and 40,800 individuals were diagnosed with cancer of the colon and rectum, respectively. For 2008, it was estimated that 148,900 new cases would be diagnosed and 49,900 people would die of the disease [12]. In 2008, new cases were diagnosed, accounting for 9% of cancer deaths in women and 8% in men. The 5-year survival is 90% for localized disease, 68% if lymph nodes are involved, but only 10% if there is evidence of metastatic spread at the time of diagnosis [2]. Worldwide, CRC represents 9.4% of all incident cancer in men and 10.1% in women. CRC, however, is not uniformly common throughout the world. There is a large geographic difference in the global distribution of CRC (Figure 2). It is mainly a disease of developed countries with a Western culture [13]. In fact, the developed world accounts for over 63% of all cases. The incidence rate varies up to 10-fold between countries with the highest rates and those with the lowest rates. It ranges from more than 40 per 100,000 people in the United States, Australia, New Zealand, and Western Europe to less than 5 per 100,000 in Africa and some parts of Asia. However, these incidence 6 7

4 rates may be susceptible to ascertainment bias; there may be a high degree of underreporting in developing countries [14]. Colorectal cancer survival is highly dependent upon stage of disease at diagnosis, and typically ranges from a 90% 5-year survival rate for cancers detected at the localized stage; 70% for regional; to 10% for people diagnosed for distant metastatic cancer (Figure 3) [15]. In general, the earlier the stage at diagnosis, the higher the chance of survival. Since the 1960s, survival for colorectal cancer at all stages have increased substantially. The relative improvement in 5-year survival over this period and survival has been better in countries with high life expectancy and good access to modern specialized health care. However, enormous disparities in CRC survival exist globally and even within regions [16]. This variation is not easily explained, but most of the marked global and regional disparity in survival is likely due to differences in access to diagnostic and treatment services [14]. Figure 2: Prevalence of colorectal cancer worldwide. Survival and Prognosis of Colorectal Cancer Figure 3: Estimated cases and death per year. 8 9

5 Colorectal Cancer Risk Factors The combination of genetic predisposition and environmental factors usually results in colon cancer. Hereditary risk contributes to approximately 20% of cases [17]. Familial adenomatous polyposis is main inherited predisposition syndromes, hereditary nonpolyposis colorectal cancers, and other types of tumor with a familial history [18]. Environmental factors account for 80% of cases. Such factors include a diet low in fibre, vegetables, and folate and high in fat, red meat (Figure 4); heavy alcohol consumption; a sedentary occupation; and cigarette smoking [19]. Although colorectal cancers may appear at different times and for different reasons, they share a common random pathway from normal epithelium through polyp to carcinoma. In this process a number of genetic changes are observed, including the inactivation of the tumor suppressor genes and the activation of specific oncogenes. Several studies have shown mutations of p53, adenomatous polyposis coli (APC), k-ras and/or changes in proteins such as APC and DNA microsatellite instability or loss of heterozygosity [20]. Mutations are present as inherited germline defects or arise in somatic cells secondary to environmental insult, which continues to contribute to the development of colon tumor. Figure 4: Expected mechanisms of red meat in colorectal cancer induction. Non modifiable Risks Factors Several risk factors are associated with the incidence of CRC. Those that an individual cannot control include age and hereditary factors. In addition, a substantial number of environmental and lifestyle risk factors may play an important role in the development of CRC [14]. Age The likelihood of CRC diagnosis increases after the age of 40, increases progressively from age 40, rising 10 11

6 sharply after age 50. More than 90% of colorectal cancer cases occur in people aged 50 or older. The incidence rate is more than 50 times higher in persons aged 60 to 79 years than in those younger than 40 years. However, CRC appears to be increasing among younger persons. In fact, in the United States, CRC is one of the 10 most commonly diagnosed cancers among men and women aged 20 to 49 years [21]. Personal History of Adenomatous Polyps Neoplastic polyps of the colorectal, namely tubular and villous adenomas, are precursor lesions of CRC. The lifetime risk of developing a colorectal adenoma is nearly 19% in the U.S. population [22]. Nearly 95% of sporadic colorectal cancers develop from these adenomas. An individual with a history of adenomas has an increased risk of developing colorectal cancer, than individuals with no previous history of adenomas [23]. A long latency period, estimated at 5 to 10 years, is usually required for the development of malignancy from adenomas. Detection and removal of an adenoma prior to malignant transformation may reduce the risk of CRC [24]. Personal History of Inflammatory Bowel Disease Ulcerative colitis and Crohn disease usually described as inflammatory bowel disease (IBD). Ulcerative colitis causes inflammation of the mucosa of the colon and rectum. Crohn disease causes inflammation of the full thickness of the bowel wall and may involve any part of the digestive tract from the mouth to the anus. These conditions increase an individual s overall risk of developing colorectal cancer. The relative risk of CRC in patients with inflammatory bowel disease has been estimated between 4- to 20-fold [25]. Family History of Colorectal Cancer or Adenomatous Polyps The majority of CRC cases occur in persons without a family history of CRC or a predisposing illness. Up to 20% of people who develop CRC have other family members who have been affected by this disease. People with a history of CRC or adenomatous polyps in one or more first-degree relatives are at increased risk [26]. The reasons for the increased risk are not clear, but it likely due to inherited genes, shared environmental factors, or some combination of these. Inherited Genetic Risk Approximately 5 to 10% of colorectal cancers are a consequence of recognized hereditary conditions. The most common inherited conditions are FAP and HNPCC, also called Lynch syndrome. Genes responsible for these forms of inherited CRC have been identified. HNPCC is associated with mutations in genes involved in the DNA repair pathway, namely the MutL homolog 1 (MLH1) and MutS protein homolog 2 (MSH2) genes, which are 12 13

7 the responsible mutations in individuals with HNPCC. FAP is caused by mutations in the tumor suppressor gene APC. The lifetime risk of CRC in people with the recognized HNPCC-related mutations may be as high as 70 to 80%, and the average age at diagnosis in their mid-40s. MLH1 and MSH2 mutations are also associated with an increased relative risk of several other cancers, including several extracolonic malignancies, namely cancer of the uterus, stomach, small bowel, pancreas, kidney, and ureter. FAP accounts for less than 1% of all CRC cases [27]. Environmental Risk Factors Colorectal cancer is widely considered to be an environmental disease including cultural, social, and lifestyle factors. As such, CRC is one of the major cancers for which modifiable causes may be readily identified, and a large proportion of cases theoretically preventable. The evidence of environmental risk comes from studies of migrants and their offspring. For example, among offspring of southern Europe migrants to Australia and Japanese migrants to Hawaii, the risk of CRC is increased in comparison with that of populations of the country of origin [28]. Stages of Colorectal Cancer Once cancer forms in the inner lining of the large intestine, it can grow into the wall of the colon or rectum. Cancer that has grown into the wall can also penetrate blood or lymph vessels. Cancer cells typically spread first into nearby lymph nodes. Cancerous cells can also be carried in blood vessels to the liver or lungs, or can spread into the pelvis and abdominal cavity to other organs and tissues. The spread of cancer cells to distant parts of the body is called metastasis [29]. Figure 5: Colorectal cancer stages. Stages of CRC can be divided into 4 stages (Figure 5). Staging is essential in determining the choices for treatment and in assessing prognosis. In situ: Cancers that have not yet begun to invade the wall of the colon or rectum; these pre-invasive lesions are not included in the cancer statistics provided in this report

8 Local: Cancers that have grown into the wall of the colon or rectum, but have not extended through the wall to invade nearby tissues. Regional: Cancers that have spread through the wall of the colon or rectum and have invaded nearby tissue, or that have spread to nearby lymph nodes. Distant: Cancers that have spread to other parts of the body, such as the liver or lung. American cancer Society [29] divided stages into: Primary Tumor (T) TX Primary tumor cannot be assessed, T0 No evidence of primary tumor Tis Carcinoma in situ: intraepithelial or invasion of lamina propria1, T1 Tumor invades submucosa, T2 Tumor invades muscularis propria, T3 Tumor invades through the muscularis propria into pericolorectal tissues, T4a Tumor penetrates to the surface of the visceral peritoneum, T4b Tumor directly invades or is adherent to other organs or structures. Symptoms of Colorectal Cancer Screening of cancer is very important due to lack of symptoms in early CRC often has no symptoms. As a tumor grows, it may bleed or obstruct the intestine. Signs may include: bleeding from the rectum, blood in the stool or in the toilet after having a bowel movement, dark or black stools, a change in the shape of the stool (e.g., more narrow than usual), cramping or discomfort in the lower abdomen, an urge to have a bowel movement when the bowel is empty, constipation or diarrhea that lasts for more than a few days, decreased appetite, unintentional weight loss. In some cases, blood loss from the cancer leads to anemia, causing symptoms such as weakness and excessive fatigue [29]. Mechanism of Colorectal Cancer CRCs can arise from one or a combination of three different mechanisms, namely chromosomal instability (CIN), CpG island methylator phenotype (CIMP) and microsatellite instability (MSI) (Figure 6). According to Fearon and Vogelstein [30], the classical CIN pathway begins with the acquisition of mutations in the APC, followed by the mutational activation of oncogene KRAS and the inactivation of the tumor suppressor gene, TP53. Aneuploidy and loss of heterozygosity (LOH) are the major players in CIN tumors, which not only constitute most of the sporadic tumors (85%) but also involve familial adenomatous polyposis cases associated with germline mutations in the APC gene [31]. The CIMP pathway is characterized by promoter hypermethylation of various tumor suppressor genes, most importantly MGMT and MLH1. This hypermethylation is often associated with BRAF mutation and microsatellite instability [32]. The MSI pathway involves the inactivation of genetic alterations in short repeated sequences. This activation occurs in CRCs in DNA mismatch repair (MMR) genes, and is a hallmark condition in familial Lynch syndrome (LS), which also appears in ~15% of the sporadic CRC cases. In addition, the hypermethylation of 16 17

9 the MMR genes may lead to MSI. This mechanism is often associated with the CIMP pathway. MSI tumors are often associated with proximal colon and poor differentiation but better prognosis [33]. Figure 6: Mechanisms of colorectal cancer. Chromosomal Instability Chromosomal instability is associated with 65%-70% of sporadic CRCs. This pathway comprises aneuploidy, which is an imbalance in the chromosome number, and LOH. Defects in chromosomal segregation, DNA damage repair, and telomere function along with specific mutations in certain oncogenes and tumor suppressor genes may be responsible for such instability. Aneuploidy arises because of defects in the mitotic checkpoint, which cause chromosome mis-segregation. Mutational upregulation or downregulation of various mitotic checkpoint players, such as hrod, hzwilch, hzw, Ding, budding uninhibited by benzimidazoles (Bub) R1, centromere-associated protein E (CENP-E), and mitotic arrest deficient (MAD) 1, can result in CIN [34]. A deleterious increase in aneuploidy may also lead to cell death. Other centromere protein genes, such as CENP-A and CENP-H, may also be overexpressed in CRCs, which leads to mislocalizations on non-centromeric chromatin positions [35]. Disruptions in the DNA damage pathway and excess telomere breakage can lead to chromosomal instability. The damaged DNA is repaired by four mechanisms, namely base excision repair (BER), double-stranded break repair (DSBR), mismatch repair (MMR), and nucleotide excision repair (NER). The polymorphisms in BER-associated genes XRCC1, OGG1, and MUTYH have been correlated with reduced oxidative DNA damage repair efficiency in CRC [36]

10 Mutational Landscape in Chromosomal Instability Mutations in the APC activate the Wnt signaling pathway by increasing β-catenin levels. β-catenin is translocated to the nucleus and enhances the transcription of various oncogenes with T-cell factor (TCF) transcription factors. High β- catenin levels are noted in gastrointestinal tumors [37]. Around 75% of CRCs have mutations or LOH in the APC gene. Most of these mutations are clustered in the mutation cluster region between codons 1282 and However, a complete inactivation is not required. Mutations sufficient for tumorigenesis differ between the proximal and distal CRCs. The effects of APC restoration in mice are demonstrated on tumor regression by the conversion of cancer cells back to normal, which indicates a similar possibility in humans. Mutations in other genes of this pathway, particularly in β-catenin, may also lead to CIN [38]. These mutations are found in 48% of CRCs without APC mutations. β-catenin activates a set of 162 Wnt pathway target genes in a colon cancer cell line. However, no conclusions could be drawn for their effect on disease prognosis [39]. Microsatellite Instability (MSI) Microsatellite instability occurs because of inactivating mutations in the DNA mismatch repair genes that are responsible for correcting DNA replication errors. The important components of the DNA mismatch repair system are ATPases hmsh2, hmsh6, hmsh3, hmlh1, hpms2, hpms1, and hmlh3 [40]. The germline mutations that may render these proteins dysfunctional can predispose to cancer as in the case of LS81. MSI is found in 15% of colorectal cancers, with only 3% associated with LS. The rest are sporadic cases caused by the hypermethylation of the MLH1 gene promoter [41]. CpG Island Methylator Phenotype (CIMP) Global DNA hypomethylation and localized promoter hypermethylation are common epigenetic events that occur in cancer. Hypomethylation refers to a marked global decrease in methylation on cytosine bases that is observed in hyperplastic and adenomatous polyps and carcinomas [42]. Hypomethylation in the repetitive DNA sequences, such as in satellite regions, can lead to genomic instability. Furthermore, loss of imprinting or promoter demethylation could reactivate the retrotransposons. The demethylation of the long interspersed nuclear element promoter has been suggested as an early event. However, demethylation has also been observed in the normal colonic mucosa of the same patients [43]. The loss of imprinting of insulin-like growth factor 2 (IGF2) is seen in almost 40% of CRC tumors, which leads to microsatellite instability in younger patients [44]

11 Other Colorectal Cancer Pathways MicroRNA Recently, micrornas (mirnas) have been found to be involved in CRC pathogenesis. mirna are a class of short (20 22 nucleotide) non-coding RNAs which regulate protein expression by inhibiting mrna translation, in particular of genes involved in cell differentiation, development, proliferation and apoptosis. The number of mir- NAs involved in CRC pathogenesis is very large and still expanding, as new mirnas are continuously being identified. They can be upregulated or downregulated in CRC, operating like oncogenes and tumor suppressor genes. For example, Bandres et al. [45] found the altered expression of 13 mirnas in patients affected by CRC and an interesting, divergent expression of mirnas in CRCs with either KRAS or BRAF mutations indicating that these altered expressions may be related to mirnas regulatory action in the RAS pathway. Upregulation of mir-31 was found to be associated with stage IV CRC [45]. Downregulation of mir-145 and mir-143 was demonstrated by other studies, showing that their expression is reduced in precancerous adenomatous polyps, as compared to normal tissue; thus, researchers suggest these mirnas play a key role in the early development of the tumors [46]. Interestingly, Lanza et al. [47] found significant upregulations of mir-17-92, mir-17-5p, mir-20, mir25, mir-92-1, mir-92-2, mir-93-1 and mir-106a in the microsatellite stable (MSS) CRC and not in MSI CRC. Furthermore, Motoyama et al. [46] demonstrated an increased expression of mir-31, mir- 183, mir-17-5p, mir18a, mir-20a and mir-92 in tumoral tissue as compared to normal colorectal mucosa, and saw an association between overexpression of mir-18a and a worse CRC prognosis. Inflammatory Pathway Chronic inflammation is a critical component of CRC initiation and progression. This is supported by finding of strong associations between IBD and CRC, and by findings supporting the positive effects of chronic NSAIDs use in CRC. Multiple different markers of inflammation predispose an individual to CRC. This happens by enhancing stimulation, by sustaining cell growth through promoting anti-apoptotic system, and by increasing DNA-damage through the activation of the mutagenic reactive oxygen and nitrogen species. Other mechanisms include the production of angiogenic and lymph angiogenic growth factors, and changes of the membrane systems to facilitate invasion and altering cell adhesion [48]. In support of the role of chronic inflammation in CRCs, researchers studied the role of the pro-inflammatory cytokine tumor necrosis factor (TNF)-α, the transcription factor; signal transducer and activator of transcription 3 (STAT3) protein, interleukin (IL)-6 and the C-reactive protein (CRP). Chronically, elevated levels of TNF-α promote tumor growth, proliferation and metastasis. IL-6 is a cytokine involved in the regulation of the acute phase of inflammation and, in its 22 23

12 own transduction pathway, stimulates the transcription of STAT3 [49]. STAT3 activation stimulates its translocation into the nucleus and then stimulates cell proliferation, differentiation, apoptosis and promotes metastasis by inducing the expression of different gene targets such as VEGFR2 (vascular endothelial growth factor receptor 2), Bcl-2, Cyclin D1, MMP2-9, ICAM-1, and COX-2 [50]. CRP is a biomarker of inflammation, both in the acute phase and in the chronic low phase of inflammation. The role of this inflammatory mediator was controversial, as researchers obtained discordant results. Chan et al. [51] investigated the influence of CRP, IL-6 and soluble tumor necrosis factor receptor 2 (TNFR-2, a TNF-α receptor superfamily member) in CRC, in a cohort of 33,000 women, found an increased risk of CRC in woman having high levels of stnfr-2 (p = 0.03), but found no correlation with the other two markers. Interestingly, those with high baseline levels of stnfr-2 who took aspirin had lower risk of developing CRC. On the other hand, Song et al [52] researched the same inflammatory markers, and did not find any correlation, only a positive association between IL-6 and increased risk of CRC in lean individuals (p = 0.03). Moreover, Knupfer et al. [53] found higher levels of IL-6 in neoplastic colorectal mucosa than in normal mucosa and strong associations between advanced CRC stage, tumor size and a worse prognosis. Moreover, Ma et al. [50] found an increased level of STAT3 in the abnormal CRC tissue compared with the normal mucosa, and its correlation with CRC metastasis and stage and also its association with cyclin D1 overexpression. Screening for Colorectal Cancer Some people who are at increased risk of CRC because of family history or certain medical conditions should begin CRC screening before age 50. Colonoscopy is the recommended screening method for most individuals in these increased and high-risk groups. Recommendations regarding age to initiate screening and rescreening intervals may differ based on individual circumstances [29]. The American Cancer Society recommendations emphasize that cancer prevention should be the primary goal of CRC screening. To achieve this goal, exams that are designed to detect both early cancer and precancerous polyps should be encouraged if resources are available and patients are willing to undergo an invasive test. The higher likelihood of polyp detection with the use of these tests substantially increases opportunities for polyp removal and CRC prevention [54]. The following options are recommended for CRC screening in men and women age 50 and older at average risk. Flexible sigmoidoscopy: A slender, flexible, hollow, lighted tube is inserted through the rectum into the colon. The sigmoidoscope is about 2 feet long (60 cm) and provides a visual examination of the rectum and lower one

13 third of the colon (sigmoid colon) [54]. Analysis of data from clinical trials, in which participants are invited to screening, indicates that sigmoidoscopy is associated with a 21% reduction in CRC incidence and a 26% reduction in CRC mortality [55]. A randomized clinical trial in the United Kingdom reported that among participants who completed a single sigmoidoscopy screening between the ages of 55 and 64, CRC incidence was reduced by 33% and mortality by 43% [56]. Colonoscopy: Like sigmoidoscopy, this procedure allows for direct visual examination of the colon and rectum. A colonoscope is similar to a sigmoidoscope, but is a much longer, more complex instrument, allowing visualization of the entire colon and removal of polyps. Studies show that colonoscopy is the most sensitive method for the detection of CRC or adenomatous polyps [57]. A recent analysis of data from the National Polyp Study found that patients who had adenomas removed during colonoscopy (with follow-up colonoscopy at one or three years) had a 53% lower risk of death from CRC than the general population. Colonoscopy also has the longest rescreening interval of all forms of testing; if normal, the exam does not need to be repeated for 10 years in averagerisk patients [58]. However, colonoscopy does have limitations. The procedure misses approximately 20% of all adenomas and 10% of large (5 mm or larger) or advanced adenomas [59]. Colonoscopy also has a higher risk of complications compared to other screening tests, including bowel tears and bleeding, especially when a polyp is removed [54]. Barium enema with air contrast: Use of this procedure, which is also called double-contrast barium enema (DCBE), has become very uncommon due to the increased availability of colonoscopy. Barium sulfate is introduced into a cleansed colon through the rectum to partially fill and open the colon. Air is then introduced to expand the colon and increase the quality of x-rays that are taken. This method is less sensitive than colonoscopy for visualizing small polyps or cancers [29]. Computed tomographic colonography (CTC): Also referred to as virtual colonoscopy, this imaging procedure was introduced in the 1990s and results in detailed, cross-sectional, 2- or 3-dimensional views of the entire colon and rectum with the use of a special x-ray machine linked to a computer [54]. Although a full bowel cleansing is necessary for a successful examination, sedation is not required. Patients with polyps of significant size (larger than 5 mm) or other abnormal results are referred for colonoscopy, optimally on the same day in order to alleviate the necessity of a second bowel preparation. Studies have shown that the performance of CTC is similar to optical colonoscopy for the detection of invasive cancer and polyps approximately 1 cm or larger in size [60]

14 Tests Primarily Effective at Detecting Colorectal Cancer Although high-sensitivity stool tests detect some precancerous polyps, the potential for prevention is both limited and incidental and cannot be the primary goal of screening with these tests. Modeling studies show that annual screening with high-sensitivity stool tests results in a mortality benefit comparable to structural exams (e.g., colonoscopy, sigmoidoscopy, or CTC), though adherence to yearly testing is a challenge in the community setting [61]. Fecal Occult Blood Rest (FOBT): Cancerous tumors and some large polyps bleed intermittently into the intestine. This blood can be detected in stool by the FOBT kit. Bleeding from CRC may be intermittent or undetectable, so accurate test results require annual testing that consists of collecting 1 to 3 samples from consecutive bowel movements. There are two types of FOBT available guaiacbased tests, which detect blood from any source (including meat in the diet), and immunochemical-based tests, which detect only human blood from the large bowel [62]. Patients who have a positive gfobt or FIT are referred for a colonoscopy to rule out the presence of polyps or cancer. Recently reported data from a large clinical trial indicated that the regular use of FOBT reduced the risk of death from CRC by 32% after 30 years of follow up. In addition, FOBT has been shown to decrease the incidence of CRC by 20% by detecting large precancerous polyps. It is important to emphasize that the effectiveness of FOBT is dependent on repeated screenings over time [63]. Stool DNA (sdna) test: The stool DNA test approved for CRC screening in 2008 is no longer commercially available. A new test has undergone extensive study and may be evaluated for inclusion as a recommended testing option in the future. This method of screening is the result of increasing knowledge about the molecular properties of cancer. Cancerous tumors and large polyps shed cells into the large bowel that contain altered DNA that can be detected in stool samples. Patients with a positive test result would be referred for a colonoscopy [64]. Any of the above recommended options are useful in screening for CRC in average-risk adults. Each of these tests has strengths and limitations related to accuracy, potential for prevention, cost, and risks. Treatment of Colorectal Cancer Most people with colon cancer will have some type of surgery to remove the tumor. Adjuvant therapy (additional treatments after surgery) may also be used. Carcinoma in situ: Surgery to remove the growth of abnormal cells may be accomplished by polypectomy (polyp removal) or local excision through the colonoscope. Resection of a segment of the colon may be necessary if the tumor is too large to be removed by local excision or if cancer cells are found at the edges of the polyp 28 29

15 after it is removed. Removing or destroying the growth of abnormal cells is all that is needed. Treatment options include polypectomy (polyp removal), local excision, or full-thickness rectal resection. This resection may be carried out through the anus. No further treatment is needed. Localized stage: Surgical resection to remove the cancer, together with a length of colon on either side of the tumor and nearby lymph nodes, is the standard treatment. Regional stage: If the cancer has only grown through the wall of the colon but has not spread to nearby lymph nodes, surgical resection of the segment of colon containing the tumor may be the only treatment needed. If the cancer is likely to come back, because of its appearance under the microscope or because it is growing into other tissues, radiation therapy and/or chemotherapy may also be recommended. If the cancer has spread to nearby lymph nodes, surgical resection of the segment of colon containing the tumor is the first treatment, usually followed by chemotherapy [29]. Distant stage: When surgery is performed, the goal is usually to relieve or prevent blockage of the colon and to prevent other local complications. If there are only a few metastases to the liver or lungs, surgery to remove these, as well as the colon tumor, may be an option. Surgery is not recommended for all patients. Chemotherapy, radiation, and biologically targeted therapies may be given alone or in combination to relieve symptoms and prolong survival. A number of targeted therapies have been approved in recent years by the US Food and Drug Administration (FDA) to treat metastatic CRC. Some of these drugs inhibit new blood vessel growth to the tumor by targeting vascular endothelial growth factor (VEGF). Others interfere with cancer cell growth by targeting the epidermal growth factor receptor (EGFR) or other proteins. Tumors with certain genetic mutations do not benefit from treatment with some of this drugs [65]. Advances in Colorectal Cancer Treatment Chemotherapy The current standard protocol for colon cancer, which is known to significantly reduce relapse rates and risks of dying from resected colon cancer, involves 6 months of adjuvant fluorouracil (5-FU) combined with leucovorin. This protocol significantly improves the 3- year diseasefree survival rate. However, only 26% of patients respond [66]. Chemotherapy treatments based on the drug fluorouracil (5-FU) have been shown to improve survival in patients with stage III or high-risk stage II disease, primarily by reducing disease recurrence. Radiation therapy may also be recommended if the cancer has grown into adjacent tissues. Adjuvant (given after surgery) chemotherapy or radiation for colon cancer is as effective in patients 30 31

16 age 70 and older (more than half of all patients) who are otherwise healthy as in younger patients, though certain drugs (i.e., oxaliplatin) may be avoided to limit toxicity. However, a recent study in California found that although chemotherapy reduced colon cancer mortality similarly across all age groups, individuals 75 and older were far less likely than younger patients to receive this treatment [67]. Drug resistance is a major problem that limits the effectiveness of chemotherapy, and its systemic toxic effects make it difficult for some patients to tolerate. Multidrug resistance (MDR) is a phenomenon by which cancer cells evade the cytotoxic effects of chemotherapeutic agents. It may occur through different mechanisms, but it often correlates with the overexpression of membrane or intracellular molecular protein, resulting in increased drug efflux, decreased drug influx, drug inactivation, alterations in drug targeting, processing of drug-induced damage, or evasion of apoptosis [68]. Penuelas et al. [69] screened the gene expression profile in HT29 human colon cancer cell resistance to methotrexate (MTX) and found that IMPDH2, IMPCH and survivin were up-regulated, while topoisomerase I and vimentin were down-regulated. One of the major goals for chemotherapy is to target tumor cells with toxic agents in a selective and specific manner, avoiding damage to normal tissues. Enzymepro drug systems are used to localize the toxic drug effects to tumor cells, and destroy tumors and metastasis but not normal tissues. Gene therapy offers the possibility of this targeted treatment, which reduces the systemic effects of chemotherapy. GDEPT (gene directed enzyme pro-drug therapy) involves gene transfer of an enzyme into tumor cells, which converts an inactive pro-drug into a toxic metabolite, leading to cell death [70]. Immunotherapy Immunotherapy is the second method for colon cancer treatment. A number of approaches using antibodies and vaccines as adjuvant therapy are being studied. Colon tumor cells can express TAA, which is able to activate the immune system. mabs directed against tumor antigens can opsonize tumor cells and promote their elimination either by activation of cellular immune effectors like NK cells (ADCC) or by activation of the complement cascade (CDC) [70]. Anti- CD3/CEA or anti-cd3/ep-cam bi-specific antibodies can cross-link T cells to colon cancer cells. Furthermore, anti-cea /B7 can convert B7-negative tumors into B7- positive tumors, provide both functional and tumor specific signals 1 and 2 for the activation of primary human T cells, and trigger their cytotoxic activity in a tumor-specific way [71]. Tumor cells can serve as vaccines to augment antitumor immunity after irradiation with x rays to prevent their outgrowth in vivo. The effect is limited, however, though it can be improved by increasing immunogenicity 32 33

17 of whole-cell vaccines based on the use of nonspecific immunostimulants such as bacillus Calmette-Guerin (BCG) and corynebacterium parvun, or on simultaneous genetransduction of co-stimulatory molecules (B7, CD40L) and multiple cytokines (IL-2, IL-12, GM-CSF) [72]. One of the mechanisms by which tumor cells evade the immune system is the lack of proper antigen-presenting cells. Improvement in host immunity against tumor cells can be achieved by promoting the differentiation of dendritic cells (DCs) from immature myeloid cells that accumulate in the bone marrow and lymphoid organs [73]. The T cell is the effector cell in anti-tumor immune response. T cells require 2 distinct signals for optimal activation, and the genetic modification of T cells ex vivo and their re-infusion into cancer patients has recently attracted considerable attention. Recent studies revealed that gene-engineered T cells expressing chimeric singlechain (scfv) receptors were capable of co-delivering CD28 costimulation and T cell receptor zeta chain (TCR-zeta) activation signals, which after reacting with the ErbB2 tumor-associated antigen of colon cancer produced high levels of cytokines, proliferated vigorously, and mediated lysis of ErbB2(+) tumors in an antigen-specific manner [74]. Anti-Angiogenesis Hypoxia and angiogenesis are two important determinants by which cancer microenvironment influences CRC development and progression. Cancer cells cannot grow when they are located at a distance greater than 110 μm from a blood vessel. Angiogenesis is thus importantly involved in CRC growth [75]. Cancer cells depend on angiogenesis to obtain nutrients and oxygen for their outgrowth and metastasis. Tumor-induced angiogenesis involves neovascularization that is regulated by angiogenic and antiangiogenic factors released from tumor cells and surrounding stromal cells. This process includes the vascular endothelial cell s migration, adhesion and invasion to extracellular matrix (ECM) and its proliferation. Anti-angiogenic therapy is a promising approach for colon cancer therapy, as it is less toxic than conventional chemotherapy and has a lower risk of drug resistance. Furthermore, anti-angiogenic agents can also transiently normalize the abnormal structure and function of tumor vasculature to make it more efficient for drug delivery and increase the efficacy of conventional therapies [76]. Notably, the level of angiogenesis is a survival predictor for CRC patients. In accordance, patients having a Doppler vascularity index >15% at the primary site have a poorer overall survival than patients with an index <15% [77]. Most of the times, angiogenesis is switched on by tumor-associated hypoxic conditions, which contributes to activate the transcription of HIF-1. In turn, HIF-1 promotes the expression of angiogenic factors by the cancer cells including VEGF, basic fibroblast growth 34 35

18 factor (bfgf) and placental derived growth factor (PLGF) [78]. Following hypoxia-induced gene expression by cancer cells, angiogenic factors interact with their receptors at the surface of endothelial cells initiating the activation of the angiogenic program. In particular, VEGF binds to VEGFR2, which triggers signaling cascades leading to the induction of vasodilation, endothelial cell migration/ proliferation and vessel assembly. The reciprocal synergistic interactions between cancer cells and endothelial cells constitute one of the best examples of the cancer cell/ microenvironment interactions. Also, pro-inflammatory cytokines (IL-6 and IL-1) released by stromal and cancer cells mutually interact to favor tumor expansion and metastasis [79]. Watanabe and colleagues discovered a novel angiogenesis inhibitor, vasohibin, an endothelium derived negative feedback regulator of angiogenesis. It inhibited migration, proliferation, and network formation by ECs as well as angiogenesis in vivo, and served as an intrinsic and highly specific feedback inhibitor of activated endothelial cells engaged in the process of angiogenesis [80]. Apoptosis Induction A balance between cell birth and cell death is necessary to sustain the colonic epithelium. Apoptosis, or programmed cell death, plays an important role in maintaining tissue homeostasis and permitting the controlled deletion of potentially harmful cells within the adult organism. Since most tumor cells have presumably disabled apoptosis to reach a malignant status, apoptosis induction represents one of the most obvious methods for cancer treatment. At the molecular level, a number of genes, molecules or signals are often changed in colon cancer and some of these are key regulators of apoptosis, such as p53 mutant, IAP, and COX2 [81]. Direct targeting of the apoptotic pathway offers a novel strategy for overcoming apoptosis resistance. Tumor suppressor p53 gene, which is the most commonly mutated gene in human cancers, plays an important role in the apoptotic pathway. Re-expression with wild-type p53 in a TP53- mutated colon-cancer cell line in a murine model can cause growth inhibition and a doubling of survival times. Furthermore, the transfer of p53 genes into tumor cells lacking functional p53 enhanced the radio responsiveness and the anti-tumor effect of common chemotherapeutic agent by promoting p53- dependent apoptosis [82]. Survivin is a member of the inhibitor of apoptosis (IAP) gene family, and is over-expressed in 53% to 64% of colorectal cancers, which is correlated with carcinogensis, angiogenesis, aggressive phenotype, and overall poor survival. Inhibition of surviving expression and function induced apoptosis and produced a phenotype of aberrant mitotic progression, characterized by supernumerary centrosomes, formation of multipolar mitotic spindles, failure of cytokinesis, and generation of multinucleated cells [83]. Cyclooxygenase-2 (COX2) is an inducible isoenzyme of cyclooxygenase, undetectable in normal colonic muco

19 sa and upregulated in 40% of adenomas and 85% of CRC. In the process of colorectal tumorigenesis, COX2 plays a key role in the various steps involved. COX-2 inhibitors can decrease the number and size of polyps and may prevent the progression from adenomatous polyp to invasive carcinoma, so that celecoxib was approved by the FDA for the treatment of polyps in patients with FAP [84]. Inflammation and recruitment of tumor-associated macrophages in CRC microenvironment. The primary colon neoplasm contains several types of bone marrowderived cells (BMDC) including tumor-associated macrophages (TAMs) [85]. The recruitment of TAMs to colon cancer is a typical response to cancer-associated inflammation and may explain why CRC responds to anti-inflammatory agents. It seems that TAMs favors CRC metastasis by secreting VEGF, thereby promoting angiogenesis at the primary site [86]. Furthermore, TAMs trigger the removal of apoptotic CRC cells that express the sulfoglycolipids SM4s. This process is associated with an important phenotypic modification of TAMs that is characterized by an increased expression of TGFβ1 and secretion of IL-6. This phenotypic modification may contribute to further activate the angiogenic process. Interestingly, neutrophils are also recruited to CRC and, as for TAMs, their recruitment is also associated with enhanced angiogenesis and metastasis. On the other hand, several studies have reported the antitumor functions of TAM. The possible anti-tumor effects of TAMs may rely on enhancing the nuclear translocation of macrophage migration inhibitory factor (MIF), and increasing the expression of GM-CSF and IL-12 [87]. Perspective Therapy for colon cancer has undergone considerable modifications in recent years. The protocol centered on fluorouracil has been joined by new drugs, including capecitabine, irinotecan, oxaliplatin, and targeted agents such as bevacizumab and cetuximab. Several new drugs are presently undergoing preclinical or clinical studies. However, the five-year survival rate from colon cancer has remained fairly static; standard therapy, however, remains non-specific; cytotoxic drugs that are effective only in some patients, yet cause side effects in most. Targeting of angiogenesis leads to minimal side effects as normal vessels are quiescent since the target is endothelial cells which are normal and genetically stable and the target of antiangiogeni inhibitors, such as kallistatin, angiostatin and vasostatin, was activated endothelial cell. Anti-angiogenic inhibitors for cancer therapy with AAV-mediated provide a new strategy to span cancer patient s life [88]. Given the clinical and pathological heterogeneity of CRC, the optimal strategies for adjuvant therapy should be individualized. With the development of analysis and definition of prognostic and predictive markers, in future, therapeutic strategies should be constructed individually for each patient based on the molecular taxonomy of tu

20 mors, which would be lower toxicity, higher therapeutic index, and a weaker tendency to induce resistant phenotypes in tumor cells [70]. Prospective Therapies for Colorectal Cancer There is hope, however, and the chance to make a change if preventive steps are taken early enough. Evidence suggests that adenomas may present for ten or more years before cancer develops [89]. Early prevention is extremely important in helping to stop polyp formation and/or the recurrence of these polyps while allowing the body to be in its optimal, healthy state to offset any abnormal changes. For many people, maintaining a healthy lifestyle and taking the steps towards wellbeing can be extremely difficult. There may be many barriers including things like stress, sleeping disorders resulting in an angry cycle of fatigue, preexisting medical conditions, decreased motivation and possibly lack of understanding or direction. Naturopathic science work in this area to help treat the root cause of the problem by using tools like dietary support, fitness modules, botanicals, supplements, acupuncture, lifestyle counseling and education [90]. Naturopathic Medicine and Colorectal Cancer Prevention is the first and foremost important step in the fight against cancer especially in those who are at a higher risk for CRC. Naturopathic medicine play a pivotal role in Colorectal Cancer Care and offers therapies that; reduce the risk of initially developing CRC. Supportive during chemotherapy, radiation and/or surgery and help to improve both the tolerance and success of conventional therapies. Also, it helps prevent recurrence once cancer has been successfully treated [90]. Diet, fitness and natural therapies are just as important in this stage or more so to maintain a healthy immune system and to create a hostile environment for the formation of cancer. This step in a continuum of health is not only needed but is imperative in helping to ensure continued treatment success [91]. Diet as a Determinant of Colorectal Cancer Microenvironment A large proportion of CRCs are related to environmental factors. In particular, CRC is highly dependent on diet, which is a major modulator of cancer microenvironment. Notably, CRC is well recognized as being associated with obesity. In fact, several epidemiological studies have associated excess body weight with an increased risk of colon cancer, particularly for men [92]. In that regard, insulin resistance associated with obesity promotes colonocyte proliferation and suppresses apoptosis. Moreover, 40 41