Linear Model of Colon Cancer Initiation

Cancer results if regulatory mechanisms of cell birth and death are disrupted. Colorectal tumorigenesis is initiated by somatic or inherited mutations in the APC tumor suppressor gene pathway. Several additional genetic hits in other tumor suppressor genes and oncogenes drive the progression from polyps to malignant, invasive cancer. The majority of colorectal cancers present chromosomal instability, CIN, which is caused by mutations in genes that are required to maintain chromosomal stability. A major question in cancer genetics is whether CIN is an early event and thus a driving force of tumor progression. We present a new mathematical model of colon cancer initiation assuming a linear flow from stem cells to differentiated cells to apoptosis. We study the consequences of mutations in different cell types and calculate the conditions for CIN to precede APC inactivation. We find that early emergence of CIN is very likely in colorectal tumorigenesis.     

Wnt pathway mutations selected by optimal beta-catenin signaling for tumorigenesis

Mutations in components of the Wnt/beta-catenin pathway are observed to be the earliest initiating event for most colorectal tumors. The majority of the mutations occur in the tumor suppressor adenomatous polyposis coli (APC), even though there are other genes that are capable of modulating the pathway activity. Moreover, the specific APC mutations associated in colon cancer indicate the possibility that the tumor selects for certain truncated forms of APC that partially retain its function, namely, inhibition of beta-catenin. We estimated the effects of various mutations in APC and other known mutations using a recent mathematical model of the Wnt pathway that was constructed to represent the conserved core molecular events. We provide evidence that APC mutations are selected not based on the maximal level of beta-catenin but rather based on distinct state of activity that appears to be optimal for the tissue-specific tumorigenesis. This optimal level is determined by balancing beta-catenin signaling and the induction of Axin2 that acts as a potent negative feedback. The predominant pattern of APC mutations may provide synergistic oncogenic effects that promote colorectal tumorigenesis: the optimal signaling for cell survival and renewal, disrupted cell adhesion, chromosomal instability, and altered asymmetric division of stem cells.     

Genomic instability and cancer

Tumorigenesis can be viewed as an imbalance between the mechanisms of cell-cycle control and mutation rates within the genes. Genomic instability is broadly classified into microsatellite instability (MIN) associated with mutator phenotype, and chromosome instability (CIN) recognized by gross chromosomal abnormalities. Three intracellular mechanisms are involved in DNA damage repair that leads to mutator phenotype. They include the nucleotide excision repair (NER), base excision repair (BER) and mismatch repair (MMR). The CIN pathway is typically associated with the accumulation of mutations in tumor suppressor genes and oncogenes. Defects in DNA MMR and CIN pathways are responsible for a variety of hereditary cancer predisposition syndromes including hereditary non-polyposis colorectal carcinoma (HNPCC), Bloom syndrome, ataxia-telangiectasia, and Fanconi anaemia. While there are many genetic contributors to CIN and MIN, there are also epigenetic factors that have emerged to be equally damaging to cell-cycle control. Hypermethylation of tumor suppressor and DNA MMR gene promoter regions, is an epigenetic mechanism of gene silencing that contributes to tumorigenesis. Telomere shortening has been shown to increase genetic instability and tumor formation in mice, underscoring the importance of telomere length and telomerase activity in maintaining genomic integrity. Mouse models have provided important insights for discovering critical pathways in the progression to cancer, as well as to elucidate cross talk among different pathways. This review examines various molecular mechanisms of genomic instability and their relevance to cancer.     

Loss of APC induces polyploidy as a result of a combination of defects in mitosis and apoptosis

Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene initiate a majority of colorectal cancers. Acquisition of chromosomal instability is an early event in these tumors. We provide evidence that the loss of APC leads to a partial loss of interkinetochore tension at metaphase and alters mitotic progression. Furthermore, we show that inhibition of APC in U2OS cells compromises the mitotic spindle checkpoint. This is accompanied by a decrease in the association of the checkpoint proteins Bub1 and BubR1 with kinetochores. Additionally, APC depletion reduced apoptosis. As expected from this combination of defects, tetraploidy and polyploidy are consequences of APC inhibition in vitro and in vivo. The removal of APC produced the same defects in HCT116 cells that have constitutively active beta-catenin. These data show that the loss of APC immediately induces chromosomal instability as a result of a combination of mitotic and apoptotic defects. We suggest that these defects amplify each other to increase the incidence of tetra- and polyploidy in early stages of tumorigenesis.     

Chromosome instability in colorectal tumor cells is associated with defects in microtubule plus-end attachments caused by a dominant mutation in APC

The attachment of microtubule plus ends to kinetochores and to the cell cortex is essential for the fidelity of chromosome segregation. Here, we characterize the causes underlying the high rates of chromosome instability (CIN+) observed in colorectal tumor cells. We show that CIN+ tumor cells exhibit inefficient microtubule plus-end attachments during mitosis, accompanied by impairment of chromosome alignment in metaphase. The mitotic abnormalities associated with CIN+ tumor cells correlated with status of adenomatous polyposis coli (APC). Importantly, we have shown that a single truncating mutation in APC, similar to mutations found in tumor cells, acts dominantly to interfere with microtubule plus-end attachments and to cause a dramatic increase in mitotic abnormalities. We propose that APC functions to modulate microtubule plus-end attachments during mitosis, and that a single mutant APC allele predisposes cells to increased mitotic abnormalities, which may contribute to tumor progression.     

Chromosome instability and tumor lethality suppression in carcinogenesis

The maintenance and survival of each organism depends on its genome integrity. Alterations of essential genes, or aberrant chromosome number and structure lead to cell death. Paradoxically, cancer cells, especially in solid tumors, contain somatic gene mutations and are chromosome instability (CIN), suggesting a mechanism that cancer cells have acquired to suppress the lethal mutations and/or CIN. Herein we will discuss a tumor lethality suppression concept based on the studies of yeast genetic interactions and transgenic mice. During the early stages of the multistep process of tumorigenesis, incipient cancer cells probably have adopted genetic and epigenetic alterations to tolerate the lethal mutations of other genes that ensue, and to a larger extent CIN. In turn, CIN mediated massive gain and loss of genes provides a wider buffer for further genetic reshuffling, resulting in cancer cell heterogeneity, drug resistance and evasion of oncogene addiction, thus CIN may be both the effector and inducer of tumorigenesis. Accordingly, interfering with tumor lethality suppression could lead to cancer cell death or growth defects. Further validation of the tumor lethality suppression concept would help to elucidate the role of CIN in tumorigenesis, the relationship between CIN and somatic gene mutations, and would impact the design of anticancer drug development.     

Reduced ATR or Chk1 Expression Leads to Chromosome Instability and Chemosensitization of Mismatch Repair–deficient Colorectal Cancer Cells

Genomic instability in colorectal cancer is categorized into two distinct classes: chromosome instability (CIN) and microsatellite instability (MSI). MSI is the result of mutations in the mismatch repair (MMR) machinery, whereas CIN is often thought to be associated with a disruption in the APC gene. Clinical data has recently shown the presence of heterozygous mutations in ATR and Chk1 in human cancers that exhibit MSI, suggesting that those mutations may contribute to tumorigenesis. To determine whether reduced activity in the DNA damage checkpoint pathway would cooperate with MMR deficiency to induce CIN, we used siRNA strategies to partially decrease the expression of ATR or Chk1 in MMR-deficient colorectal cancer cells. The resultant cancer cells display a typical CIN phenotype, as characterized by an increase in the number of chromosomal abnormalities. Importantly, restoration of MMR proficiency completely inhibited induction of the CIN phenotype, indicating that the combination of partial checkpoint blockage and MMR deficiency is necessary to trigger CIN. Moreover, disruption of ATR and Chk1 in MMR-deficient cells enhanced the sensitivity to treatment with the commonly used colorectal chemotherapeutic compound, 5-fluorouracil. These results provide a basis for the development of a combination therapy for those cancer patients.     

Mutation analysis of p53, K-ras, and BRAF genes in colorectal cancer progression

Gene mutations in APC, K-ras, and p53 are thought to be essential events for colorectal cancer development. Recent data seem to indicate that K-ras and p53 mutations rarely co-exist in the same tumor, indicating that these alterations do not represent a synergistic evolutionary pathway. Moreover, an inverse relation between K-ras gene activation and BRAF mutations has been demonstrated, suggesting alternative pathways for colorectal cancer transformation. To reconstruct the chronological modulation of these gene mutations during cell transformation and colorectal cancer progression, mutations of p53, K-ras, and BRAF genes were analyzed by Single Strand Conformation Polymorphism (SSCP) or sequencing analysis in 100 colorectal cancer samples, evenly distributed among different Dukes' stages. We found mutations in p53, K-ras, and BRAF genes in 35%, 30%, and 4% of tumors, respectively, and observed a minimal or no co-presence of these gene alterations. Moreover, the frequency of molecular p53 mutations increased as tumor stage increased, suggesting an important role for this gene in the progression of colorectal cancer. Conversely, K-ras or BRAF genes were not related to tumor stage or location. These data seem to indicate the absence of a co-presence of the genes, highlighting the possibility of multiple pathways for colorectal tumor progression. Moreover, mutations in p53, K-ras, and BRAF are not present in about one-third of colorectal cancers and therefore other gene mutations need to be investigated to better understand molecular mechanisms at the basis of cell transformation and the progression of colorectal cancer.     

Progression of colorectal cancer is associated with multiple tumor suppressor gene defects but inhibition of tumorigenicity is accomplished by correction of any single defect via chromosome transfer.

Carcinogenesis is a multistage process that has been characterized both by the activation of cellular oncogenes and by the loss of function of tumor suppressor genes. Colorectal cancer has been associated with the activation of ras oncogenes and with the deletion of multiple chromosomal regions including chromosomes 5q, 17p, and 18q. Such chromosome loss is often suggestive of the deletion or loss of function of tumor suppressor genes. The candidate tumor suppressor genes from these regions are, respectively, MCC and/or APC, p53, and DCC. In order to further our understanding of the molecular and genetic mechanisms involved in tumor progression and, thereby, of normal cell growth, it is important to determine whether defects in one or more of these loci contribute functionally in the progression to malignancy in colorectal cancer and whether correction of any of these defects restores normal growth control in vitro and in vivo. To address this question, we have utilized the technique of microcell-mediated chromosome transfer to introduce normal human chromosomes 5, 17, and 18 individually into recipient colorectal cancer cells. Additionally, chromosome 15 was introduced into SW480 cells as an irrelevant control chromosome. While the introduction of chromosome 17 into the tumorigenic colorectal cell line SW480 yielded no viable clones, cell lines were established after the introduction of chromosomes 15, 5, and 18. Hybrids containing chromosome 18 are morphologically similar to the parental line, whereas those containing chromosome 5 are morphologically distinct from the parental cell line, being small, polygonal, and tightly packed. SW480-chromosome 5 hybrids are strongly suppressed for tumorigenicity, while SW480-chromosome 18 hybrids produce slowly growing tumors in some of the animals injected. Hybrids containing the introduced chromosome 18 but was significantly reduced in several of the tumor reconstitute cell lines. Introduction of chromosome 5 had little to no effect on responsiveness, whereas transfer ot chromosome 18 restored responsiveness to some degree. Our findings indicate that while multiple defects in tumor suppressor genes seem to be required for progression to the malignant state in colorectal cancer, correction of only a single defect can have significant effects in vivo and/or in vitro.     

The many faces of the tumor suppressor gene APC

Inactivation of the tumor suppressor adenomatous polyposis coli (APC) protein is a critical early step in the development of familial and sporadic colon cancer. Close examination of the function of APC has shown that it is a multifunctional protein involved in a wide variety of processes, including regulation of cell proliferation, cell migration, cell adhesion, cytoskeletal reorganization, and chromosomal stability. Tantalizing clues to the different functions of APC have been provided by the identification of proteins interacting with several discrete motifs within APC. Each of these putative functions could link APC inactivation with tumorigenesis. Here, we will summarize recent findings regarding the diverse role of APC. We will emphasize the interaction of APC with different binding partners, the role of these complex interactions for normal functioning of the cell, and how disruption of these interactions may play a role in tumor development. The rapid progress made recently shows the many faces of APC, leading to a constant reappreciation of this multitasking tumor suppressor protein.     

miRNA profiling in colorectal cancer highlights miR-1 involvement in MET-dependent proliferation

Altered expression of miRNAs is associated with development and progression of various human cancers by regulating the translation of oncogenes and tumor suppressor genes. In colorectal cancer, these regulators complement the Vogelstein multistep model of pathogenesis and have the potential of becoming a novel class of tumor biomarkers and therapeutic targets. Using quantitative real-time PCR, we measured the expression of 621 mature miRNAs in 40 colorectal cancers and their paired normal tissues and identified 23 significantly deregulated miRNAs. We subsequently evaluated their association with clinical characteristics of the samples and presence of alterations in the molecular markers of colorectal cancer progression. Expression levels of miR-31 were correlated with CA19-9 and miR-18a, miR-21, and miR-31 were associated with mutations in APC gene. To investigate the downstream regulation of the differentially expressed miRNAs identified, we integrated putative mRNA target predictions with the results of a meta-analysis of seven public gene expression datasets of normal and tumor samples of colorectal cancer patients. Many of the colorectal cancer deregulated miRNAs computationally mapped to targets involved in pathways related to progression. Here one promising candidate pair (miR-1 and MET) was studied and functionally validated. We show that miR-1 can have a tumor suppressor function in colorectal cancer by directly downregulating MET oncogene both at RNA and protein level and that reexpression of miR-1 leads to MET-driven reduction of cell proliferation and motility, identifying the miR-1 downmodulation as one of the events that could enhance colorectal cancer progression.     

New perspectives on APC control of cell fate and proliferation in colorectal cancer

Aberrant Wnt/β-catenin signaling following loss of the tumor suppressor adenomatous polyposis coli (APC) is thought to initiate colon adenoma formation. Considerable evidence for this model has come from mouse models of Apc truncation where nuclear β-catenin is detectable soon after loss of Apc. However, examination of tumors from familial adenomatous polyposis coli (FAP) patients has failed to confirm the presence of nuclear β-catenin in early lesions following APC loss despite robust staining in later lesions. This observation presents the possibility that colon adenomas arise through a β-catenin-independent function of APC. Additionally, there is a well established role for inflammation and specifically COX-2 and prostaglandin E2 in the progression of colorectal cancer. Here we review the current literature regarding the functions of APC in regulating WNT/β-catenin signaling as well as its control of intestinal cell fate and differentiation. Further, we provide a brief commentary on our current understanding of the role that inflammation plays in colorectal tumorigenesis and how it fits in with APC dysfunction. Though there are currently contrasting models to explain colon tumorigenesis, our goal is to begin to reconcile data from multiple different model systems and provide a functional view into the initiation and progression of colon cancer.     

Mutations in the APC tumour suppressor gene cause chromosomal instability

Two forms of genetic instability have been described in colorectal cancer: microsatellite instability and chromosomal instability. Microsatellite instability results from mutations in mismatch repair genes; chromosomal instability is the hallmark of many colorectal cancers, although it is not completely understood at the molecular level. As truncations of the Adenomatous Polyposis Coli (APC) gene are found in most colorectal tumours, we thought that mutations in APC might be responsible for chromosomal instability. To test this hypothesis, we examined mouse embryonic stem (ES) cells homozygous for Min (multiple intestinal neoplasia) or Apc1638T alleles. Here we show that Apc mutant ES cells display extensive chromosome and spindle aberrations, providing genetic evidence for a role of APC in chromosome segregation. Consistent with this, APC accumulates at the kinetochore during mitosis. Apc mutant cells form mitotic spindles with an abundance of microtubules that inefficiently connect with kinetochores. This phenotype is recapitulated by the induced expression of a 253-amino-acid carboxy-terminal fragment of APC in microsatellite unstable colorectal cancer cells. We conclude that loss of APC sequences that lie C-terminal to the beta-catenin regulatory domain contributes to chromosomal instability in colorectal cancer.     

Mutation-selection networks of cancer initiation: tumor suppressor genes and chromosomal instability

In this paper, we derive analytic solutions of stochastic mutation-selection networks that describe early events of cancer formation. A main assumption is that cancer is initiated in tissue compartments, where only a relatively small number of cells are at risk of mutating into cells that escape from homeostatic regulation. In this case, the evolutionary dynamics can be approximated by a low-dimensional stochastic process with a linear Kolmogorov forward equation that can be solved analytically. Most of the time, the cell population is homogeneous with respect to relevant mutations. Occasionally, such homogeneous states are connected by 'stochastic tunnels'. We give a precise analysis of the existence of tunnels and calculate the rate of tunneling. Finally, we calculate the conditions for chromosomal instability (CIN) to precede inactivation of the first tumor suppressor gene. In this case, CIN is an early event and a driving force of cancer progression. The techniques developed in this paper can be used to study arbitrarily complex mutation-selection networks of the somatic evolution of cancer.     

Chromosomal instability and human cancer

Genetic instability is a defining feature of human cancer. The main type of genetic instability, chromosomal instability (CIN), enhances the rate of gross chromosomal changes during cell division. CIN is brought about by mutations of CIN genes, i.e. genes that are involved in maintaining the genomic integrity of the cell. A major question in cancer genetics is whether genetic instability is a cause and hence a driving force of tumorigenesis. A mathematical framework for studying the somatic evolution of cancer sheds light onto the causal relations between CIN and human cancer.     

The Wnt connection to tumorigenesis

Wnt signaling has been identified as one of the key signaling pathways in cancer, regulating cell growth, motility and differentiation. Because of its widespread activation in diverse human tumor diseases, the Wnt pathway has gained considerable and growing interest in tumor research over recent years. Evidence that altered Wnt signaling is important for human tumor development came from three major findings: (i) the tumor suppressor adenomatous polyposis coli (APC) binds to the Wnt pathway component beta-catenin and is involved in its degradation, (ii) mutations of APC in colon tumors lead to stabilization of the beta-catenin protein and (iii) tumor-associated mutations of beta-catenin in colorectal cancer as well as in other tumor types lead to its stabilisation, qualifying beta-catenin as a proto-oncogene. Here we will describe the biochemical interactions which shape the Wnt pathway and focus on its role in tumorigenesis.     

SNP-Array genotyping and spectral karyotyping reveal uniparental disomy as early mutational event in MSS- and MSI-colorectal cancer cell lines

In this study nine colorectal cancer cell lines were analysed by 10K SNP-arrays and spectral karyotyping (SKY). Complex chromosomal alterations and breakpoints of deleted or translocated fragments found by SKY could further be characterized by SNP-array analysis. Interestingly many monoallelic regions identified by SNP-array analysis display no copy number alterations, representing uniparental disomy (UPD). It was demonstrated that UPD seems to be involved in activation of early-acting tumor suppressor genes in MSS- (APC, CDKN2A) and MSI- (MLH1, MSH2, APC, CDKN2A) colorectal cancer cell lines. Genes involved later on in the adenoma-carcinoma sequence (i.e. TP53/SMAD4) were not found to be inactivated by UPD. Furthermore, identified amplified monoallelic regions may include oncogenes activated by allele-specific-amplification (i.e. Cyclin D1). However, at present, the majority of the monoallelic regions located in the present study have not yet been associated with known tumor suppressor genes and oncogenes. Further studies are warranted to identify relevant genes in the respective regions and to further verify the results presented here.     

The impact of p53 and p73 on aneuploidy and cancer

Initiation, progression and evasion are sequential steps in cancer formation, with autonomous cell proliferation as a final outcome. Genetic or epigenetic alterations of key regulatory genes of the cell cycle are frequently associated with these phenomena. Recently, chromosomal instability, a long-supposed driving force of tumorigenesis, was associated with dysregulation of mitotic genes, providing advantages to tumor cells. Numerous molecules thus provide a key link in the chain of relationships between chromosomal instability and cancer. Here, we discuss emerging evidence revealing that two p53 family members, p53 and p73, might be key regulatory genes at the heart of the relationship between chromosomal instability and cancer. We argue that the role of members of the p53 family as tumor suppressor proteins, their impact on the control of cellular ploidy, and their newly emerging connection with mitotic checkpoint regulatory genes support the suggestion that p73 and p53 could be two of the missing links among chromosomal instability, the mitotic checkpoint and cancer.     

Progressing toward a molecular description of colorectal cancer development.

During the past decade, the powerful techniques of molecular biology and genetics have been applied to the study of human cancer. Colorectal tumors have proved to be an excellent system in which to search for and study the mutations involved in the development and progression of a common human cancer because of their natural history and several inherited syndromes that strongly predispose to colorectal cancer. A current view is that colorectal cancer results, at least in part, from the accumulation of multiple mutations in oncogenes and tumor suppressor genes in an affected cell. Although the genetic alterations often occur in a preferred sequence, the total accumulation of changes, rather than their order with respect to one another, appears to be a critical determinant of the biological properties of the tumor cell. Study of the inherited and somatic mutations in colorectal tumor cells may provide insights not only into the pathogenesis of cancer, but may also lead to future understanding of the mechanisms of the origin of mutations and the role of environmental and dietary factors in colorectal tumor development.