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Chromosomal instability is a major pathway of sporadic colon carcinogenesis. Chromosome arm 1p appears to be one of the “hot spots” in the non-neoplastic mucosa that, when deleted, is associated with the initiation of carcinogenesis. Chromosome arm 1p contains genes associated with DNA repair, spindle checkpoint function, apoptosis, multiple microRNAs, the Wnt signaling pathway, tumor suppression, antioxidant activities, and defense against environmental toxins. Loss of 1p is dangerous since it would likely contribute to genomic instability leading to tumorigenesis. The 1p deletion-associated colon carcinogenesis pathways are reviewed at the molecular and cellular levels. Sporadic colon cancer is strongly linked to a high-fat/low-vegetable/low-micronutrient, Western-style diet. We also consider how selected dietary-related compounds (eg, excess hydrophobic bile acids, and low levels of folic acid, niacin, plant-derived antioxidants, and other modulatory compounds) might affect processes leading to chromosomal deletions, and to the molecular and cellular pathways specifically altered by chromosome 1p loss.
Chromosomal instability is a major feature of sporadic colon carcinogenesis.1–11 Eighty-five percent of colorectal cancers are aneuploid, the remaining 15% being diploid.5 Chromosome 1p deletions in colon tumors have been reported by laboratories from at least 15 countries around the world.12–49 Chromosome 1p deletions occur at an early stage of colon carcinogenesis,21,24,26–28,30,31,33,37,39,41–45 and are strongly linked to karyotypic evolution during colon cancer development.43
Many reports in the literature indicate that the macroscopically normal mucosa proximal or distal to a colon cancer exhibit aneuploidy (loss or gain of chromosomes or parts of chromosomes). Relevant to this review, Cianciulla et al44 reported that deletions of chromosome 1p were simultaneously found in both the distant normal-appearing mucosa of 76% of patients who also harbored 1p deletions in their cancer. These findings indicate that the loss of chromosome 1p may be one of the “hot spots” among the numerous defects in the non-neoplastic mucosa associated with the possible initiation of colon carcinogenesis.50–70
The pioneering work of Paraskeva et al71–75 indicated the likely involvement of chromosome 1p loss in in vitro immortalization72,74 and in the progression of adenomas to carcinomas.75 The functional importance of loss of distal 1p in colon tumorigenesis was demonstrated in 1993 by Tanaka et al76 who introduced chromosomal band 1p36 into colon carcinoma cells and found that their tumorigenicity was suppressed.
Chromosome 1p deletions can affect distinct pathways of sporadic colon carcinogenesis, including both chromosomal instability and chromosomal instability-negative pathways. The underlying mechanisms associated with the loss of chromosome 1p that may contribute to genomic instabilty and drive colon carcinogenesis are loss of genes associated with DNA repair, spindle checkpoint function, apoptosis, multiple microRNAs (miRNAs), the Wnt signaling pathway, tumor suppression, antioxidant activities, and defense against environmental toxins.77,78 Since centromeric instability and resulting telomeric fusions have been proposed as a mechanism for the loss of chromosome 1p,79 the loss of genes located on chromosome 1p that function to ensure centromeric stability and telomere integrity, in turn, can exacerbate chromosomal instability throughout the genome. These 1p deletion-associated pathways that may lead to colon carcinogenesis will be reviewed at the molecular and cellular levels, and dietary factors that affect these pathways (eg, excess hydrophobic bile acids, and low levels of folic acid, niacin, plant-derived antioxidants, and other modulatory compounds) will be explored. It is likely that certain dietary factors prevent, initiate, or exacerbate genomic instability in colon epithelial cells and thus have importance for colon carcinogenesis.
Chromosome 1, the longest human chromosome, is gene-dense with 3141 genes.80 The genes located on chromosome 1 were identified with the assistance of the Weizmann Institute of Science websites:
GeneLoc (www.genecards.weizmann.ac.il/geneloc/index.shtml) and GeneCards – The Human Gene Compendium (www.genecards.org). Genes located on the p arm of chromosome 1 that are associated with protection against oxidative stress, DNA damage, mitotic perturbations, excessive cellular proliferation, development of apoptosis resistance, aberrant colonic cell differentiation, and environmental toxicity have been tabulated and the function of the gene products described (Tables 1–8). Since many of these genes have tumor suppressive capabilities, the simultaneous loss caused by a 1p deletion could initiate the formation of neoplastic clones and enhance tumorigenesis through Darwinian selection.8
Cells with DNA damage, spindle damage, and dysfunctional telomeres signal DNA damage responses.81–84 These DNA damage responses include the activation of numerous checkpoints that arrest the damaged cells in the G1, S, G2, or M-phase of the cell cycle, depending upon the nature of the damage or dysfunction and the stage of the cell cycle of the target cell. DNA-damage checkpoints are activated following direct damage to DNA.85–91 Spindle assembly checkpoints are activated following damage to the mitotic machinery,85,92–98 or as a result of DNA damage during mitosis.99 Telomere checkpoints are activated by defective telomeres.100–106 These checkpoints prevent the damaged cell from completing DNA replication and mitosis until all damage is repaired (Figure 1), and thus prevent 1) mutations that could be formed by replicating a damaged DNA template, 2) aneuploidy that could result from chromosome mis-segregation, and 3) telomere fusions that result in anaphase bridges, broken chromosomes, and translocations as a consequence of the well-known breakage–fusion–bridge cycles.107–114
However, cells with excessive direct DNA damage,115–122 massive chromosome loss or chromosomal imbalances,123 prolonged activation or inhibition of the spindle checkpoint pathways,122–127 or excessively shortened or dysfunctional telomeres,128–140 initiate a cascade of molecular events that ultimately leads to either caspase-dependent cell death,141–143 caspase-independent cell death,144 or a special form of apoptosis referred to as mitotic catastrophe145–148 (Figure 2). (Brightfield micrographs are shown in Figure 3 illustrating the cellular alterations that accompany apoptosis [Figure 3A], mitotic perturbation [Figure 3B], mitotic catastrophe [Figure 3C], and micronuclei formation [associated with aneuploidy] [Figure 3D]). The cell-destructive and cell-protective pathways are downstream of a common signal transduction network that responds to DNA damage.149 The repair/survival and non-repair/cell death pathways are probably activated simultaneously.149 The repair, checkpoint, and cell death response to DNA damage are, however, well co-ordinated,150 the interplay of positive and negative regulatory loops resulting in a delayed death response to DNA damage.149
The genes on chromosome 1p associated with DNA repair or the DNA damage response (DDR) include CLSN, DCL-RE1B (APOLLO), DDI2, GADD45α, MSH4, MUTYH, RAD54L, and TP73. The functions of these gene products are described in Table 1. The pathways that lead to the prevention of genomic instability are diagrammatically shown in Figure 4. DNA damage elicits a well orchestrated and highly interactive series of events called the DDR, which causes cells to undergo growth arrest so that DNA damage can be adequately repaired. Although p53 mutation or loss of heterozygosity (LOH) is a late event in colon carcinogenesis,151 the loss of p73 (found on chromosome 1p) through chromosomal deletion events may act early in colon carcinogenesis. P73 is an important isoform of the p53 family, since it performs many of the transcriptional functions of p53, and may even target the same genes as p53 during the DDR. In addition, TP73 has distinct transcriptional targets and harmonizes with p53 and p63 to maintain genomic stability.152–158 In addition to its role in growth arrest after DNA damage to allow DNA repair to take place, p73 plays an active role in spindle dynamics, mitotic exit and chromosomal stability. The PSRC1 (proline/serine-rich coiled-coil 1) gene found on chromosome 1p (see Table 2) encodes a protein which is a direct transcriptional target of both p53 and p73.159 PSRC1 functions as a microtubule destabilizing protein that controls spindle dynamics and mitotic progression by recruiting and regulating microtubule depolymerases.160 Through its transcriptional activity, p73 is important for the M-to-G1 transition during mitosis.161 Functional knock-out of p73 gene expression by small interfering RNAs alters mitotic progression, resulting in an increase of ana-telophase cells, the accumulation of aberrant late mitotic figures, and the appearance of abnormalities in the subsequent interphase.161 This novel pathway involves the p73-mediated transcription of Kip2/p57, a cyclin-dependent kinase inhibitor, and the coordination of mitotic exit and transition to G1.161,162 Like p53, p73 has been confirmed to be a tumor suppressor.163–167 Therefore, a loss of p73 should have a major impact in the development of genomic instability during carcinogenesis.
Since base excision repair (BER) removes damage that would otherwise be mutagenic in mammalian cells,168–170 BER is one of the most important DNA repair pathways in the gastrointestinal tract. BER ameliorates environmentally induced DNA damage in addition to the alkylation, oxidation, and deamination events that occur during normal metabolic processes.171,172 A critical enzyme in the base excision repair pathway is MUTYH (MutY homolog or A/G-specific adenine DNA glycosylase), whose germline mutation is a known cause of MAP (MutYH-associated polyposis), a recently described autosomal recessive colorectal adenoma predisposition syndrome with a very high risk of colorectal cancer.173 Myh deficiency enhances intestinal tumorigenesis in multiple intestinal neoplasia (ApcMin/+) mice.174 Interestingly, Myh deficiency in mice has a larger effect on tumor initiation than on progression in the small bowel.174 Since 1p deletions are observed in the human non-neoplastic mucosa of patients with colon cancer,44 it is possible that Myh-deficient field defects may initiate the process of colon carcinogenesis in humans as it does in the mouse model. Since MUTYH-null mouse embryonic stem cells exhibit a mutator phenotype,175 the loss of MUTYH can affect multiple pathways associated with colon carcinogenesis. The role of MUTYH in the repair of oxidative DNA damage begins with the formation of 8-oxo-guanine (8-oxoG) (see Figure 4), which then causes a mispairing of the oxidized guanine base with adenine upon DNA replication. Mismatch repair processes are activated and MUTYH excises adenine leaving an apurinic (AP) site resulting, after AP endonuclease action, in a DNA single strand (ss) break.176–180 The activity of MUTYH, in conjunction with other glycosylases and the spontaneous generation of AP sites, may be quite extensive, since about 9000 AP sites/cell occur daily.168 The AP site is then correctly repaired by the sequential action of several enzymes which catalyze template-directed insertion of one or a few nucleotides at the previously damaged site.172
In addition to their role in DNA repair or the DDR, MUTYH and p73 play important roles in the death of cells that experience either excessive oxidative DNA damage or chromosomal instability. The MUTYH-mediated cell death pathway is described in the next section followed by a section on the p73-mediated cell death pathway, which utilizes part of the MUTYH pathway in its mediation of cell death in response to excessive mitotic perturbation.
MUTYH-mediated cell death has, as a central player, the activation of PARP-1 [poly(ADP-ribose) polymerase-1] (Figure 5). Excessive DNA ss breaks caused by the action of MUTYH and AP endonuclease in the nucleus results in the activation of PARP-1, which attaches polymers of ADP-ribose to proteins, thereby opening up the chromatin to allow access of DNA repair proteins.181,182 PARP initially serves as a survival protein facilitating the rapid repair of DNA strand breaks, and also prevents DNA degradation, in part, by inhibiting the activity of deoxyribonucleases through the process of poly(ADP) ribosylation.183 Since the synthesis of ADP-ribose polymers consumes nicotinamide adenine dinucleotide (NAD+),184 and NAD+ is largely found in mitochondria where it participates in the production of ATP (bottom right side of Figure 5), sustained PARP activation will consume energy reserves, resulting in cell death, usually through the process of necrosis.185–188 A marked deficiency in energy reserves may cause the ATP-dependent Na+/K+ transport proteins, which maintain ionic balance, to fail, resulting in cell swelling and lysis of the cell,189 one of the hallmarks of necrosis.190
In addition to the above energy catastrophe caused by excessive PARP activity in the nucleus, persistent single-stranded gaps in newly replicated DNA initiated by the action of MUTYH in mitochondria can result in the fragmentation and depletion of mitochondrial DNA (mtDNA)191,192 accompanied by the loss of mitochondrial function culminating in cell death191,193 (bottom right side of Figure 5). Dysfunctional mitochondria can release Ca++ into the cytosol which can activate calpains, causing Bax activation, lysosomal rupture, and the release of cathepsins into the cytosol191,194 resulting in a caspase-independent mode of cell death. Calpain activation can also result in Bax activation, followed by Bax oligomerization and mitochondrial damage, resulting in the loss of the mitochondrial membrane potential.
There is another unique mechanism that can lead to PARP-mediated cell death after excessive MUTYH activity, in addition to the fragmentation of mtDNA, energy catastrophe and calpain/lysosomal rupture/cathepsin pathways of mitochondrial failure described above. The main product of PARP-1 activity is the generation of polymers of ADP-ribose (PAR). Although these polymers are usually covalently bound to proteins, free PAR polymers are themselves toxic195–197 and function as a death signal.197–199 The PAR polymers bind to mitochondria and induce the release of tAIF (truncated apoptosis-inducing factor) from the mitochondria into the cytosol199 (lower left side of Figure 5). tAIF is then translocated to the nucleus where it binds to DNA,200–202 causes DNA condensation203 and recruits DNA degrading factors (eg, endogenous endo- and exo-nucleases) resulting in DNA degradation198,204 (upper left side of Figure 5). This series of events is part of an intricate program of caspase-independent cell death,203–213 and is currently an active area of research.
Several mechanisms have been proposed to explain how tAIF is released from the mitochondria into the cytosol.210,214 Prior to truncation, AIF is embedded in the inner mitochondrial membrane,215 and the release of AIF requires its cleavage215,216 from a 62 kDa AIF mitochondrial form to a truncated 57 kDa soluble AIF form (tAIF).217,218 Calpain-I, which is activated by Ca++,219 and Ca++-independent cathepsins B, L, and S218,220 can cleave intramitochondrial AIF.221–223 The calpains and cathepsins can truncate AIF in the same position at Gly102/Leu103.218 Calpain-I, however, appears to be the critical enzyme regulating AIF processing in which the AIF pathway is important for cell death.219 Oxidative modifcation of AIF markedly increases the susceptibility of AIF to calpain-I-mediated processing, most probably through the exposure of a normally hidden calpain cleavage site.219 Since the PAR polymer is a highly negatively charged molecule, it could depolarize mitochondria leading to opening of the mitochondrial membrane permeability transition pore (MPTP) followed by the release of tAIF.197,199 PAR polymers of increasing complexity and molecular weight are more toxic than simple PAR polymers of low molecular weight.197 The PAR polymer could also bind to PAR polymer binding proteins associated with mitochondria, which then release AIF.199,224–226 This results in AIF cleavage producing a tAIF, which is soluble and enters the cytosol. The release of tAIF may also be caused by a significant but not excessive decrease in NAD+ (as a result of PARP activity), ATP, and the mitochondrial membrane potential, resulting in the opening of the MPTP (mitochondrial permeability transition pore).186,196,211 The release of tAIF may also be caused by other caspase-independent pathways involving molecules that are often found in the downstream execution phase of apoptosis, such as tBid (truncated Bid),227–229 Bax oligomers (formed after activation of Bax by Ca++-dependent calpains),211,217 Bak,230 and Bim-EL.231,232 The activation of PARP also activates other stress-response pathways such as the RIP/TRAF2/JNK pathway,233–235 which may be responsible, in part, for generation of tBid228 and the phosphorylation of Bim-EL. The phosphorylation of Bim-EL releases Bim-EL from sequestration by the microtubular dynein motor complex,236 allowing it to bind to bcl-2,231 thereby enhancing the cell death process.
Mechanisms that interfere with tAIF release include the 1) degradation of the PAR polymer by PARG (PAR glycohydrolase),237 2) inhibition of tAIF translocation to the nucleus by Bcl-2, Bcl-xl, HSP70, or Iduna, and 3) interference of transcription of the AIF gene by BNIP3.238 PARG, Bcl-2, Bcl-xl, HSP70, Iduna, and BNIP3 have been shown to be upregulated during carcinogenesis, consistent with the development of tumor cell resistance to cell death. In addition, pro-cell death molecules involved in this MUTYH/PARP/AIF pathway, such as AIF, Bid, Bax, Bak, and Bim-EL, have been reported to be downregulated during carcinogenesis. Thus, overall, MUTYH likely has an important role in the death of cells exposed to excessive reactive oxygen species/reactive nitrogen species (ROS/RNS)-induced DNA damage, and interference with the MUTYH cell death pathway is associated with carcinogenesis.
Like p53, p73 is responsible for the induction of apoptosis in response to excessive DNA damage that cannot be repaired.239 P73 has the ability to upregulate the transcription of numerous classic apoptosis-related genes such as caspases 3, 6, and 8, Bcl-2 family members, and death receptors (Figure 6). In order for p73 to function as a transcription factor, it must be phosphorylated. The c-Abl kinase, activated by DNA damage, phosphorylates and activates p73 on tyrosine 99.240 The stress-induced mitogen-activated protein kinase, p38 MAPK, phosphorylates and activates p73 on threonine residues.239 The degradation of p73 by the E3 ubiquitin-like protein, Itch, is prevented by the Yes-associated protein, YAP. E2F1, p53, and c-jun (located on chromosome 1p; Figures 4 and and6)6) may also have a role in p73 activation in different cell types.241,242 One mechanism by which p73 induces apoptosis includes the transcription of PUMA (p53 upregulated modulator of apoptosis), which in turn causes Bax translocation to the mitochondria with the release of cytochrome c.243 A second mechanism involves the transcription of scotin, which causes endoplasmic reticulum (ER) stress and subsequent apoptosis.244,245 Unlike p53, a direct role of p73 in the apoptotic process (eg, mitochondrial translocation and perturbation) has not been verified. The role of p73 in the regulation of the miRNA processing complex will be discussed in the section “MiRNAs and miRNA processing”. As noted above, loss of p73 through chromosome 1p deletion occurs early in colon carcinogenesis, contrary to the loss of p53 which is a late event.
There are 24 genes on chromosome 1p whose gene products affect many different aspects of the mitotic process, and include kinases, phosphatases, centromere proteins, centrosome proteins, cyclins, regulatory mitotic proteins, motor spindle proteins, regulators of chromosomal condensation, a mitosis-related transcription factor, a deacetylase, and a major spindle checkpoint protein (Table 2). The large number of mitosis-related genes that are lost if there is a chromosome 1p deletion could potentially be responsible for colon cancer initiation and progression, since cancer epidemiology studies show that abnormal expression of mitosis-related genes is frequent in different tumor types.246,247 Mitotic checkpoints, and specifically the spindle assembly checkpoint, are major targets for tumor-associated alterations.247 The mitotic spindle assembly checkpoint is essential for ensuring that all chromosomes are properly aligned on the metaphase plate, with every chromosome attached to a spindle microtubule by its kinetochore to prevent aneuploidy.97 If these processes fail to occur and the cell undergoes a prolonged mitotic arrest (Figure 2), the cell may be eliminated through caspase-dependent or caspase-independent cell death mechanisms147 to ensure genomic stability (Figure 7).
Oxidative stress is a major factor that can induce disturbances in spindle organization,248,249 induce centrosome amplification, cause proteolysis of the anaphase inhibitor securin and mitotic cyclins,250 affect components of the anaphase-promoting complex,251 and override the spindle checkpoint,250 thereby affecting chromosomal stability. During the process of mitosis, direct oxidative damage to chromosomes resulting in double-strand breaks, or oxidative damage to telomeres can activate p53 (Figure 7) or p73 (Figure 6), major DNA damage response proteins that elicit apoptosis through multiple caspase-dependent mechanisms. In addition, caspase-independent mitotic cell death can also occur during a mitotic catastrophe (Figure 3C, Figure 7), which is a prestage to distinct modes of cell death that may be caspase-dependent or caspase-independent.148
The length of time that a spindle is destabilized may determine the mode and timing of cell death after mitotic exit.123,124,126 It has been suggested that prolonged mitotic delay can lead to the decay of anti-apoptotic messenger RNAs (mRNAs)252,253 and/or the gradual accumulation of pro-apoptotic signals.252,254 Of the 24 mitosis-related genes (Table 2), the products of 7 genes have dual-role mitosis/pro-apoptotic functions. These dual-role mitosis/pro-apoptotic genes include APITD1, CCNL2, CDC2L2, CDC42, E2F2, KIF1B, and PLK3 (Table 2). Cells may become genomically unstable if they evade mitotic checkpoints through a process referred to as mitotic slippage, mitotic arrest slippage, or mitotic checkpoint slippage255–263 (Figure 7). With mitotic slippage, the cell exits mitosis prematurely, carrying broken chromosomes, abnormal numbers of chromosomes, and unrepaired DNA damage into the daughter cells. In addition to loss of pro-apoptotic proteins, it has been reported that the gradual loss of the checkpoint effector, cyclin B, releases the mitotic arrest induced by spindle disruptive agents, despite the continued presence of spindle damage and upstream checkpoint proteins.14,258,260 In order for a DNA-damaged cell to survive after mitotic slippage, it must evade both apoptosis in the subsequent G1 phase of the cell cycle124 (Figure 7) and reproductive cell death that can follow centrosome amplification and the generation of tetraploid cells264 (Figure 7).
Thus, a decrease in pro-apoptotic mitotic/cell cycle-related genes located on chromosome 1p (APITD1, CCNL2, CDC2L2, CDC42, E2F2, KIF1B, PLK3) (Table 2) may result in resistance to cell death, a critical event that drives tumorigenesis.52,54,265–267
Seven genes associated with apoptosis are located on chromosome 1p. Bcl-10 and Bcl2L15 are Bcl-2 family members, THAP3 is a zinc-coordinating DNA-binding protein, DNA fragmentation factor A (DFFA) and B (DFFB) are the two subunits of DFF, caspase-9 is a major initiator caspase in the apoptotic proteolytic cascade, and TNFRSF25 is a death domain-containing receptor related to TNFR-1 and CD95 (Apo-1/Fas). The deletion of 3 of these genes would have important implications for carcinogenesis through the increase in apoptosis resistance, and will be discussed in some detail.
DFF is a heterodimeric protein composed of a catalytically active 40 kD subunit, DFFB (CAD [caspase-activated DNase]), and an inhibitory 45 kD subunit, DFFA (ICAD [inhibitor of CAD]).268,269 When bound to DFFB, DFFA inhibits the nuclease activity of DFFB.268,269 During apoptosis, caspase-3 cleaves DFFA at amino acids 117 and 224 and dissociates it from DFFB, thereby releasing the inhibition of DFFB.270 DFFB activity results in chromatin condensation271 and the formation of the typical crescents and margination of chromatin that are characteristic of classic apoptotic cells at the ultrastructural level.190,266,272–276 Characteristic ultrastructural features of apoptotic cells treated with a ROS-generating and DNA-damaging agent are shown in Figure 8. At the molecular level, the action of DFF on DNA results in the initial cleavage of DNA into 50- to 300-kb long fragments,277,278 representing the dismemberment of the higher order organization of chromatin into chromosomal loop domains, and the fragmentation of DNA into oligonucleosomal sized fragments that form a “ladder” on agarose gel electrophoresis.279 The importance of DFF in suppressing tumorigenesis280 was demonstrated by Yan et al281 using DFF40-null mice. DFF-deficient cells exhibit significant increases in mutation, chromosomal instability, and survival compared with wild-type control cells.281 This is probably a result of the inhibition of cell death of DNA-damaged cells resulting from the failure to undergo DNA fragmentation.282,283 DFF is reported to avoid chromosome instability in a p53-independent manner.284 Irradiation of cells with a caspase-resistant form of DFFA led to increased clonogenic survival of cells with increased chromosomal aberrations and aneuploidy.284 The ability of DFF to maintain chromsosomal stability appears to be the result of the DNA fragmentation-induced death of cells with excessive DNA damage.284 Although DFFB has intrinsic DNAse activity, both DFFA and DFFB are required to generate DNase activity,140,269 and must be co-expressed.280 DFFA has been postulated to stabilize the synthesis of DFFB,270,271 or mediate the correct folding and chromatin localization of DFFB.271 The absence of DFF results in an increased frequency of cell transformation and enhanced susceptibility to radiation-induced carcinogenesis, indicating that DFF is a tumor suppressor.280 Recently, it has been reported that the expression of DFFA protein, but not DFFA mRNA, is regulated by a specific miRNA, miR-145, suggesting a mechanism of translational regulation.285 The regulation of DFFB by miRNA has not been investigated, and, so far, none of the miRNAs found on chromosome 1p (Table 4) have been determined to have DFFA or DFFB as target mRNAs for translational regulation.
Caspase-9 is a member of the family of cysteine-aspartic acid-specific proteases (caspases), and is also referred to as Apaf-3 (apoptotic protease-activating factor 3). In the presence of cytochrome c and dATP, Apaf-1 binds to procaspase-9286 via a CARD (caspase activation recruitment domain),287 forming a complex referred to as the apoptosome.286,288,289 The cellular oxidative state can affect apoptosome formation by promoting an interaction between caspase-9 and Apaf-1 via disulfide formation.290 In the apoptosome, caspase-9 is activated to process other downstream caspases, including caspase-3 and caspase-2.291 Caspase-9 plays an important role in apoptosis induced by genotoxic stress.292,293 The caspase-9-induced apoptotic pathway can result from mitochondrial membrane depolarization, formation of the apoptosome, and the activation of multiple caspases, including caspase-3 and caspase-2.294 Loss of caspase-9 is therefore important to carcinogenesis, since it can result in apoptosis resistance and the propagation of DNA-damaged cells.295 If caspase-9 is lost, caspase-3 cannot be activated, and thus cannot cleave many substrates including DFFA, an essential endonuclease in apoptosis (see previous page). Similarly, if caspase-9 is lost, caspase-2 may not be activated. Caspase-2 plays a specific role in genotoxic stress-induced apoptosis in some cell types.296,297 (However, there is another pathway for activation of caspase-2. Activation of p53 by DNA damage can result in the p53-mediated transcription of the death domain protein PIDD [p53-induced protein with a death domain], which, together with RAIDD or RIP1, can form a multiprotein complex called the PIDDosome298–300 which then activates caspase-2298). DNA damage can also activate caspase-2 through the activation of c-Abl.301 C-Abl binds directly to caspase-9, phosphorylates it on Tyr-153, which then results in the autocleavage and activation of caspase-9 resulting in the apoptosis of excessively DNA-damaged cells.301 Caspase-9 also mediates apoptosis caused by ER stress.302 ER stress first activates caspase-12,302 which is located on the outer membrane of the ER;303 caspase 12 then activates caspase-9 through a cytochrome c-independent mechanism.302 In some cells, ER stress can result in caspase-8 activation, formation of tBid, mitochondrial damage, release of cytochrome c and the activation of caspase-9 through the formation of the apoptosome.304 Therefore, ER stress can activate caspase-9 through both mitochondrial-independent and -dependent mechanisms.
miRNAs are evolutionarily conserved, endogenous, small (21 to 24 nucleotides) non-coding RNAs cleaved from 70 to 100 nucleotide hairpin-shaped precursors that reduce translation and stability of target mRNAs through RISC (RNA interference effector complex)-mediated mRNA degradation and translational suppression via sequence-recognition interactions with the 3′ untranslated region of their targeted mRNAs.305–315 The diverse cellular functions affected by miRNAs306,316,317 is underscored by the prediction that thousands of genes are potential miRNA targets.318–320 At least 800 different miRNAs predicted by computational scanning in the human genome have been documented (http://microrna.sanger.ac.uk). Individual miRNAs have the potential to downregulate large numbers of target mRNAs with seed region complementary sites in their 3′ untranslated regions.321–323 It has been speculated that miRNAs could regulate ~30% of the human genome.306 MiRNAs function in proliferation, cell cycle control, the prevention of replicative stress, differentiation, and apoptosis.324–333 More than half of the known human miRNAs are located at fragile sites, as well as at sites of LOH, amplification, and common breakpoint regions, which are particular genomic regions that are prone to alteration in cancer cells.327 The overexpression or underexpression of miRNAs as a result of chromosomal additions or deletions, respectively, in individual cells can have dramatic effect on hundreds to thousands of target genes. It is, therefore, not surprising that aberrant expression of miRNAs is associated with cancerous tissues,334–340 and that characteristic miRNA expression profiles are features of certain human cancers.341–350 Impaired miRNA processing enhances cellular transformation and tumorigenesis,351,352 and certain miRNAs are even classified as tumor suppressors and oncogenes.353–355 Alterations in a series of specific miRNAs have been associated with the age of onset of colon cancer, the growth of colon cancer cells, and certain stages of colon carcinogenesis.344,356–369 Human colon cancer profiles from 80 colon tumors and 28 samples of normal mucosa show differential miRNA expression depending on mismatch repair status and are characteristic of undifferentiated proliferative states.367 Examination of the genomic regions containing differentially expressed miRNAs revealed that they were also differentially methylated in colon cancer at a far greater rate than would be expected by chance.367 MiRNA profiles could accurately predict microsatellite status in a set of 39 colon cancer studied by Lanza and colleagues.370 This is probably a reflection of the presence or near absence of chromosomal instabilty in the respective microsatellite stable vs unstable cancers.371
There are 20 miRNAs and 3 components of the miRNA processing complex (Argonaute proteins 1,3,4) encoded on chromosome 1p (Table 4). One of the 20 miRNAs, miR-34a, is known to be regulated by p53.309,330,372–376 Tarasov et al375 evaluated the differential regulation of 74 miRNAs by p53; 50 miRNAs were either positively or negatively regulated by p53, miR-34a showing the highest fold increase (33.4 fold). Although the 20 miRNAs found on chromosome 1p can have pleiotropic effects on cells, miR-34a is the most well studied for its role in cell cycle arrest and apoptosis in response to DNA damage.309,330,374,377,378 The miR-34 family of miRNAs is one of only 18 mammalian miRNA families379 that are present in flies and worms.309 It is probable that links between p53 and the miRNA-34 family may have arisen early in the evolution of the stress-related p53 network.309 Because of its central role in preventing carcinogenesis, miR-34a has been classified as a tumor suppressor.372,377 MiR-34a has numerous downstream targets, including bcl-2 (major anti-apoptotic protein), NOTCH1, Delta1 (ligand for NOTCH1), NOTCH2 (found on chromosome 1p), CDK4, CDK6, Cyclin D1, Cyclin E2, c-Met, MYCN, SIRT1 and E2F3.319,362,374,375,377,380–384 The inhibition of NOTCH1 by miR-34a would enhance apoptosis since NOTCH1 is known to inhibit p53 activity385,386 and to have an anti-apoptotic role387,388 in tumorigenesis. The inhibition of SIRT1 by miR-34a contributes to p53-dependent apoptosis389 through deacetylating and stabilizing p53 leading to an increase in p21 and PUMA.384 The E2F3 transcription factor is not known to have a role in apoptosis; however, it is a novel repressor of the ARF/p53 pathway390 and a potent transcriptional inducer of cell-cycle progression.377 Therefore, the downregulation of E2F3 by miRNA-34a would have a growth inhibitory effect.362,374 MYCN has important roles in both cell proliferation and apoptosis, and MYCN amplification is almost always associated with the loss of chromosome 1p36.382 It is probable that the effects of miR-34a on cellular molecular pathways is widespread, since enforced expression of 34A shows a dramatically altered gene expression profile with upregulation of 532 mRNA transcripts and downregulation of 681 mRNA transcripts highly enriched for those genes that regulate cell-cycle progression, apoptosis (BCL2, BIRC3 [baculoviral IAP repeat-containing 3], DcR3 [decoy receptor 3]), DNA repair, and angiogenesis.330 In conclusion, although p53 is a late event in colon carcinogenesis, the deletion of a major downstream target of p53, miR-34a, as a result of chromosomal 1p deletion, could have dramatic effects on colon tumorigenesis.
MiR-101 is a miRNA that, like 34a, is pro-apoptotic391 and considered to be a tumor suppressor.391,392 The nomenclature of miR-101-1 (Table 4) and miR-101-2 is based on the fact that miR-101-1 is produced from a genomic locus on chromosome 1p31 and miR-101-2 from a genomic locus on chromosome 9p24.392 Loss of heterozygosity at both 1p and 9p are known to be associated with cancer.392 The mechanism by which miR-101 induces apoptosis is by targeting and decreasing the expression of the multifaceted anti-apoptotic protein Mcl-1 (myeloid cell leukemia sequence 1).391 Mcl-1 undergoes rapid turnover which may serve as a convergence point for signals that affect global translation, thereby coupling translation to cell survival and the apoptotic machinery.393 (The DNA damage response can also result in Mcl-1 destruction and the initiation of apoptosis.394,395) Mcl-1 specifically inhibits apoptosis, in part, by sequestering the pro-apoptotic Bim, Bak, tBid, and Noxa, in an inactive state. Since Mcl-1 can interact with tBid and inhibit its induction of cytochrome c release, it plays an important role in resistance to TRAIL and TNFα-induced apoptosis.396,397 Therefore, Mcl-1 can inhibit apoptosis induced by both the death receptor (extrinsic) and mitochondrial (intrinsic) pathways. Mcl-1 is targeted for proteasome-mediated degradation by the E3 ubiquitin ligase MULE398 and is rapidly degraded with a half-life of 30 minutes to 3 hours.393 Its short half-life relates to the presence of a long proline-, glutamic acid-, serine-, and threonine-rich (PEST) region upstream of the Bcl-2 homology domains.398 The inhibition of translation with cycloheximide can cause the rapid degradation of Mcl-1 within 30 minutes, thereby triggering the apoptotic machinery through the release of Bim and the activation of Bak and Bax.393 Although full-length Mcl-1 does not interact with Bax, the caspase-mediated cleavage of Mcl-1 at Asp127 generates a fragment that induces apoptosis through direct interaction with Bax.399 Phosphorylation of Mcl-1 can affect its function and degradation.400 The phosphorylation of Mcl-1 is prominent in cells that accumulate in the G2/M phase of the cell cycle as a result of exposure to microtubule disrupting agents, and in synchronized cells passing through this phase.401 This phosphorylation, especially at serine 64, enhances the anti-apoptotic function of Mcl-1,400 thereby allowing cells to properly align their chromosomes prior to anaphase. In colorectal mucosa, the Mcl-1 protein is found in the apical cells of the crypt,402,403 whereas the distribution is more diffuse in the malignant cells.403
In addition to the development of apoptosis resistance, the loss of miR-101 also leads to cancer progression through the overexpression of histone methytransferase EZH2 (enhancer of zeste homolog 2), a polycomb group member, with concomitant dysregulation of epigenetic pathways.392,404 MiR-101 also represses the expression of FOS (v-fos FBJ murine osteosarcoma viral oncogene homolog) oncogene, a key component of the AP-1 (activator protein-1) transcription factor, MYCN (a gene amplified in many tumors), and COX-2, an enzyme involved in the production of prostaglandins from the metabolism of arachidonic acid.405 Enhanced expression of miRNA-101 also has an effect on the late stages of cancer, since it inhibits invasion and migration.
The p53/p63/p73 family of tumor suppressors are known to regulate the major components of the miRNA processing complex,164,406 which include Drosha-DGCR8, Dicer-TRBP2, and Argonaute proteins. Drosha (RNASEN) is an RNAse III endonuclease; DGCR8 is a double stranded RNA binding protein; DICER contains an RNA helicase motif required for the formation of RISC (RNA induced silencing complex); TRBP2 (trans-activation-responsive RNA binding protein 2) is a component of the miRNA loading complex (composed of DICER1, AGO2, and TRBP2) required for the formation of RISC. Argonaute proteins are endonucleases that aid in the maturation of pre-miRNAs of 60 to 70 nucleotides to mature miRNAs of 21 to 24 nucleotides; the tethering to mRNA mimics the miRNA-mediated repression of protein synthesis.164,407,408 There are 8 members of the Argonaute family in the human genome;409 4 belong to the PIWI subfamily and are expressed mainly in the testis, whereas the other 4 belong to the elF2C/AGO subfamily and are expressed in a variety of adult tissues. Ago1 and Ago2 (catalytic engine of RISC) reside in 3 complexes with distinct DICER and RNA-induced proteins involved in RNA metabolism.410 Three of the 4 members of the elF2C/AGO subfamily are found in a tandem cluster of closely related Argonaute non-nucleolytic proteins,411 Ago1, Ag3, and Ago4 on chromosome 1p (Table 4). Therefore, loss of chromosome 1p should have a major impact on the process of miRNA processing in the affected cells.
A family of miRNAs on chromosome 1p of particular interest to colon carcinogenesis is the miR-200 family, which includes miR-200a, -200b, and -429 (Table 4). These 3 family members are all encoded on a 7.5-kb polycistronic primary miRNA transcript and help determine the epithelial phenotype of cancer cells through the regulation of the Wnt/β-catenin signaling pathway.412,413 Wnt growth factors activate a cascade of intracellular events, known as the canonical Wnt pathway, which ultimately leads to a coordinated proliferation, differentiation, and sorting of the epithelial cell population that forms the colonic crypts.414 In colorectal cancer, epithelial cells that acquire mutations in the Wnt/β-catenin signaling pathway gain inappropriate proliferative capabilities mimicking the effect of a permanent Wnt stimulation.414 Beta-catenin is a transcription factor that translocates to the nucleus and activates target genes involved in stimulation of the cell cycle and inhibition of apoptosis. E-cadherin binds directly to β-catenin in the cytoplasm, which restricts the movement of β-catenin to the nucleus. ZEB1 and ZEB2 are proteins that repress the transcription of E-cadherin. Members of the miR-200 family were found to directly target the mRNA of ZEB1 and ZEB2,412,415–418 upregulate E-cadherin expression in cancer cell lines, and reduce cellular motility.412 Conversely, downregulation of one miR-200 family member that was tested, miR-200a, was shown to promote tumor growth by reducing E-cadherin and activating the Wnt/β-catenin signaling pathway.413 Cancer progression has some similarities with embryonic development and wound healing, in which a process of epithelial-to-mesenchymal transition (EMT) occurs.419 Although the EMT normally occurs as a process of stem cell differentiation, the EMT that occurs during carcinogenesis involves a change from a differentiated tumor to a more invasive dedifferentiated tumor.412,419,420
The loss of the miR-200 family of miRNAs, coupled with the loss of 4 proteins associated with the Wnt/β-catenin signaling pathway (Table 5 below), and the loss of the pro-apoptotic miR-34a and the miRNA transcriptional protein, p73, should have a significant impact on the initiation and progression of colon cancer.
The Wnt signaling pathway is critical for the differentiation and sorting of the epithelial cell population necessary for the organization of the colonic crypts and for the regulation of crypt cell renewal and homeostasis.414,421 Wnt signaling is initiated by the binding of extracellular Wnt factors to receptors on the cell surface, which triggers a signaling cascade that leads to the accumulation of β-catenin.414,422 In the absence of Wnt signals, β-catenin is degraded by a multicomplex complex composed, in part, of APC (adenomatous polyposis coli), GSK3β (glycogen synthase kinase-3-beta), and the scaffold proteins Axin1 and Axin2/conductin,423–425 forming the β-catenin destruction box. This destruction box is responsible for the GSK3β-mediated phosphorylation of β-catenin and its subsequent degradation by the ubiquitin-proteasome pathway. The Wnt signals block this phosphorylation and degradation, resulting in the accumulation of β-catenin. Cytoplasmic β-catenin accumulation and translocation to the nucleus allows β-catenin to associate with TCF/LEF (T cell factor/lymphocyte enhancer factor) transcription factors which target genes that enhance cell survival and proliferation (ie, c-myc, cyclin D1).426–428 Mutations in APC, β-catenin, Axin1, or ICAT (inhibitor of beta-catenin and Tcf-interacting protein) result in the deregulated accumulation of β-catenin and the constitutive activation of Wnt signaling,429–431 a major cause of cancer, including colorectal cancer.418,424,425,432
There are 4 genes located on chromosome 1p that are directly involved in the Wnt signaling pathway (CTNNBIP1, DVL1, WNT2B, and WNT4) (Table 5). WNT2B and WNT4 are secreted signaling factors and Dvl1 is a cytoplasmic molecule that associates with Frat-1 to activate the Wnt signaling pathway. The loss of these positive regulators of the Wnt signaling pathway as a result of a chromosomal 1p deletion may contribute to the dysregulation of crypt organization that could initiate the carcinogenic process.433 CTNNBIP1/ICAT (Table 5), on the other hand, is a negative protein regulator of the Wnt signaling pathway. ICAT disrupts β-catenin–TCF interactions,434–436 thereby downregulating gene expression associated with proliferation and cell survival. The crystallographic structure of ICAT indicates the mechanism by which ICAT interferes with β-catenin function. The NH2-terminal domain of ICAT binds to armadillo repeats 10–12 of β-catenin, whereas the COOH-terminal domain of ICAT binds to the groove formed by armadillo repeats 5–9.435,437 The armadillo repeats 5–9 are crucial for the binding of β-catenin to both TCF and E-cadherin.438 The importance of ICAT in the prevention of carcinogenesis is underscored by the fact that ICAT is a multipotent inhibitor of β-catenin438 by interfering with the binding of β-catenin to TCF, cadherins, and APC, with consequences for transcription, cell adhesion, and cytoskeletal function.438–440 The cytoplasmic and nuclear location of ICAT, using an immunohistochemical approach, is consistent with a broader role for ICAT than previously reported.440
In addition to the effects on transcription and cell adhesion, ICAT can function as a pro-cell death molecule in certain situations. Overexpression of ICAT in colorectal tumor cells results in growth arrest and cell death, and serves to eliminate cells with a constitutively activated Wnt signaling pathway.441 Using flow cytometry, the cell death was evidenced by a sub-G1 peak of the cell cycle, and the forced entry of cells into an illegitimate DNA synthetic phase without having undergone a prior mitosis (enhanced trypan exclusion of >4N cells).441 Transgenic mice expressing ICAT also make activated T cells (dependent on β-catenin–TCF signaling for survival442,443) highly susceptible to apoptosis (using annexin V staining), by reducing the expression of BclxL below a critical threshold.436 The mechanism by which ICAT reduces BclxL expression is not known at the present time.
Since chromosomal instability is a major feature of colon carcinogenesis, it is appropriate to consider the role of the Wnt signaling pathway in mitotic control and aberrant Wnt signaling in the generation of chromosomal aberrations. A precedent for exploring the role of aberrant Wnt signaling in chromosomal instability are the findings that 1) multiple signaling pathways converge to orient the mitotic spindle in Caenorhabditis elegans embryos;444 2) APC and EB1 (a microtubule-associated protein) have the ability to maintain proper spindle positioning in the developing nervous system of Drosophila;445,446 3) binding of APC protein to microtubules increases microtubule stability and is regulated by GSK3β;447 4) APC has a role in chromosome segregation;448 5) β-catenin is a component of the mammalian mitotic spindle and functions to ensure proper centrosome separation and subsequent establishment of a bipolar spindle;449 6) GSK3β has a role in mitotic spindle dynamics and chromosome alignment,450 and localizes to the centrosome and specialized cytoskeletal structures;451 7) dishevelled genes are involved in mitotic progression in cooperation with polo-like kinase 1;452 and 8) conductin/axin2 and Wnt signaling regulates centrosome cohesion.453 It is now well established that aberrant Wnt/β-catenin signaling can induce chromosomal instability in cancer, including colon cancer.454–458 An understanding of the mechanisms by which specific components of the Wnt signaling pathway affect mitosis, mitotic slippage and other aspects of the cell cycle, including interaction with spindle checkpoint proteins, needs to be experimentally determined.
Experiments involving somatic cell fusion and chromosome segregation established the concept that certain genes are capable of suppressing tumorigenesis.459,460 Tumor suppressors are genes whose miRNA or protein products reduce the formation of tumors and prevent malignant progression by decreasing proliferation, regulating the cell cycle, maintaining chromosome integrity, enhancing DNA repair, inducing apoptosis, and, by reducing angiogenesis, invasion, migration, and cell adhesion. Classic tumor suppressor genes that, when deleted or mutated, contribute to tumorigenesis in many types of tumors include p53, RB, INK4a (p16), and ARF.461 In colorectal cancer, mutations and LOH of the tumor suppressor, APC, can affect both the initiation and progression of cancer, whereas the loss of p53 is a late event. Therefore, when the loss of chromosome 1p became associated with many types of cancer, including colon cancer, several groups began the quest to identify the specific tumor suppressor gene or genes located on 1p.462–467 Several genomic loci were identified as “hot spots” for tumor suppressor genes, which included 1p36 and 1p34. It became evident that many genes, both inside and outside of these “hot spots”, could be classified as tumor suppressors; 26 tumor suppressor genes, their genomic loci, and the function of their gene products are listed in Table 6. (Note: 11 genes classified as tumor suppressors in Table 6 are not listed in other tables [Tables 1–5 and and77]).
Several tumor suppressors are haploinsufficient,468 and cell cycle regulatory tumor suppressor genes seem especially dosage-sensitive.469 These findings indicate that the loss of only one copy of a gene in a diploid cell could have a biologic effect.469 Such a loss could contribute to cellular transformation, with the process of selection driving clonal expansion of pre-neoplastic cells.8
Certain tumor suppressors play a more prominent role in tumorigenesis than others in particular tissue types. However, it is probable that the loss of numerous tumor suppressor genes as a result of a chromosomal deletion probably plays a prominent role in the initiation and progression of cancer through a “combination” of different and/or complementary adverse cellular and molecular events.461,467
Four genes on chromosome 1p are associated with defense against oxidative stress (Table 7). Two of these (peroxiredoxin 1 [PRDX1] and endoplasmic reticulum protein ERP19 [TXNDC12]) utilize reducing equivalents provided through the thioredoxin system, and 2 (glutamate-cysteine ligase [modifier subunit] or GCLM and glutathione peroxidase 7 [GPX7]) utilize glutathione. One of the most important genes associated with oxidative stress is glutamate-cysteine ligase (GCL) (also called gamma-glutamylcysteine synthetase), the first rate limiting enzyme of glutathione synthesis.470,471 This enzyme requires coupled ATP hydrolysis to form an amide bond between the γ-carboxyl group of glutamate and the amino group of cysteine to form γ-glutamylcysteine. The enzyme consists of a heavy catalytic subunit (73 kDa) and a light (31 kDa) regulatory subunit (GCLM); the light chain or modifier subunit is found on chromosome 1p. It has been known for the past 2 decades that the ultimate formation of glutathione is required for intestinal function.472 The long-term ingestion of reduced glutathione has recently been shown to suppress the accelerating effect of a beef tallow diet on colon carcinogenesis in rats.473 The specific importance of GCLM to protection against oxidative stress is underscored in GCLM (−/−) knock-out mice, which are severely compromised in the oxidative stress response.474
GCL can be increased by oxidative stress or glutathione depletion475,476 through the inhibition of SHP-1477 and the activation of jun N-terminal kinase (JNK).477,478 The increase in GCL can protect against mitochondrial injury and numerous cellular processes that are depend on the generation of glutathione, such as cell cycle progression, inhibition of caspases (protection against apoptosis), activity of detoxification enzymes (see GSTM genes in Table 8; discussed below), and DNA repair.479–482 Recent studies indicate that a reduced state of proteins in the nucleus is an important environment that induces heterochromatin formation482 and the regulation of histones and PARP activities.483
Chromosome 1p contains 19 genes associated with protection against toxins/carcinogens derived from the environment, dietary/cooking-derived components, and metabolism (Table 8). These genes consist of 2 arylacetamide deacetylase-like enzymes, 4 members of the aldo-keto reductase family, 6 members of the cytochrome P450 family of polypeptides, all 5 members of the mu class of glutathione-S-transferases (GSTs), and 2 metal response element binding transcription factors. A compilation of the 10 most significant transcripton factors capable of targeting the 5′-upstream promoter regions of these 19 genes (GeneCards [SABiosciences’ database; UCSC Genome Browser]) indicates the possible involvement of 95 distinct transcription factors that control their expression. In addition, the Wnt/beta-catenin signaling pathway has been shown to activate various P450 family and GST mu class enzymes in mouse models.484 Since transcription factors respond to different cellular demands and stresses, the presence of these genes on chromosome 1p indicates that the loss of this chromosome arm could compromise the cell’s ability to respond to a variety of environmental toxins/carcinogens that could damage DNA.
It is of interest that all 5 genes of the mu class of GSTs are located on chromosome 1p. The 5 genes are arranged in tandem in the physical order 5′-M4-M2-M1-M5-M3-3′.485,486 The M4-M2-M1-M5 sequence in the gene cluster is oriented in a head-to-tail orientation, whereas the M3 gene is oriented tail-to-tail with respect to the adjacent M5 gene, and is therefore transcribed in the reverse orientation relevant to the other 4 GST mu genes.485 This GST mu gene cluster functions in the detoxification of electrophilic compounds by conjugating glutathione to a wide number of endogenous and exogenous toxins/carcinogens.487 Genetic polymorphisms in GSTM1 increase susceptibility to gastric and colorectal adenocarcinomas.488 In addition, about 70% of human loci is deleted for GSTM1 and 50% of the human population is homozygous deleted for GSTM1.485 This deletion is a result of unequal crossing-over between the two 2.3 kb repeated regions in the intergenic regions that flank the GSTM1 gene. Homozygous deletion of GSTM1 results in increased baseline chromosomal aberrations in lymphocytes among smokers, indicating the role of epoxides and other reactive metabolites of polycyclic aromatic hydrocarbons in inducing genomic instability in these compromised cells.489 All 5 GSTM genes have distinct promoter regions that respond to a different array of transcription factors. Therefore, the loss of chromosome 1p would compromise cellular defenses against toxins/carcinogens, especially in individuals harboring the GSTM1 deletion or other specific polymorphisms.
We have described in this review how the combination of the persistent damage to a cell’s genome with the inability of that cell to adequately repair the damage or die in response to the excessive damage, is a dangerous situation which can result in clonal selection and the development of colon carcinogenesis. The molecular and cellular mechanisms that are associated with the death of cells are most complex, and include both caspase-dependent and caspase-independent processes. Listed in Tables 1–7 are 27 pro-apoptotic/pro-cell death genes found on chromosome 1p, whose simultaneous loss caused by a chromosome 1p deletion could have a major impact on the development of resistance to cell death. In Table 9, we extract from those tables the specific genes whose products contribute to cell death. Caspase-9 and both subunits of DNA fragmentation factor are on the downstream execution phase of apoptosis, and the consequences of their loss are obvious. However, the loss of other gene products (eg, TP73, miR-34a) can have pleiotropic effects on cell death pathways because of multiple transcriptional or translational targets. In addition, TP73, KIF1B, and E2F2 are classified as haploinsufficient genes, with loss of function implied with the presence of only 1 allele.490 Some gene products have dual DNA repair/pro-cell death functions (eg, MUTYH) and dual mitosis/pro-cell death functions (KIF1B). One can see (Table 9) that, in addition to classic pro-apoptotic genes, there are dual role cell survival/pro-cell death genes, DNA damage-response genes, various tumor suppressor genes, genes associated with mitosis, miR-NAs, Wnt signaling, and protection against the generation of peroxides. The mechanism of action of these 27 genes in the control of cell fate is an active area of investigation and beyond the scope of this review. This detailed study of the implications of the loss of chromosome 1p serve as an example of how specific chromosomal deletions can have a major impact on carcinogenesis.
In this section we first address what alteration in specific dietary factors can lead to the loss of chromosome segments or entire chromosome arms in general to produce loss of heterozygosity. Second, we will consider how the consequences of the loss of genes located on chromosome 1p might be affected by pro-carcinogenic and anti-carcinogenic dietary factors. Our approach is to show how specific dietary factors may influence the molecular and cellular processes affected by chromosome 1p loss that were described in previous sections. Links of diet to any of the specific genes lost by the 1p deletion (see Tables 1–8) are listed in Table 10.
Diets high in fat,473,539–547 but low in fiber,540,548–551 low in vegetable intake,552–555 and micronutrient deficient556–560 induce oxidative stress and DNA damage and adversely affect many molecular pathways that prevent genomic instability and apoptosis resistance, 2 major processes that, together, enhance the development of sporadic colon cancer.
The effects of diet likely occur early in the carcinogenesis process, since an altered vegetable intake is known to affect pivotal carcinogenesis pathways in the colonic mucosa from adenoma patients and controls.561 Although 2 alleles are associated with each gene, and the loss of 1 allele may be compensated for by the other, many genes are reported to be haploinsufficient, including those associated with the mitotic checkpoint.562 It is relevant that TP73, KIF1B, and E2F2, found on chromosome 1p, have also been reported to be haploinsufficient,490,563,564 and could have dramatic consequences for colon tumorigenesis if only 1 allele is expressed in colonic epithelial cells. It is possible that many other genes may be found to be haploinsufficient in the future, since a map of 1079 probable haploinsufficient genes has been compiled by systematic identification of genes unambiguously and repeatedly compromised by copy number variation among 8458 apparently healthy individuals.565 Those genes with a high probability of exhibiting haploinsufficiency were enriched among genes implicated in human dominant diseases and among genes causing abnormal phenotypes in heterozygous knockout mice.565 In addition, the loss of several genes on the same chromosome arm that affect a particular molecular pathway (see Tables 1–8) may together have a significant effect on that pathway, although the loss of a single gene may have little effect. Specific dietary factors may decrease the protein levels of certain genes through post-translational mechanisms (eg, proteasomal degradation), thereby inducing a functional pseudo-biallelic loss of a gene, one through a physical loss of the chromosomal segment harboring that gene, and the other an actual degradation of the gene product.
Although dietary factors may affect many processes associated with carcinogenesis, we will evaluate specific factors associated with oxidative stress/inflammation, since these genotoxic processes are known to have major effects on the initiation and progression of cancer, including colon cancer.566–578 Direct damage to DNA, assessed by immunohistochemical staining of 8-oxoG, correlates with poor survival in colorectal cancer.579 ROS can cause excessive DNA double strand breaks, resulting in the loss of chromosome segments or entire arms, depending on the location of the break. In addition, several DNA repair proteins are degraded through an oxidative mechanism,580,581 thereby affecting DNA repair and increasing susceptibility to cancer.582 Oxidative stress can affect spindle organization, induce centrosome amplification, cause proteolysis of components of the anaphase-promoting complex, and override the spindle checkpoint, thereby affecting chromosomal stability. Therefore, oxidative stress can induce a mutator phenotype in affected cells.583 The big question is what dietary factors contribute directly to oxidative DNA damage and aneuploidy (alteration in the number of whole chromosomes or chromosomal segments). We now address several dietary factors that may be associated with these forms of genomic instability. Although the literature on dietary factors associated with genomic instability is substantial, we have chosen to discuss the effects of a high-fat diet, folate deficiency, and niacin deficiency, since the molecular and cellular mechanisms associated with the overabundance or deficiency of these factors have been especially well studied.
A high-fat diet derived from beef tallow or corn oil (eg, linoleic acid, palmitic acid) is one of the major causes of sporadic colon cancer. Long-chain nonesterified (“free”) fatty acids (FFA) and some of their derivatives and metabolites can modify the intracellular production of ROS, in particular superoxide anions and hydrogen peroxide, in part, through their interference with the mitochondrial electron transport chain.584 FFA can also interfere with the glutathione system and stimulate the generation of superoxide anions from phagocytic NADPH oxidases.584 Chronic exposure of cells to FFA (eg, palmitic acid) can also alter miRNA expression (eg, miR-34a, miR-146).585
The genotoxicity associated with a high-fat diet is also caused, in part, by high concentrations of hydrophobic bile acids released into the gastrointestinal tract in response to high-fat meals where they act as detergents to aid in the digestion of fats. Our research group showed that deoxycholic acid (a major hydrophobic bile acid in the human colon) induces ROS586–589 in vitro, and oxidative DNA damage,590 sessile adenomas,591 and colon cancer592 in dietary-related mouse models. In addition to the bile acid-induced formation of 8-oxoG in guanine bases of DNA and the induction of DNA strand breaks (activation of γ-H2AX593 and PARP594), we have shown that deoxycholic acid affects genomic instability at the chromosomal level.595 Evidence indicating the induction of chromosomal damage by deoxycholic acid include the formation of micronuclei and aberrant mitoses, attenuation of activation of the nocodazole-induced spindle checkpoint, and decrease in protein expression of major spindle checkpoint proteins (eg, Mad2, BubR1, securin). The dramatic effect of deoxycholic acid on the process of mitosis is underscored by the finding that deoxycholic acid modulates 71 mitosis-related genes at the mRNA and/or protein levels in vitro and in vivo using mouse models.8 The induction by hydrophobic bile acids of both DNA and chromosomal damage indicates that hydrophobic bile acids are endogenous carcinogens that, at high pathophysiologic concentrations, are capable of contributing to the initiation and progression of colon cancer.8,189,595–597 In addition to causing genomic instability, deoxycholic acid can activate survival pathways (eg, NF-κB594 and autophagy598), which allow for the survival and selection of cells with genomic instability.8,599
Coffee drinkers have a lower incidence of cancer, including that of the colon and rectum.600–603 One coffee compound that we found to prevent the formation of bile acid-induced proximal colon cancer in a mouse model is chlorogenic acid (CGA), the ester of caffeic acid with quinic acid.592 CGA is one of the most abundant polyphenols in the human diet, with coffee, fruits (eg, blueberry, strawberry, raspberry, apple), and vegetables (eg, eggplants, potato, carrot, tomato) as its major sources.493,604 CGA and its metabolites are likely responsible, in part, for the lower risk of rectal cancer associated with the consumption of decaffeinated coffee in 2 large prospective cohort studies.603 One possible mechanism by which polyphenols can reduce colon cancer in this model is through the reduction in deoxycholic acid levels.605 In this study, Han et al605 report that when rats on a high-fat diet (30% beef tallow) received dietary curcumin (component of the Indian spice turmeric) or caffeic acid (metabolite of CGA), the fecal concentration of deoxycholic acid was substantially reduced. In addition, dietary supplementation of this high-fat diet with caffeic acid, catechin (plant polyphenol), rutin (citrus flavonoid glycoside), and ellagic acid (plant polyphenol) significantly reduced the levels of fecal lithocholic acid, a second major hydrophobic bile acid and risk factor for colon cancer.605
The induction of double-strand breaks is a major cause of the production of chromosomal fragments and the deletion of hundreds to thousands of genes. An important DNA repair protein in preventing large chromosomal deletions is Parp-1606 (Figure 5). DNA strand breakage is directly caused by ROS (which would be enhanced due to the loss of genes encoding antioxidant proteins in the chromosome 1p deletion [Table 7]) or as a result of the activity of base excision repair enzymes (see Figure 5). Strand breakage activates Parp-1, which is involved with opening up chromatin and allowing DNA repair processes to occur, including base excision repair, single-strand and double-strand repair (Figure 5). Shibata et al606 carried out mutation analysis using Parp-1 knockout (Parp−/−) mice, and found that PARP deficiency enhanced deletion mutations, especially >1 kbp. A dietary micronutrient whose deficiency has a major effect on PARP activity is niacin (vitamin B3) obtained from meat and corn. The term niacin refers to nicotinic acid and nicotinamide, which are both used by humans to form NAD+. PARP-1 utilizes NAD+ to make poly(ADP-ribose) needed for poly(ADP-ribosyl)ation of proteins. In keeping with the protective effect of PARP, we determined that pre-treatment of cells in vitro with nicotinic acid and nicotinamide protected against bile acid-induced apoptosis,607 presumably by enhancing PARP-mediated DNA repair of bile acid-induced DNA damage and replenishing the NAD+ levels in mitochondria. In addition, we showed that pre-treatment of cells with nicotinic acid and nicotinamide upregulated the mRNA levels of the glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glucose-6-phosphate dehydrogenase (G6PD).608 GAPDH and G6PD may protect against oxidative stress, in part through the generation of the reduced pyridine nucleotides, NADH and NADPH, respectively, from NAD+.608 Niacin supplementation was even reported to improve pellagra (severe niacin deficiency) in a patient with Crohn’s disease,609 a pre-cancerous inflammatory condition610 associated with oxidative DNA damage.611 Pellagra most probably developed in these Crohn’s disease patients through a combination of intestinal malabsorption of niacin/nicotinic acid612,613 and the high demand for NAD+ that accompanies DNA damage-induced PARP-1 activity (see Figure 5). Work from our laboratory indicated that CGA and its metabolites, caffeic acid, m-coumaric acid, and 3-(m-hydroxyphenyl) propionic acid, increased PARP-1 protein expression.493 The modulation of PARP-1 protein levels by CGA may explain, in part, the colon cancer preventive properties of CGA when added as a supplement to the bile acid-induced colon cancer mouse model.592
The mechanisms by which chromosome segments are deleted and translocated can be most complex. Deletions and translocations can arise from centromeric instability and telomeric instability.7,614 and have been proposed as possible mechanisms for chromosomal aberrations associated with chromosome 1.615–617 Centromeric instability can result from hypomethylation or acetylation of pericentromeric heterochromatin, resulting in decondensation/uncoiling/disruption of the centromere618–620 and loss of the affected chromosome arms. Telomeric instability is characterized by telomeric fusions, formation of anaphase bridges during mitosis, broken chromosomes upon the stress of cell division, and fusion of chromosomal fragments to chromosome ends. This cycle of chromosomal abberations is referred to as breakage–fusion–bridge cycles.107–114,621 Six genes found on chromosome 1p (APITD1, CCDC28B, CDCA8, HDAC1, KIF2C, RCC2) are associated with centomeres (see Table 2), and whose loss would affect centromeric instability. A deficiency of HDAC1, for example, has been reported to disrupt pericentromeric heterochromatin.622 In addition to its role in the repair of interstrand cross-links,623 APOLLO (aka DCLRE1B [DNA cross-link repair 1 B]) is also involved in the protection of telomeres (see Table 1). APOLLO is stabilized when bound to the telomere-binding protein TRF2, and protects human telomeres in S phase624 (Figure 4). A reduced level of APOLLO results in an increased number of telomere-induced DNA damage foci and telomeric fusions in S-phase,624 suggesting that APOLLO contributes to a processing step associated with the replication of chromosome ends. Hydrophobic bile acids, probably through the generation of oxidative stress, can modulate 71 genes associated with mitosis8 and decrease the protein expression of 3 major spindle checkpoint proteins (eg, Mad2, BubR1, securin).8 These alterations in gene expression, coupled with direct oxidative damage to components of the mitotic apparatus, may be responsible, in part, for the observed bile acid-induced mitotic aberrations.595 It is, therefore, possible that bile acids may contribute to the loss of chromosome 1p through its effects on centromere instability and telomeric fusions.
Another mechanism by which large chromosomal deletions can occur is through folic acid deficiency.625,626 Folic acid can attenuate the loss of heterozygosity of the DCC tumor suppressor gene in the colonic mucosa of patients with colorectal adenomas,625 indicating that folic acid deficiency can affect allelic deletion and associated micronuclei formation.627,628 Folates are a group of water-soluble B vitamins (obtained from leafy, green vegetables, the whole grain quinoa, and lentils) whose deficiency contributes to colon cancer.629–633 Folates maintain DNA stability through their ability to donate one-carbon units for cellular metabolism and particularly for DNA biosynthesis, repair, and methylation.629,633 Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in one-carbon metabolism. MTHFR catalyzes a unidirectional reaction that determines the balance between cellular availability of 5,10-methylenetetrahydrofolate, used for thymidylate and purine synthesis, and methyltetrahydrofolate used for biological methylation.629 Folate deficiency, therefore, enhances carcinogenesis by impairing normal methylation and nucleotide synthesis, and creates an imbalance between the partitioning of cellular folates into these two pathways. Inhibition of folate metabolism results in excessive uracil misincorporation into DNA633,634 with approximately 4 million uracil bases/cell.559 The repair of 2 adjacent uracil residues on opposite strands of DNA can result in a double-strand break, leading to chromosomal breakage and aneuploidy.558,629,634 Folate deficiency also induces hypomethylation and inhibits DNA excision repair in immortalized normal human colon epithelial cells633 and in the rat colon.635
Recent studies have implicated folate deficiency in the modulation of miRNA expression.497,636 Using microarrays of 385 known human miRNAs, it was determined that folate deficiency in vitro in cultured cells induced a statistically significant fold-change in 24 miRNAs.636 One of these miRNAs was miR-34a, which is found on chromosome 1p and involved in p53-mediated signaling (see Table 4 and the section on MiRNA and MiRNA Processing). MiRNAs were also determined to be altered in patients on a folate-deficient diet.636 In addition to folate deficiency, polymorphisms of MTHFR and altered folate levels are associated with colon cancer risk.637–640 The fact that MTHFR is located on chromosome 1p at 1p36.22 indicates that the loss of this chromosome arm, coupled with folate deficiency, can have major effects on genomic instability.
In this section we have considered how dietary factors such as niacin, folic acid, and a low-fat diet associated with low bile acid levels, together with antioxidants that protect against oxidative DNA damage (Table 10), might affect the processes relevant to carcinogenesis that are altered by chromosome 1p loss. In addition to a deficiency in dietary factors that prevent oxidative DNA damage, a deficiency of certain dietary factors that modulate DNA repair proteins, miRNA expression, antioxidant enzymes, defenses against environmental toxicity, and the Wnt signaling pathway (Table 10) can exacerbate the effects of the loss of chromosome 1p. An understanding of the complex molecular and cellular pathways that are affected by dietary factors is an enormous undertaking, but one that has become a focus of colon cancer prevention.
This work was supported in part by NIH 5 R01 CA119087, Arizona Biomedical Research Commission Grant #0803, VA Merit Review Grant 0142 of the Southern Arizona Veterans Affairs Health Care System and Biomedical Diagnostics and Research, Inc., Tucson, Arizona.
The authors declare no conflicts of interest.