Previous work from the authors’ laboratory indicated that persistent exposure of apoptosis-sensitive colon epithelial cells to DOC, a hydrophobic bile acid, for ~40 weeks led to the selection of cells that were stably resistant to DOC.5
These resistant cells were characterized at the molecular and cellular levels to understand some of the pathways and key proteins that may have been modulated to account for their resistance to apoptosis.5
In one study, two-dimensional gel electrophoresis and mass spectroscopy of the HCT-116RC cell line, revealed a dramatic increase in maspin protein expression.5
The DOC-induced maspin expression may have been the result of DOC-induced DNA damage and/or oxidative stress, since the maspin
promoter contains binding sites for major transcription factors that respond to DNA damage and/or oxidative stress, such as NF-kappaB,47
Figure 7 Molecular signaling pathways that may be activated after persistent exposure of cells to hydrophobic bile acids. Since hydrophobic bile acids can perturb cellular membranes, surface molecules can be activated that generate reactive oxygen and nitrogen (more ...)
Maspin is a multifunctional protein whose expression in different anatomic locations may affect different aspects of carcinogenesis.56
Although maspin acts as a tumor suppressor in the breast and has a pro-apoptotic role in that tissue, the authors have shown for the first time that maspin acts as an anti-apoptotic protein in colon epithelial cells. This may explain its role as a stress-response protein in the colon (), since cells need to evade cell death so that oxidative DNA damages can be adequately repaired.10
In fact, knockout of both maspin genes is embryonically lethal,57
indicating its importance to cell survival or function. However, extended upregulation of anti-apoptotic proteins can have deleterious consequences. Apoptosis-resistant cells that harbor unrepaired DNA damages and enter the pool of proliferating cells may propagate mutations, some of which may lead to cancer58
The mechanisms by which maspin inhibits apoptosis in the colon are not known.
The maspin protein has 8 cysteines60
and exists in several molecular configurations that are dependent upon degree of oxidative stress and the extent of intramolecular disulfide bridge formation.61
In addition to the generation of oxidative stress, reactive oxygen species can have physiological effects on cells because of their ability to positively or negatively affect protein function,62
thereby affecting different signaling pathways63
(). Since maspin can interact with at least 20 different molecules,56
the effect of oxidative stress on maspin and the downstream maspin–protein interactions can ultimately affect cell survival and carcinogenesis. Since caspases have cysteines as a critical amino acid affecting their pro-apoptotic function,65
it is possible that maspin may physically interact with and inhibit caspases through disulfide bridge formation (). Studies in different cell types indicate that maspin can interact with specific proteins that may reduce cellular stresses and cell death, such as glutathione S-transferase (GST)66
and heat shock protein 70 (HSP70) ().66
GST is a major antioxidant defense protein that protects epithelial cells in aberrant crypt foci from DOC-induced apoptosis,67
and the activity of GST is increased by its interaction with maspin.66
HSP70 serves to protect against cell death since it sequesters apoptosis-inducing factor (AIF) in the cytoplasm,68
thereby preventing the translocation of AIF to the nucleus and binding to DNA.74
When translocation of AIF to the nucleus occurs, it induces DNA condensation and the recruitment of endogenous endoand exonucleases, resulting in DNA fragmentation.76
The signaling pathways that lead to apoptosis are most complex, and maspin may interfere, through physical interactions, with the action or activity of apoptosis regulatory proteins (). The survival of cells depends not only on inhibiting pro-cell death pathways, but the activation of prosurvival pathways. Since the authors previously reported that the NF-κB and autophagy pathways are constitutively activated and contributed to the survival of the resistant HCT-116RC cells,5
maspin may also interact with key proteins in these prosurvival pathways (). A full understanding of maspin’s role as an anti-apoptotic protein will benefit from technologies such as yeast 2-hybrid analyses, co-immuno-precipitations, cDNA microarrays, proteomic analyses and transmission electron microscopy after siRNA knockdown, and confocal microscopy for organelle localizations under different experimental conditions.
To understand maspin’s role in colon carcinogenesis, an immunohistochemical analysis of maspin expression was performed in the epithelial cells of macroscopically normal colonic mucosa, polyps, adenomas and adenocarcinomas. It was found that maspin is highly expressed at the polyp and adenocarcinoma stages of colon carcinogenesis, which is consistent with others’ results.29
High maspin expression, however, was not observed as “field defects” in the non-neoplastic colonic mucosa,78
either directly adjacent to a tumor or at different distances proximal or distal to the tumor. In contrast, “field defects” were observed in the decreased expression of the DNA repair proteins Pms2, ERCC1, Xpf, and Ku86,41
and in an apoptosis-related mitochondrial protein, cytochrome c oxidase.40
It is possible that a dramatic and sustained increase in maspin expression at the polyp stage may drive sporadic colon tumorigenesis through its anti-apoptotic function and interaction with other survival signaling pathways in the colon. The anti-apoptotic function of maspin might also allow cells in a field of genomic instability within polyps and adenocarcinomas to proliferate and undergo further clonal expansion ().
Although DOC was previously thought to be a promoter of colon cancer, the authors have previously documented that DOC is a true carcinogen.80
The authors found that prolonged dietary feeding of DOC to mice (to produce the colonic level of DOC found in the colons of humans on a high-fat diet) was responsible for the formation of both adenomas81
in the mouse proximal colon. The DOC-induced increase in maspin expression previously documented5
may explain the increase in maspin expression in ampullary adenocarcinomas that occur near the ampulla of Vater,82
through which copious amounts of bile are released into the small intestine (see endoscopy image in Rana et al83
). DOC contained in the secreted bile may be responsible for the formation of these rare adenocarcinomas that occur in the small intestine.
An interesting question has emerged from the authors’ studies concerning the role of maspin expression during colon carcinogenesis. Since maspin expression first appears significantly increased at the polyp stage of sporadic colon carcinogenesis, but is significantly increased in the non-neoplastic mucosa of patients with inflammatory bowel disease, what is the role of inflammation in sporadic colon cancer? An increase in maspin expression as a “field defect” was not observed in the colon of patients with sporadic colon cancer, and distinct areas of inflammatory infiltrates, as observed in UC, were absent in the tissues examined in the present study. There appear to be multiple pathways to colon cancer, including inherited genetic mutations, inflammatory conditions of the colon (eg, UC, Crohn’s disease), and a high-fat/low-vegetable diet. The development of sporadic colon cancer appears to result, in large part, from dietary factors. Since increased hydrophobic bile acids are associated with a high fat diet, persistent exposure to these bile acids over decades can induce multiple stresses on colon cells, causing DNA damage, oxidative/ nitrosative stress, and mitotic perturbations,34
resulting in the gradual selection of clones with a growth advantage (). The increase in maspin at the polyp stage in sporadic colon cancer may then reflect the proliferation of persistently stressed clones of cells. Since maspin is clearly a stress-response protein, it is possible that it responds to specific stresses associated with the polyp stage of colon carcinogenesis, which may be accentuated by a decrease in key DNA repair proteins, for example. Since the tissues of the polyps/cancers with high maspin expression also do not show an apparent inflammatory infiltrate, the association between maspin and inflammation in sporadic colon cancer cannot be made in the present study. In inflammatory bowel disease, large areas of the colon may be in a “highly stressed state,” as a result of multiple deleterious factors secreted from inflammatory cells, resulting in persistently increased maspin expression (and increased apoptosis resistance) in the non-neoplastic mucosa. The normal mucosa of patients with UC can harbor significant DNA damage, oxidative damage, and even mutations in cancer-related oncogenes,91
a situation that may increase the probability of development of neoplasia under these severe inflammatory conditions.
In future studies, it will be important to understand the mechanisms by which maspin functions as an anti-apoptotic protein, and to determine its role in early preneoplastic lesions. It may also serve as a biomarker to evaluate colon cancer risk and tumor progression, perhaps in conjunction with other biomarkers, such as increased 8-oxoguanine and loss of DNA repair proteins.