Where Do the Cancers Arise

Grow Younger Blood

Longevity Health and Wellness Protocol

Get Instant Access

Liver cancer

The assessment of DNA alterations in tumour cells allows a precise determination of their clonality. Where HBV is involved, there is no doubt that the integration of HBV-DNA into the hepatic genome is a significant event in hepatocarcinogenesis.1,35,36 Moreover inspection of viral integration sites amongst tumour cells clearly indicates that each tumour is monoclonal, i.e. derived from a single cell.37-40 Likewise, studies of HCC clonality based on restriction fragment length polymorphisms of X-linked genes such as the androgen receptor gene (HUMARA) come to the same conclusion.41 The important question is, which cell is involved in cancer initiation? As discussed above, in the liver there are many cells endowed with longevity and long-term repopulating potential, suggesting that there may be more than one type of carcinogen target cell. Irrespective of which target cell is involved, what is clear is that cell proliferation at the time of carcinogen exposure is pivotal for "fixation" of the genotoxic injury into a heritable form.

Animal models

Many models of liver cancer utilise a brief exposure to an initiating carcinogen at a time when the liver is in a proliferative state, either during the period of postnatal growth or shortly after a PH or necrogenic insult. For example, Craddock42 clearly demonstrated the carcinogenic effects of dimethylni-trosamine (DMN) on the rat liver when it was administered one day after a PH (when some 30% to 40% of hepatocytes would be in S phase), whereas the same compound, at the same dose, was not carcinogenic to normal adult rats.

Taking this view, Sell43-45 has opined that in models of experimental hepatocarcinognesis as a whole, there may be at least four distinct cell lineages susceptible to neoplastic transformation (see Fig. 3). This supposition is based on the fact that there is considerable heterogeneity in the prolif-erative responses that ensue after injury in the many different models of hepatocarcinogenesis. Thus, hepatocytes are implicated in some models of

Fig. 3. Schematic diagram of various lineages that respond to specific cell damaging insults and therefore are likely founder cells for the tumours that develop subsequently. (1) The cells that normally respond to hepatocyte loss are the hepatocytes themselves; (2) potential stem cells may reside in the canal of Hering and they or their progeny (oval cells/HPCs) may give rise to most HCCs; (3) the interlobular bile duct epithelia may give rise to CCs associated with fluke infection; and (4) periductular cells are associated with experimental hepatocarcinogenesis associated with ethionine and a choline-deficient diet. Largely based on an idea by Stewart Sell.

Fig. 3. Schematic diagram of various lineages that respond to specific cell damaging insults and therefore are likely founder cells for the tumours that develop subsequently. (1) The cells that normally respond to hepatocyte loss are the hepatocytes themselves; (2) potential stem cells may reside in the canal of Hering and they or their progeny (oval cells/HPCs) may give rise to most HCCs; (3) the interlobular bile duct epithelia may give rise to CCs associated with fluke infection; and (4) periductular cells are associated with experimental hepatocarcinogenesis associated with ethionine and a choline-deficient diet. Largely based on an idea by Stewart Sell.

HCC, direct injury to the biliary epithelium implicates essentially unipotent cholangiocytes in some models of CC, whereas HPC/oval cell activation accompanies many instances of liver damage irrespective of aetiology, making such cells most likely to be carcinogen targets. A fourth cell type that might be susceptible to neoplastic transformation is the so-called nondescript periductular cell that responds to periportal injury; the suggestion that such a cell may be of bone marrow origin would be experimentally verifiable in the context of a sex-mismatch bone marrow transplantation (see above) and the appropriate carcinogenic regimen.

So, for example, after diethylnitrosamine (DEN) exposure, there is little oval cell proliferation but the emergence of «-fetoprotein (AFP)-positive hepatocytes, followed by AFP+ foci and eventually AFP+ HCC, suggests that HCC develop from hepatocytes in this model.45 On the other hand, direct injury to the bile ducts induced by furan leads to bile duct hyperplasia and intestinal metaplasia, and prolonged furan exposure results in CCs with a smaller number of HCCs, observations consistent with abile duct cell origin of these tumours. Likewise, fluke infection can lead to marked bile duct hyperplasia and subsequent exposure to DMN leads to the rapid development of CC. Many models of hepatocarcinogenesis are characterised by a striking proliferation of oval cells, particularly the so-called "Solt-Farber" model, where a single exposure to DEN is followed by a course of 2-AAF designed to block the regenerative ability of normal hepatocytes; thus, when a PH is performed only those cells "initiated" and therefore resistant to the antiproliferative effects of 2-AAF can respond — hence the "resistant hep-atocyte" model of carcinogenesis (reviewed in Allison etal.46). Despite the name of the model, Sell concludes that the sequence of hepatocyte foci, to nodules of increasing size to HCC is most likely to have its origins in bipotential oval cells. In animal models of metabolic liver disease such as Wilson's disease, hepatocyte destruction and inflammation is accompanied by a prominent oval cell reaction (Fig. 4), and, perhaps not surprisingly, a high incidence of both HCC and CC. A fourth type of cell that may be involved in carcinogenesis is the periductular oval cell47 that proliferates and expresses AFP in response to carcinogens such as ethionine in a choline-deficient diet (CDE diet).

The direct involvement of hepatocytes in hepatocarcinogenesis has been clearly established in rats. Gournay et al.48 found that some preneoplastic foci (expressing gamma glutamyl transpeptidase and the placental form of glutathione S-transferase) were directly descended from hepatocytes. This was achieved by stably labelling hepatocytes at one day after a 2/3 PH with ¡-galactosidase using a recombinant retroviral vector containing the ¡-galactosidase gene; subsequent feeding with 2-AAF lead to foci, some of which were composed of ¡-galactosidase-expressing cells. Using the same labelling protocol, Bralet et al.49 found that 18% of hepatocytes expressed ¡-galactosidase at the completion of regeneration after a 2/3 PH; subsequent chronic treatment with DEN resulted in many HCCs, 17.7% of which also expressed ¡-galactosidase, leading to the conclusion that a random clonal origin of HCC from mature hepatocytes was operative in the model.

Extensive polyploidy is associated with terminal differentiation and cell senescence, and in many models of chronic injury precedes overt oval cell development. Moreover, the carcinogenic process in animals and man is associated with the presence of more diploid cells — consistent with an

Fig. 4. An oval cell reaction in a Long Evans Cinnamon (LEC) rat. These animals develop HCC and CC with a high frequency.

expansion of oval/HPCs during ongoing liver injury, cells that eventually give rise to so-called small cell dysplasia, a likely precursor lesion for HCC (see below). If tumours do arise from oval/HPCs, then this would suggest a block in oval cell differentiation, a process called stem cell maturation arrest by Sell and Pierce.50 Direct evidence for the involvement of oval cells in the histogenesis of HCC was provided by Dumble et al.51 who isolated oval cells from p53 null mice; when these cells were transplanted into athymic nude mice they produced HCCs.

Human studies

As in rodents, HCC appears to evolve from focal precursor lesions that reflect the stages of multi-step carcinogenesis. Usually in a setting of chronic inflammation with liver cell damage and concurrent regeneration, activation of HPCs invariably follows,15,19,21,52 and the first lesions are thought to be either foci of small cell dysplasia or low-grade dysplastic nodules.53 Further rounds of mutation and clonal expansion eventually led to HCC (Fig. 5).

Fig. 5. A multi-step model for the progression to HCC in human liver. Oval cells (HPCs) may be involved in the histogenesis of many HCCs and initially give rise to foci of small cell dysplasia. Further rounds of mutation and clonal expansion may give rise to other pre-cancerous lesions such as low-grade dysplastic nodules and high-grade dysplastic nodules before HCC develops.

Fig. 5. A multi-step model for the progression to HCC in human liver. Oval cells (HPCs) may be involved in the histogenesis of many HCCs and initially give rise to foci of small cell dysplasia. Further rounds of mutation and clonal expansion may give rise to other pre-cancerous lesions such as low-grade dysplastic nodules and high-grade dysplastic nodules before HCC develops.

It seems highly likely that mature polyploid hepatocytes are not the cells of origin of most HCCs, but rather that most HCCs have their origin in HPCs.

An origin of HCC from HPCs is often inferred from the fact that many tumours contain an admixture of mature cells and cells phenotypically similar to HPCs. This would include small oval-shaped cells expressing OV-6, CK7 and CK19, and chromogranin-A, along with cells with a phenotype intermediate between HPCs and the more mature malignant hepatocytes54 (Fig. 6). Cells resembling HPCs (OV.1+ or OV-6+) have also been noted in hepatoblastoma;52 this tumour, the most common liver tumour in childhood is widely believed to be stem cell-derived given that there can be

Fig. 6. HPCs (arrowheads) in a hepatic adenoma immunoreactive for CK19 (A), OV-6 (B), and chromogranin-A (C). Intermediate hepatocyte-like cells (arrows) positive for OV-6 and chromogranin-A surround the HPCs (courtesy of Professor Tania Roskams).

both epithelial and mesenchymal tissue components. Cells with an HPC phenotype have also been noted in a relatively rare subset of hepatic malignancies where there are clearly two major components, an HCC component and a CC component, again suggestive of an origin from a bipotential progenitor.55

Colon cancer

Numerous steps are involved in the progression of normal tissue through dysplasia to malignancy, and based on the observation that the accumulation of molecular alterations seemed to parallel the clinical progression of tumours, Vogelstein et al.1 proposed a stepwise model of colorectal tumourigenesis. The molecular pathogenesis of FAP has shed much light on the initial mutations required in this step-like progression. FAP results in the formation of multiple bowel adenomas in the second and third decades of life. Colonic cancer is inevitable in these patients who therefore require prophylactic colectomy. The heritable nature of FAP was first recognised at the end of the 19th century; however, it was not until 1986 that an interstitial deletion of chromosome 5q was observed in an FAP patient.56 This prompted linkage analysis studies which co-demonstrated tight linkage of the condition to markers on chromosome 5q21.51 The gene responsible was Adenomatous Polyposis Coli (APC),58 which encodes alarge (approximately 2800 amino acids), multi-functional cytoplasmic protein. APC binds and down-regulates ^-catenin and is vital in control of Wnt signalling. Mutations in APC are also found in 63% of sporadic adenomas59 and up to 80% of sporadic colorectal cancers.60 Mutations in APC are present in very early adenomas59 and are sufficient to promote small adenoma growth in the absence of microsatellite instability, K-ras or ^-catenin mutation or allelic loss of lp.61 Thus, APC inactivation provides a stem cell with a selective growth advantage by allowing unregulated activation of Wnt signalling. Mutations in ^-catenin, preventing its breakdown can also promote adenoma initiation; however, small adenomas with ^-catenin mutations alone do not progress to larger adenomas or carcinomas as frequently as adenomas with APC mutations.62 Therefore, although the role of APC in the regulation of Wnt signalling is most important in the prevention of tumour initiation, its involvement in apoptosis and chromosomal stability also have an effect on the progression of the adenoma growth (reviewed in Fodde et al.6"3).

Clonal expansion of mutated cells — niche succession and crypt fission

There are three possibilities that can result from a stem cell division:

• The production of one stem cell and one daughter cell — asymmetric division.

• Symmetric division with self-replication, where two stem cells are produced.

• Symmetric division with stem cell loss, where both daughter cells go on to differentiate.

The majority of divisions are thought to be asymmetric and there is some evidence supporting the retention of template DNA strand within the stem cells located in the niche — the so-called immortal strand hypothesis, ensuring any DNA replication errors are passed on to the differentiating, short-lived daughter cells, affording a mechanism of stem cell genome protection.64 Park et al.65 used ethyl nitrosurea (ENU) to induce mutations in the X-linked gene for glucose-6-phosphate dehydrogenase (G6PD) to demonstrate the expansion of a mutated clone within the crypt. G6PD gene mutation resulted in loss of staining in affected cells. After ENU treatment, they initially observed crypts that were only partially stained for G6PD, which eventually disappeared with the contemporaneous emergence of fully mutated crypts (monoclonal conversion or crypt purification). These eventually gave rise to patches of crypts that failed to stain with

G6PD. More recently, Taylor et al.66 used mitochondrial DNA mutations in colonic crypt cells to demonstrate the presence of partially mutated crypts in the human colon. They observed that human colonic crypt cells accumulate sufficient mitochondrial DNA (mtDNA) mutations with age to cause a biochemical defect in the mtDNA-coded subunits of COX. This defect can also be stained for with immunohistochemistry. Normal colonic tissue shows numerous completely COX-deficient crypts, but also a few partially stained crypts. Serial sections of these partial crypts allowed them to reconstruct three-dimensional images of the crypt revealing a ribbon of COX negative cells extending from the base of the crypt to the top. The ribbon of mutated, COX negative cells appear to be the progeny of one of the small number of stem cells in the niche, and that the partially negative crypts are likely to be intermediate steps in the expansion of the mutated clone with eventual formation of a completely clonal COX-deficient crypt.

Yatabe et al.67 used CpG-methylation patterns in three non-expressed genes to study the dynamics of the stem cells within the niche, and proposed the niche succession model. They showed that the differences in methylation tag sequence between cells in adjacent crypts (intercrypt variation) were more pronounced than the tag variation seen between cells in the same crypt (intracrypt variation). They proposed that intracrypt variation was a consequence of multiple, yet related stem cells within each crypt. Stochastic extinction or amplification of one stem cell line by occasional symmetrical division results in a "bottleneck" effect, wherein all cells in the crypt are descended from an original stem cell. Mathematical modelling suggested that this bottleneck occurs once every 8.2 years in the normal human colon. In normal appearing crypts from FAP patients; however, there was a greater intracrypt variation in methylation tags suggesting slower niche succession, probably as a consequence of enhanced stem cell survival. The longevity of APC+/- stem cells in FAP patients increases the chances of receiving or selecting for a second hit in the gene.68 Once APC protein function is impaired, a growth advantage is bestowed on the cell and clonal expansion would then occur much more quickly.

Niche succession is a way by which a single stem cell line can "hitchhike" its way to clonal dominance of a single crypt,69 but then how does a stem cell line expand into adjacent tissue? As described above, clonality experiments in both mice and humans have shown clustering of mutated, phenotypically similar crypts together in patches.65'66 It is thought that a process called crypt fission, whereby crypts undergo basal bifurcation followed by longitudinal division, with the ultimate formation of two daughter

Fig. 7. A model for the development of early colorectal cancer (CRC). Mutation occurs in a stem cell located near the base of the crypt and mutated cell progeny occupy part of the crypt. Through a stochastic process (called niche succession or monoclonal conversion) the affected crypt becomes wholly occupied by dysplastic cells — the monocryptal adenoma. Further expansion can occur by the dysplastic crypt undergoing crypt fission and budding leading to an oligocryptal adenoma (aberrant crypt focus). Key: normal colonocytes are brown and mutated colonocytes are blue.

Fig. 7. A model for the development of early colorectal cancer (CRC). Mutation occurs in a stem cell located near the base of the crypt and mutated cell progeny occupy part of the crypt. Through a stochastic process (called niche succession or monoclonal conversion) the affected crypt becomes wholly occupied by dysplastic cells — the monocryptal adenoma. Further expansion can occur by the dysplastic crypt undergoing crypt fission and budding leading to an oligocryptal adenoma (aberrant crypt focus). Key: normal colonocytes are brown and mutated colonocytes are blue.

crypts, is responsible for the clustering of apparently related crypts. This process is central in the massive increase in crypt number (in both the small and large intestine) in the postnatal period and in the regenerative phase following radiation. The crypt cycle — crypts born by crypt fission gradually increasing in size until they, themselves, divide by crypt fission — takes approximately 108 days in the mouse jejunum and 9-18 years in the human large intestine. It was originally suggested that a crypt would be prompted to go into fission once it had reached a threshold size; however, attention has now focused on the stem cell number being the important factor. The rate of crypt fission is increased in many pathological conditions and is undoubtedly the mechanism by which dysplastic crypts multiply to form microadenomas or dysplastic aberrant crypt foci (ACF)70 (see Fig. 7). ACF are morphologically and genetically distinct lesions that are the precursors of adenomas and cancers.71

Top-down or bottom-up?

Studies have shown that dysplastic ACF are clonal populations,72 and expand by crypt fission;73 however, the expansion of a mutated clone from a single cell to form small adenomas is contentious, with two main theories — the top-down and bottom-up models (Fig. 8).

The top-down model is based upon the frequent observation of dysplas-tic cells solely at the luminal surface of the crypts,74 along with apparent retrograde migration of adenomatous cells from the surface to the base of the crypt. Shih et al.74 examined the morphology and molecular characteristics of small (1-3 mm), well-orientated specimens from sporadic adenomas. Using digital single nucleotide polymorphism (SNP) analysis

Bottom up Top down

Bottom up Top down

Fig. 8. Top-down or bottom-up growth of colorectal adenomas? Bottom-up: the stem cell, located in the crypt base undergoes APCmutation (A). The mutated cell proliferates (B) and spreads to the top of a crypt to form a monocryptal adenoma (C). Initial further expansion is by crypt fission (based on Preston et al.79). Top-down: the initial transformation event occurs in a cell in the intracryptal zone (A) and then spreads laterally and downwards (B) eventually filling the whole crypt (C) (adapted from Shih et al?4).

Fig. 8. Top-down or bottom-up growth of colorectal adenomas? Bottom-up: the stem cell, located in the crypt base undergoes APCmutation (A). The mutated cell proliferates (B) and spreads to the top of a crypt to form a monocryptal adenoma (C). Initial further expansion is by crypt fission (based on Preston et al.79). Top-down: the initial transformation event occurs in a cell in the intracryptal zone (A) and then spreads laterally and downwards (B) eventually filling the whole crypt (C) (adapted from Shih et al?4).

of four SNPs within the APC gene, they assessed for LOH of APC in cells in the upper portion of the crypts, most of which had truncating APC mutations on nucleotide sequence analysis. These were not seen in the histologically normal crypt bases. Only these upper crypt cells showed prominent proliferative activity and nuclear localisation of ^-catenin. These observations were not easily reconciled with the conventional view of the stem cell origin of cancer, and the authors proposed two possible explanations to explain their findings. First, they considered a relocation of the stem cell area to the intracryptal zone, and second they suggested that a mutated stem cell migrates from the base of the crypt to the luminal surface before expanding laterally and downwards. Lamprecht and Lipkin75 adjusted the latter model slightly to suggest that APC mutations occur within a transit-amplifying cell, preventing it from terminally differentiating and altering the migration dynamics of the cell, and allowing it to remain in the mucosa as an incipient aberrant clone. The bottom-up model involves the recognition of the earliest lesion in tumour development, the monocryptal adenoma, where the dysplastic cells occupy an entire single crypt. These lesions are common in FAP,76 and although rare in non-FAP cases, have been described previously.77 Clonality studies in the XO/XY FAP patient have shown that monocryptal adenomas are clonal populations.78 Analysis of tiny (<3 mm) adenomas in FAP patients showed increased proliferative activity and nuclear (-catenin translocation in morphologically dysplastic cells from the crypt base to the luminal surface. Additionally, there was a sharp cut-off between the dysplastic surface epithelium with nuclear (-catenin activity, and the normal mucosa in a neighbouring unaffected crypt. The observation of an increased, asymmetrical crypt fission index in adenomatous tissue led the researchers to propose the bottom-up model — an abnormal stem cell clone with a growth advantage expands from the stem cell niche at the crypt base to fill an entire crypt. Thereafter, initial spread is by crypt fission to form ACF, with top-down spread undoubtedly occurring in slightly larger lesions.79

Was this article helpful?

0 0
Staying Young

Staying Young

Discover The Secrets To Staying Young Forever. Discover How To Hinder The Aging Process On Your Body And In Your Life. Do you feel left out when it comes to trying to look young and keeping up with other people your age? Do you feel as though your body has been run down like an old vehicle on its last legs? Those feelings that you have not only affect you physically, but they can also affect you mentally. Thats not good.

Get My Free Ebook


Post a comment