ERBB3 Positively Correlates with Intestinal Stem Cell Markers but Marks a Distinct Non Proliferative Cell Population in Colorectal Cancer

Several studies have suggested ERBB3/HER3 may be a useful prognostic marker for colorectal cancer. Tumours with an intestinal stem cell signature have also been shown to be more aggressive. Here, we investigate whether ERBB3 is associated with intestinal stem cell markers in colorectal cancer and if cancer stem cells within tumours are marked by expression of ERBB3. Expression of ERBB3 and intestinal stem cell markers (LGR5, EPHB2, CD44s and CD44v6) was assessed by qRT-PCR in primary colorectal tumours (stages 0 to IV) and matched normal tissues from 53 patients. The localisation of ERBB3, EPHB2 and KI-67 within tumours was investigated using co-immunofluorescence. Expression of ERBB3 and intestinal stem cell markers were significantly elevated in adenomas and colorectal tumours compared to normal tissue. Positive correlations were found between ERBB3 and intestinal stem cell markers. However, co-immunofluorescence analysis showed that ERBB3 and EPHB2 marked specific cell populations that were mutually exclusive within tumours with distinct proliferative potentials, the majority of ERBB3+ve cells being non-proliferative. This pattern resembles cellular organisation within normal colonic epithelium where EPHB2 labelled proliferative cells reside at the crypt base and ERBB3+ve cells mark differentiated cells at the top of crypts. Our results show that ERBB3 and intestinal stem cell markers correlate in colorectal cancers. ERBB3 localises to differentiated cell populations within tumours that are non-proliferative and distinct from cancer stem cells. These data support the concept that tumours contain discrete stem, proliferative and differentiation compartments similar to that present in normal crypts.     

The adenomatous polyposis coli‐associated exchange factors Asef and Asef2 are required for adenoma formation in ApcMin/+mice

Sporadic and familial colorectal tumours usually harbour biallelic adenomatous polyposis coli (APC)‐associated mutations that result in constitutive activation of Wnt signalling. Furthermore, APC activates Asef and Asef2, which are guanine‐nucleotide exchange factors specific for Rac1 and Cdc42. Here, we show that Asef and Asef2 expression is aberrantly enhanced in intestinal adenomas and tumours. We also show that deficiency of either Asef or Asef2 significantly reduces the number and size of adenomas in Apc^Min/+ mice, which are heterozygous for an APC mutation and spontaneously develop adenomas in the intestine. We observed that the APC–Asef/Asef2 complex induces c‐Jun amino‐terminal kinase‐mediated transactivation of matrix metalloproteinase 9, and is required for the invasive activity of colorectal tumour cells. Furthermore, we show that Asef and Asef2 are required for tumour angiogenesis. These results suggest that Asef and Asef2 have a crucial role in intestinal adenoma formation and tumour progression, and might be promising molecular targets for the treatement of colorectal tumours.     

A bacterial driver-passenger model for colorectal cancer: beyond the usual suspects

Cancer has long been considered a genetic disease. However, accumulating evidence supports the involvement of infectious agents in the development of cancer, especially in those organs that are continuously exposed to microorganisms, such as the large intestine. Recent next-generation sequencing studies of the intestinal microbiota now offer an unprecedented view of the aetiology of sporadic colorectal cancer and have revealed that the microbiota associated with colorectal cancer contains bacterial species that differ in their temporal associations with developing tumours. Here, we propose a bacterial driver-passenger model for microbial involvement in the development of colorectal cancer and suggest that this model be incorporated into the genetic paradigm of cancer progression.     

The retinoblastoma protein (Rb) as an anti-apoptotic factor: expression of Rb is required for the anti-apoptotic function of BAG-1 protein in colorectal tumour cells

Although the retinoblastoma-susceptibility gene RB1 is inactivated in a wide range of human tumours, in colorectal cancer, the retinoblastoma protein (Rb) function is often preserved and the RB locus even amplified. Importantly, we have previously shown that Rb interacts with the anti-apoptotic Bcl-2 associated athanogene 1 (BAG-1) protein, which is highly expressed in colorectal carcinogenesis. Here we show for the first time that Rb expression is critical for BAG-1 anti-apoptotic activity in colorectal tumour cells. We demonstrate that Rb expression not only increases the nuclear localisation of the anti-apoptotic BAG-1 protein, but that expression of Rb is required for inhibition of apoptosis by BAG-1 both in a γ-irradiated Saos-2 osteosarcoma cell line and colorectal adenoma and carcinoma cell lines. Further, consistent with the fact that nuclear BAG-1 has previously been shown to promote cell survival through increasing nuclear factor (NF)-κB activity, we demonstrate that the ability of BAG-1 to promote NF-κB activity is significantly inhibited by repression of Rb expression. Taken together, data presented suggest a novel function for Rb, promoting cell survival through regulating the function of BAG-1. As BAG-1 is highly expressed in the majority of colorectal tumours, targeting the Rb–BAG-1 complex to promote apoptosis has exciting potential for future therapeutic development.     

Mutations in the APC tumour suppressor gene cause chromosomal instability

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

Two modes of microsatellite instability in human cancer: differential connection of defective DNA mismatch repair to dinucleotide repeat instability

Microsatellite instability (MSI) is associated with defective DNA mismatch repair in various human malignancies. Using a unique fluorescent technique, we have observed two distinct modes of dinucleotide microsatellite alterations in human colorectal cancer. Type A alterations are defined as length changes of ≤6 bp. Type B changes are more drastic and involve modifications of ≥8 bp. We show here that defective mismatch repair is necessary and sufficient for Type A changes. These changes were observed in cell lines and in tumours from mismatch repair gene-knockout mice. No Type B instability was seen in these cells or tumours. In a panel of human colorectal tumours, both Type A MSI and Type B instability were observed. Both types of MSI were associated with hMSH2 or hMLH1 mismatch repair gene alterations. Intriguingly, p53 mutations, which are generally regarded as uncommon in human tumours of the MSI⁺ phenotype, were frequently associated with Type A instability, whereas none was found in tumours with Type B instability, reflecting the prevailing viewpoint. Inspection of published data reveals that the microsatellite instability that has been observed in various malignancies, including those associated with Hereditary Non-Polyposis Colorectal Cancer (HNPCC), is predominantly Type B. Our findings indicate that Type B instability is not a simple reflection of a repair defect. We suggest that there are at least two qualitatively distinct modes of dinucleotide MSI in human colorectal cancer, and that different molecular mechanisms may underlie these modes of MSI. The relationship between MSI and defective mismatch repair may be more complex than hitherto suspected.     

Enhanced expression of the complement regulatory protein CD55 predicts a poor prognosis in colorectal cancer patients

This study prospectively correlated the level of expression of CD55 on tumours with 7-year survival in 136 colorectal cancer patients. Patients with tumours expressing high levels of CD55 had a significantly worse survival (24%) than patients with low CD55 levels (50%, p<0.02). A similar difference was seen for patients (Duke's B or C) with a high risk of recurrence (29% vs 58%, p<0.05). Furthermore, there was a progressive deterioration in prognosis with increasing antigen expression (p=0.01). It remains unclear if CD55 is overexpressed by tumours to protect them from complement or if it is related to the recent observation that CD55 is a ligand for the T-cell activation antigen CD97. However, it is a marker of aggression, as colorectal cancer patients whose tumours overexpress CD55 have a significantly reduced 7-year survival.     

Treatment of chemically induced autochthonous rat mammary and colorectal carcinomas with interleukin-2

Summary The antineoplastic efficacy of human interleukin-2 (IL-2) in autochthonous methylnitrosourea-induced mammary carcinoma and in acetoxymethly-methylnitrosamine-induced colorectal carcinoma of Sprague Dawley rats has been investigated. Under the conditions applied, IL-2 was non-toxic. In the mammary carcinoma IL-2 was therapeutically inactive. In the colorectal carcinoma, 1200 U IL-2/day exhibited significant antitumour activity in established tumours as well as in tumours treated prophylactically before their manifestation (P <0.05). The effect of IL-2 seemed to be more pronounced when given before manifestation of colorectal tumours (T/C = 8.7% vs 17.8% in established tumours). The differential sensitivity of the autochthonous mammary and colorectal carcinoma may be explained by differences in their proliferation rates and differences in volumes at the beginning of IL-2 therapy. IL-2 seems to be preferentially active in small tumours with a low proliferation rate, a feature typical of colon tumours     

Casein kinase II is elevated in solid human tumours and rapidly proliferating non-neoplastic tissue

Protein kinase CKII (i.e. casein kinase II, CKII, NII) is expressed at a higher level in rapidly proliferating tissues and in solid human tumours (e.g. colorectal carcinomas) when compared to the corresponding non-neoplastic colorectal mucosa. This could be shown by (a) Western blotting of cellular extracts from solid tumours followed by immunostaining with an anti-CKII polyclonal antibody, (b) immunohistochemical staining of cells from tissue sections and (c) by activity measurements using the CKII-specific synthetic peptide (RRRDDDSDDD). The maximum observed activity in the colorectal carcinomas investigated was up to eightfold higher in the tumour specimens than in the non-neoplastic tissue (i.e. colorectal mucosa). The activity range was between 33-350 U/mg protein and in the case of colorectal mucosa 13-106 U/mg protein. The amount of CKII determined in the individual tumours was in the range 0.4-1.6 nmol/g tissue.     

Vindesine as a stathmokinetic agent in human rectal tumours in organ culture

Organ culture, using human colorectal mucosa and tumours, is a good system in which to test a new stathmokinetic agent such as vindesine. Using this system we have found that vindesine has similar metaphase-arresting properties to vincristine, including at least a 6-fold dose response difference in its ability to arrest mitosis in mucosa and tumour, mucosa being the more sensitive. Vindesine is a satisfactory stathmokinetic agent, but in view of its greater cost offers no particular advantages over vincristine.     

Vindesine As A Stathmokinetic Agent In Human Rectal Tumours In Organ Culture

Organ culture, using human colorectal mucosa and tumours, is a good system in which to test a new stathmokinetic agent such as vindesine. Using this system we have found that vindesine has similar metaphase-arresting properties to vincristine, including at least a 6-fold dose response difference in its ability to arrest mitosis in mucosa and tumour, mucosa being the more sensitive. Vindesine is a satisfactory stathmokinetic agent, but in view of its greater cost offers no particular advantages over vincristine.     

Acromegaly and cancer

In recent years, it has become increasingly recognized that acromegaly is associated with an increased prevalence of colorectal cancer and pre-malignant tubular adenomas. The aetiology of these tumours is unknown but is likely to reflect increased levels of both insulin-like growth factor I (IGF-I), which is implicated in the development of sporadic colorectal cancer, and environmental factors, such as the bile acid deoxycholic acid. There is also evidence to suggest that the prevalence of breast and perhaps haematological malignancies might be increased in acromegaly, although these associations have been based on mostly small epidemiological surveys and clarification will come in the future once large-scale epidemiological studies have been completed.     

Up-regulation of 12(S)-lipoxygenase induces a migratory phenotype in colorectal cancer cells

12(S)-Lipoxygenase (LOX) and its product 12(S)-hydroxyeicosatetraenic (HETE) acid have been implicated in angiogenesis and tumour invasion in several tumour types while their role in colorectal cancer progression has not yet been studied. We have analysed 12(S)-LOX expression in colorectal tumours and found gene expression up-regulated in colorectal cancer specimens for which the pathology report described involvement of inflammation. Using cell line models exposed to 12(S)-HETE or over-expressing 12(S)-LOX malignant cell growth as well as tumour cell migration was found to be stimulated. Specifically, Caco2 and SW480 cells over-expressing 12(S)-LOX formed fewer colonies from sparse cultures, but migrated better in filter-migration assays. SW480 LOX cells also had higher anchorage-independent growth capacity and a higher tendency to metastasise in vivo. Knock-down or inhibition of 12(S)-LOX inhibited cell migration and anchorage-independent growth in both 12(S)-LOX transfectants and SW620 cells that express high endogenous levels of 12(S)-LOX. On the cell surface E-cadherin and integrin-β1 expression were down-regulated in a 12(S)-LOX-dependent manner disturbing cell-cell interactions. The results demonstrate that 12(S)-LOX expression in inflammatory areas of colorectal tumours has the capacity to induce an invasive phenotype in colorectal cancer cells and could be targeted for therapy.     

A double immunochemical method for detecting faecal haemoglobin and albumin in rectal screening

Undoubtedly, colonoscopy is the "gold standard" in the diagnosis of colorectal cancers. Sophisticated bowel preparation and risk of bowel perforation and bleeding, as well as the patient's discomfort during examination lead to low compliance in screening. Therefore, alternative non-invasive screening methods tend to come into the fore. In this study we compared the sensitivity and specificity of the double immunochemical FECA test for the haemoglobin + albumin content of the faeces with those of control colonoscopy in the detection of colorectal neoplasms. In a 3-year period 154 patients (69 males and 85 females) were scheduled for colonoscopy with previously collected stool samples. The sensitivity and specificity of the double immunochemical test for faecal haemoglobin + albumin content were determined in colorectal neoplasms of different severity. Colonoscopy served as a control examination. Colonoscopy identified in 77 cases benign lesions, and in 10 cases malignant tumours. The double immunochemical test for faecal blood and protein successfully used in model screening population showed in our present study 52.7% sensitivity and 92.3% specificity for significant neoplastic lesions (high-risk polyps and tumours). When the evaluation was limited to the high-risk polyps, the sensitivity was modified to 45.5% and the specificity to 92.3% and in case of invasive tumours to 90% and 100%, respectively. If only faecal haemoglobin content was measured, the overall sensitivity for polyps of any size and sort was 15.7% which, however, increased to 27.63% if faecal albumin was also measured. Based on relevant literature, the sensitivity of the FECA test for colorectal polyp and cancer is more favourable than that of other FITs. However, the increased sensitivity of the double faecal protein test falls short of the standard colonoscopy. Therefore, in certain cases the latter might be considered as a primary screening method.     

Specific in vitro and in vivo drug localisation to tumour cells using a hybrid-hybrid monoclonal antibody recognising both carcinoembryonic antigen (CEA) and vinca alkaloids

Summary By using a bispecific monoclonal antibody recognising both carcinoembryonic antigen (CEA) and the cytostatic vinca alkaloid drugs we have been able to show specific tumour localisation of vinca alkaloids. In vitro studies with sections of human colorectal tumours have demonstrated that the hybrid-hybrid 28.19.8 monoclonal is able to specifically localise vindesine to cells expressing CEA. Furthermore, the hybrid-hybrid 28.19.8 localises in vivo preferentially to tumour tissues in nude mice bearing the MAWI human xenograft tumour. This tumour-bound hybrid-hybrid monoclonal antibody induces profound changes in the bio-distribution of vinca alkaloid drugs, targeting them specifically to tumour tissues.     

Identification of a human ribosomal protein mRNA with increased expression in colorectal tumours

A human ribosomal protein cDNA was selected from a normal colon cDNA library on the basis of overexpression in familial adenomatous polyposis. Nucleotide sequence analysis was used to identify this cDNA as corresponding to the human equivalent of the rat ribosomal protein L31 (HL31). We have quantified the expression of HL31 mRNA in colorectal tumours and found overexpression in 23 out of 23 cases. Our results indicate that HL31 is associated with a malfunction of normal growth regulatory mechanisms in these tumours, and suggest a role for HL31 in proliferation and neoplasia.     

Suppression of mucin 2 enhances the proliferation and invasion of LS174T human colorectal cancer cells

Altered expression of MUC2 (mucin 2) is related to tumour development in colorectal cancer. Colorectal mucinous carcinomas are positive for MUC2 expression, whereas MUC2 is down-regulated in non-mucinous adenocarcinomas. In the present study, we down-regulated MUC2 expression by RNAi (RNA interference) and investigated the in vitro and in vivo effects on the proliferation and invasion/migration potential of the LS174T human colorectal cancer cells. The LS174T cell line is a goblet-cell-like colorectal cancer cell line that continuously produces high levels of MUC2. Inhibition of MUC2 expression in vitro by transfection of LS174T cells with the recombinant plasmid pcDNA6.2-GW/EmGFP-miR-MUC2 led to the production of a stably transfected MUC2-RNAi LS174T cell line. The proliferation and invasion/migration of MUC2-RNAi cells in vitro were significantly higher than those in control cells, as assessed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], colony formation and transwell assays. Subcutaneous injection of MUC2-RNAi LS174T cells into nude mice resulted in the development of subcutaneous tumours visible to the naked eye after 1 week. The growth rate of tumours derived from MUC2-RNAi LS174T cells was greater than that of tumours derived from control cells. Ki67 and matrix metalloproteinase-9 proteins were detected by immunohistochemistry in the xenografts. The expression levels of these proteins were higher in the MUC2-RNAi-derived xenografts than in xenografts derived from control cells. Although the role of MUC2 in colorectal tumorigenesis is not fully understood, these results strongly suggest a relationship between the proliferation and invasion of LS174T cells and the expression of MUC2.     

The tumour suppressor gene product APC blocks cell cycle progression from G0/G1 to S phase.

The APC gene is mutated in familial adenomatous polyposis (FAP) as well as in sporadic colorectal tumours. The product of the APC gene is a 300 kDa cytoplasmic protein associated with the adherence junction protein catenin. Here we show that overexpression of APC blocks serum-induced cell cycle progression from G0/G1 to the S phase. Mutant APCs identified in FAP and/or colorectal tumours were less inhibitory and partially obstructed the activity of the normal APC. The cell-cycle blocking activity of APC was alleviated by the overexpression of cyclin E/CDK2 or cyclin D1/CDK4. Consistent with this result, kinase activity of CDK2 was significantly down-regulated in cells overexpressing APC although its synthesis remained unchanged, while CDK4 activity was barely affected. These results suggest that APC may play a role in the regulation of the cell cycle by negatively modulating the activity of cyclin-CDK complexes.