Intestinal tumours induced by the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in multiple intestinal neoplasia mice have truncation mutations as well as loss of the wild-type Apc(+) allele.

C57BL/6J-Min/+ (multiple intestinal neoplasia) is a murine model for familial adenomatous polyposis (FAP), where the mice are heterozygous for a nonsense Apc(Min) (adenomatous polyposis coli) mutation, and therefore develop numerous spontaneous adenomas in the small intestine and colon. Neonatal exposure of Min/+ mice to the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (eight subcutaneous injections of 25 or 50 mg/kg PhIP to pups or 50 mg/kg PhIP to lactating dams) markedly increased (2--9-fold) the number of intestinal tumours, especially in the small intestine. We examined whether the Apc gene was affected in small intestinal and colonic tumours induced by PhIP. In spontaneous tumours formed in these mice, the main mechanism for tumour induction is loss of the wild-type Apc(+) allele, i.e. loss of heterozygosity (LOH). Also in the PhIP-induced tumours, this is a major mechanism, since large fractions of PhIP-induced tumours had LOH in APC: However, mechanisms other than LOH must also prevail, since a lower frequency of LOH was found in the small intestinal tumours from male mice exposed to PhIP either via breast milk (65%) or by direct injection (68%), compared with the untreated controls (92%). Tumours that had retained the wild-type Apc(+) allele were further analysed for presence of truncated Apc proteins with in vitro synthesized protein (IVSP) assay. Truncated Apc proteins, indicating truncation mutations in exon 15 of the Apc gene, were detected in 20% (8 of 40) of the tumours not showing LOH from the small intestine after PhIP exposure, all in segment 2 (codons 686--1217). Seventeen percent (2 of 12) of the colonic tumours had a truncated Apc protein in segment 3 (codons 1099--1693). Importantly, no truncated proteins were detected in tumours from unexposed mice with apparently retained wild-type Apc(+) allele. These results show that PhIP induces intestinal tumours in the Min/+ mice both by causing LOH and truncation mutations in the wild-type Apc(+) allele.     

Expression patterns of stromal MMP‐2 and tumoural MMP‐2 and ‐9 are significant prognostic factors in invasive ductal carcinoma of the breast

Matrix metalloproteinases (MMPs) are matrix‐degrading enzymes that play a pivotal role in aggressive behaviours, such as rapid tumour growth, invasion, and metastasis, of several types of solid tumours. In particular, stromal MMP‐2 plays important roles in the progression of malignant tumours, but most clinical studies have focused on tumoural MMP‐2 and ‐9 expression, and not stromal MMP‐2 expression. One hundred and seventy‐seven cases diagnosed as invasive ductal carcinoma of the breast between 2000 and 2005 were included in this study. Expressions of tumoural MMP‐2 and ‐9 and stromal MMP‐2 were analysed by immunostaining on a tissue microarray. Subsequently, the associations between those results and various clinicopathological parameters were evaluated. Stromal MMP‐2 expression correlated significantly with clinicopathological parameters such as advanced T category, larger tumour size, high histological grade, tumour necrosis, ER‐ and PR‐negative, and HER‐2‐positive (all p < 0.05). In univariate and multivariate analyses, overall survival was linked with stromal MMP‐2 expression as well as dual expression of stromal MMP‐2 and tumoural MMP‐2 and ‐9 (all p < 0.05). Stromal MMP‐2 expression may play a crucial role in predicting aggressive clinical behaviour in breast cancer patients.     

Comparative analysis and clinical value of the expression of metalloproteases and their inhibitors by intratumour stromal mononuclear inflammatory cells and those at the invasive front of breast carcinomas

González L O, González‐Reyes S, Marín L, González L, González J M, Lamelas M L, Merino A M, Rodríguez E, Pidal I, del Casar J M, Andicoechea A & Vizoso F
(2010) Histopathology 57, 862–876 Comparative analysis and clinical value of the expression of metalloproteases and their inhibitors by intratumour stromal mononuclear inflammatory cells and those at the invasive front of breast carcinomas Aims: Matrix metalloproteases (MMPs) and their inhibitors (TIMPs) play an essential role in the degradation of stromal connective tissue and basement membrane components. The aim of this study was to determine whether the dynamic analysis of these components can help to predict tumour aggressiveness. Methods and results: An immunohistochemical study was performed using tissue arrays and specific antibodies against MMPs ‐1, ‐2, ‐7, ‐9, ‐11, ‐13 and ‐14 and TIMPs ‐1, ‐2 and ‐3. More than 5000 determinations on cancer specimens from 124 patients with invasive breast cancer were performed on the tumour centre core as well as on the invasive front. Immunostaining for MMPs/TIMPs on mononuclear inflammatory cells (MICs) was evaluated. To identify specific groups of tumours with distinct expression profiles, data obtained from both MICs populations were analysed by unsupervised hierarchical cluster analysis. When compared with MICs at the invasive front, intratumour MICs more frequently showed expression of MMP‐7 and ‐1 and TIMP‐3, but less frequently expression of MMP‐9 and ‐11 and TIMP‐2. Conclusions: Our data led us to consider the need of further studies in order to identify subsets of MICs and other protein elements of the microenvironment as attractive targets for new therapeutic strategies against cancer.     

Expansion of a mutated clone: from stem cell to tumour

Intestinal stem cells are adult, tissue-based stem cells located at the base of the intestinal crypt and are capable of regenerating all intestinal cell types. The progeny of mutated stem cells can expand to fill an entire crypt as a consequence of genetic drift, selective advantage or hitchhiking-eventually forming a clonal crypt population by a process called "niche succession". Cancer is believed to be a disease of stem cells. The digestive tract has a very high cancer prevalence partly due to rapid epithelial cell turnover and exposure to dietary toxins. Work on the hereditary cancer syndromes, including familial adenomatous polyposis (FAP), has led to significant advances, including the adenoma-carcinoma sequence. The initial mutation involved in this stepwise progression is in the "gatekeeper" tumour suppressor gene adenomatous polyposis coli (APC). In FAP somatic, second hits in this gene are non-random events, selected for by the position of the germline mutation. The early growth of adenomas is contentious, with two main theories, the "top-down" and "bottom-up" hypotheses, attempting to explain the spread of dysplastic tissue in the bowel. Initial X chromosome inactivation studies suggested that colorectal tumours were monoclonal; however, work on a rare XO/XY human patient with FAP and chimeric Min mice showed that approximately 76% of adenomas were polyclonal. A reduction in tumour multiplicity in the chimeric mouse model has been achieved by the introduction of a homozygous tumour resistance allele. This model has been used to suggest that short-range interaction between adjacent initiated crypts, not random polyp collision, is responsible for tumour polyclonality.     

Identification of the modifier of Min 2 (Mom2) locus, a new mutation that influences Apc-induced intestinal neoplasia.

Min (Multiple intestinal neoplasia) mice carry a dominant mutation in the adenomatous polyposis coli (Apc) gene and develop multiple adenomas throughout their intestinal tract (Moser et al. 1990; Su et al 1992). Polyp multiplicity in Min mice is greatly influenced by genetic background. A modifier locus, Mom1 (Modifier of Min 1), was identified and localized to distal mouse chromosome 4 (Moser et al. 1992; Dietrich et al. 1993), and accounts for some of the genetic variance in polyp multiplicity. Mom1 is a semidominant modifier of polyp size and multiplicity in Min mice (Gould and Dove 1997), and encodes the secretory type II nonpancreatic phospholipase A2 (Pla2g2a) gene (MacPhee et al. 1995; Cornier et al. 1997, 2000). We now report the identification of a second Modifier of Min 2 (Mom2) locus that is the result of a spontaneous mutation. One resistant Mom2 allele can suppress 88%-95% of polyps detected in Apc(Min)/+ mice, indicating that Mom2 acts in a dominant fashion. Linkage analysis has localized Mom2 to distal mouse chromosome 18. The effects of the Mom2 locus on reducing polyp multiplicity are stronger than the effects of the Mom1 locus, in both the small and large intestines. Some Apc(Min)/+ mice that carried one resistant Mom2 allele were tumor-free at 21 weeks of age, even in the absence of a resistant Mom1 allele. Thus, the resistant Mom2 allele can, in some cases, completely suppress the penetrance of the Apc(Min) mutation.     

Mutated K-rasAsp12 promotes tumourigenesis in ApcMin mice more in the large than the small intestines, with synergistic effects between K-ras and Wnt pathways

K‐ras mutations are found in 40–50% of human colorectal adenomas and carcinomas, but their functional contribution remains incompletely understood. Here, we show that a conditional mutant K‐ras mouse model (K‐ras^Asp12/Cre), with transient intestinal Cre activation by β‐Naphthoflavone (β‐NF) treatment, displayed transgene recombination and K‐ras^Asp12 expression in the murine intestines, but developed few intestinal adenomas over 2 years. However, when crossed with Apc^Min/+ mice, the K‐ras^Asp12/Cre/Apc^Min/+ offspring showed acceleration of intestinal tumourigenesis with significantly changed average lifespan (P < 0.05) decreased to 18.4 ± 5.4 weeks from 20.9 ± 4.7 weeks (control Apc^Min/+ mice). The numbers of adenomas in the small intestine and large intestine were significantly (P < 0.01) increased by 1.5‐fold and 5.7‐fold, respectively, in K‐ras^Asp12/Cre/Apc^Min/+ mice compared with Apc^Min/+ mice, with the more marked increase in adenoma prevalence in the large intestine. To explore possible mechanisms for K‐ras^Asp12 and Apc^Min co‐operation, the Mitogen‐activated protein kinase (Mapk), Akt and Wnt signalling pathways, including selected target gene expression levels, were evaluated in normal large intestine and large intestinal tumours. K‐ras^Asp12 increased activation of Mapk and Akt signalling pathway targets phospho‐extracellular signal‐regulated kinase (pErk) and pAkt, and increased relative expression levels of Wnt pathway targets vascular endothelial growth factor (VEGF), gastrin, cyclo‐oxygenase 2 (Cox2) and T‐cell lymphoma invasion and metastasis 1 (Tiam1) in K‐ras^Asp12/Cre/Apc^Min/+ adenomas compared with that of Apc^Min/+ adenomas, although other Wnt signalling pathway target genes such as Peroxisome proliferator‐activated receptor delta (PPARd), matrix metalloproteinase 7 (MMP7), protein phosphatase 1 alpha (PP1A) and c‐myc remained unchanged. In conclusion, intestinal expression of K‐ras^Asp12 promotes mutant Apc‐initiated intestinal adenoma formation in vivo more in the large intestine than the small intestine, with evidence of synergistic co‐operation between mutant K‐ras and Apc involving increased expression of some Wnt‐pathway target genes.     

Tumor suppressor gene adenomatous polyposis coli downregulates intestinal transport

Loss of function mutations of the tumor suppressor gene adenomatous polyposis coli (APC) underly the familial adenomatous polyposis. Mice carrying an inactivating mutation in the apc gene (apc (Min/+)) similarly develop intestinal polyposis. APC is effective at least in part by degrading β-catenin and lack of APC leads to markedly enhanced cellular β-catenin levels. β-Catenin has most recently been shown to upregulate the Na+/K+ ATPase. The present study, thus, explored the possibility that APC could influence intestinal transport. The abundance and localization of β-catenin were determined utilizing Western blotting and confocal microscopy, the activity of the electrogenic glucose carrier (SGLT1) was estimated from the glucose-induced current in jejunal segments utilizing Ussing chamber experiments and the Na+/H+ exchanger (NHE3) activity from Na+ -dependent re-alkalinization of cytosolic pH (ΔpH(i)) following an ammonium pulse employing BCECF fluorescence. As a result, β-catenin abundance in intestinal tissue was significantly higher in apc (Min/+) mice than in wild-type mice (apc (+/+)). The β-catenin protein was localized in the basolateral membrane. Both, the glucose-induced current and ΔpH(i) were significantly higher in apc (Min/+) mice than in apc (+/+) mice. In conclusion, intestinal electrogenic transport of glucose and intestinal Na+/H+ exchanger activity are both significantly enhanced in apc (Min/+) mice, pointing to a role of APC in the regulation of epithelial transport.     

Helicobacter hepaticus infection promotes colon tumorigenesis in the BALB/c-Rag2(-/-) Apc(Min/+) mouse

Adenomatous polyposis coli (APC) mutations are linked to human and mouse colorectal cancers. The Apc multiple intestinal neoplasia (Min) mouse mutation causes adenomas to develop throughout the small and large intestines. The BALB-Min (C.B6-Apc(Min/+)) congenic strain was generated by backcrossing into BALB/c the Apc(Min) allele from C57BL/6J-Apc(Min/+) mice. BALB-Min mice have a low tumor multiplicity (27.4 small intestine tumors/mouse) and a relatively long life span (>1 year) that makes them amenable to long-term studies. To investigate the interplay of the adaptive immune system and intestinal tumorigenesis, the immunodeficient compound mutant strain BALB-RagMin (C.Cg-Rag2(-/-) Apc(Min/+)) was generated. BALB-RagMin mice had a significant increase in tumors in the small, but not large, intestine relative to their BALB-Min counterparts (43.0 versus 24.0 tumors/mouse, respectively). The results suggest that the adaptive immune system plays a role in either the elimination or the equilibrium phase of cancer immunoediting in the small intestine in this model. We investigated the effect of the enterohepatic bacterial pathogen Helicobacter hepaticus on liver and intestine tumorigenesis in BALB-RagMin mice. H. hepaticus-infected BALB-RagMin mice developed moderate hepatitis, moderate typhlitis, and mild colitis. There were no differences in small intestine and cecal tumor multiplicity, regionality, or size relative to that in uninfected mice. However, H. hepaticus-infected BALB-RagMin mice had a significant increase in colon tumor incidence relative to uninfected BALB-RagMin mice (23.5% versus 1.7%, respectively). The data suggest that H. hepaticus, which is present in many research colonies, promotes colon tumorigenesis in the BALB-RagMin mouse and that it has the potential to confound colon tumorigenesis studies.     

The C-terminus of Apc does not influence intestinal adenoma development or progression

Adenomatous polyposis coli (APC ) mutations are found in most colorectal tumours. These mutations are almost always protein-truncating, deleting both central domains that regulate Wnt signalling and C-terminal domains that interact with the cytoskeleton. The importance of Wnt dysregulation for colorectal tumourigenesis is well characterized. It is, however, unclear whether loss of C-terminal functions contributes to tumourigenesis, although this protein region has been implicated in cellular processes--including polarity, migration, mitosis, and chromosomal instability (CIN)—that have been postulated as critical for the development and progression of intestinal tumours. Since almost all APC mutations in human patients disrupt both central and C-terminal regions, we created a mouse model to test the role of the C-terminus of APC in intestinal tumourigenesis. This mouse (Apc(ΔSAMP)) carries an internal deletion within Apc that dysregulates Wnt by removing the beta-catenin-binding and SAMP repeats, but leaves the C-terminus intact. We compared Apc(ΔSAMP) mice with Apc(1322T) animals. The latter allele represented the most commonly found human APC mutation and was identical to Apc(ΔSAMP) except for absence of the entire C-terminus. Apc(ΔSAMP) mice developed numerous intestinal adenomas indistinguishable in number, location, and dysplasia from those seen in Apc(1322T) mice. No carcinomas were found in Apc(ΔSAMP) or Apc(1322T) animals. While similar disruption of the Wnt signalling pathway was observed in tumours from both mice, no evidence of differential C-terminus functions (such as cell migration, CIN, or localization of APC and EB1) was seen. We conclude that the C-terminus of APC does not influence intestinal adenoma development or progression.     

E1AF expression is associated with extra‐prostatic growth and matrix metalloproteinase‐7 expression in prostate cancer

E1AF is associated with malignant aggressiveness via regulation of matrix metalloproteinases (MMPs), which play pivotal roles in invasion through the degradation of extracellular matrix of tissues surrounding tumors. However, the clinical significance of E1AF and MMPs in patients with prostate cancer is not fully understood. We reviewed 50 tissue samples from patients with T2‐3N0M0 prostate cancer who had undergone radical operation. Expression levels of E1AF, MMP‐1, ‐3, ‐7, ‐9 and ‐14 were determined semiquantitatively by immunohistochemistry. The mean ± SD percentage of E1AF‐stained cancer cells was 8.56 ± 5.22, and it was significantly higher (p < 0.001) than the E1AF‐immunostaining index of normal cells (1.17 ± 0.61). E1AF immunostaining index in pT3 (12.74 ± 4.80) was significantly higher (p < 0.001) than that in pT2 (5.78 ± 3.31). Although E1AF expression correlated with that of MMP‐7 and MMP‐9 (r = 0.47, p < 0.001 and r = 0.41, p = 0.004, respectively), multivariate analysis showed that E1AF correlated with only MMP‐7 expression (OR = 5.81, 95% CI = 1.27–26.59, p = 0.023). Our results demonstrated that increased expression of E1AF is involved in tumor aggression of prostate cancer. This finding may be influenced by regulation of MMP‐7. We speculate that E1AF is a possible target in treatment and prevention of tumor growth in prostate cancer.     

A urokinase-type plasminogen activator deficiency diminishes the frequency of intestinal adenomas in ApcMin/+ mice

The interaction of urokinase-type plasminogen activator (uPA) and its receptor, uPAR, on cell surfaces facilitates the generation of cell-bound plasmin, thus allowing cells to establish a proteolytic front that enables their migration through protein barriers. This complex also activates cell signalling pathways that influence cell functions. Clinical studies have identified uPA as an indicator of poor overall survival in patients with colorectal cancer. In the current study, a mouse model of colon cancer, Apc(Min/+), with an additional deficiency of uPA (Apc(Min/+)/Plau-/-) was used to determine the effects of uPA on tumour initiation and growth. Utilizing this model, it was found that the number of tumours was diminished in these mice relative to Apc(Min/+) mice, which correlated with the decreased leukocyte infiltration in the tumours. However, tumour growth was not impeded in Apc(Min/+)/Plau-/- mice, and proliferation and tumour vascularization were, in fact, enhanced in Apc(Min/+)/Plau-/- mice. These latter effects are consistent with a mechanism involving up-regulation of COX-2 expression and Akt pathway activation in Apc(Min/+)/Plau-/- mice. The results from this study suggest that uPA plays dual and opposing roles in regulating lesion development: one early, during the transition from normal epithelia to dysplastic lesions, and another later during tumour growth.     

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in thyroid C-cells and medullary thyroid carcinomas

Aims : Hyperplastic C-cells of the thyroid and medullary thyroid carcinomas (MTCs) were studied immunocytochemically for calcitonin, carcinoembryonic antigen (CEA), chromogranin A, matrix metalloproteinase (MMP)-2 and -9, and tissue inhibitor of metalloproteinase (TIMP)-1 and -2. Methods and results : Non-neoplastic C-cells were positive for all these substances whereas MTC tumour cells were relatively weaker stained, and thyroid follicular cells were negative for all the substances studied. We have recently reported that pancreatic islet cells and islet cell tumours were positive for MMPs and TIMPs, and, in addition, anterior pituitary cells and pituitary adenomas and parathyroid gland and its adenomas were also positively stained. The MMP- and TIMP-positive endocrine cells correspond to Pearse's APUD cells, derived from neural crest and endodermal cells. Conclusions : MMPs and TIMPs may well be added as newly recognized markers for neuroendocrine cells. The possible function of MMP-TIMP homeostasis in C-cells and MTCs is discussed.     

Genetic determinants modulate susceptibility to pregnancy-associated tumourigenesis in a recombinant line of Min mice

Min mice provide a good model of human familial adenomatous polyposis. Recently, we have reported on two recombinant inbred lines (I and V) and the location of a modifier (Mom3) close to Apc, which altered polyp numbers in our mice possibly by modifying the frequency of wild-type (WT) allele loss at Apc; mice with severe disease (line V) showed elevated rates of loss. We now show that in line I only, a single pregnancy caused a significant increase in adenoma multiplicity compared with virgin controls (P<0.001) and that an additional pregnancy conferred a similar risk. Pregnancy was linked to both adenoma initiation and enhanced tumour growth in line I mice, and interline crosses indicated that susceptibility to pregnancy-associated adenomas was under genetic control. We found no evidence for the involvement of oestrodial metabolizing genes or the oestrogen receptors (Esr1 and 2) in tumour multiplicity. Importantly, a significantly elevated frequency of WT allele loss at Apc was observed in adenomas from parous mice (line and backcrossed) carrying the line I Min allele relative to equivalent virgin controls (P=0.015). Our results provide the first experimental evidence for genetic determinants controlling pregnancy-associated tumourigenesis; analogous genetic factors may exist in humans.     

Matrix metalloproteinases in lung: multiple, multifarious, and multifaceted

The matrix metalloproteinases (MMPs), a family of 25 secreted and cell surface-bound neutral proteinases, process a large array of extracellular and cell surface proteins under normal and pathological conditions. MMPs play critical roles in lung organogenesis, but their expression, for the most part, is downregulated after generation of the alveoli. Our knowledge about the resurgence of the MMPs that occurs in most inflammatory diseases of the lung is rapidly expanding. Although not all members of the MMP family are found within the lung tissue, many are upregulated during the acute and chronic phases of these diseases. Furthermore, potential MMP targets in the lung include all structural proteins in the extracellular matrix (ECM), cell adhesion molecules, growth factors, cytokines, and chemokines. However, what is less known is the role of MMP proteolysis in modulating the function of these substrates in vivo. Because of their multiplicity and substantial substrate overlap, MMPs are thought to have redundant functions. However, as we explore in this review, such redundancy most likely evolved as a necessary compensatory mechanism given the critical regulatory importance of MMPs. While inhibition of MMPs has been proposed as a therapeutic option in a variety of inflammatory lung conditions, a complete understanding of the biology of these complex enzymes is needed before we can reasonably consider them as therapeutic targets.