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Elevated cancer metabolism releases lactic acid and CO 2 into the under-perfused tumor microenvironment, resulting in extracellular acidosis. The surviving cancer cells must adapt to this selection pressure; thus, targeting tumor acidosis is a rational therapeutic strategy to manage tumor growth. However, none of the major approved treatments are based explicitly on disrupting acid handling, signaling, or adaptations, possibly because the distinction between acid-sensitive and acid-resistant phenotypes is not clear. Here, we report pH-related phenotypes of sixty-eight colorectal cancer (CRC) cell lines by measuring i) extracellular acidification as a readout of acid production by fermentative metabolism and ii) growth of cell biomass over a range of extracellular pH (pHe) levels as a measure of the acid sensitivity of proliferation. Based on these measurements, CRC cell lines were grouped along two dimensions as "acid-sensitive"/"acid-resistant" versus "low metabolic acid production"/"high metabolic acid production." Strikingly, acid resistance was associated with the expression of CEACAM6 and CEACAM5 genes coding for two related cell-adhesion molecules, and among pH-regulating genes, of CA12 . CEACAM5/6 protein levels were strongly induced by acidity, with a further induction under hypoxia in a subset of CRC lines. Lack of CEACAM6 (but not of CEACAM5) reduced cell growth and their ability to differentiate. Finally, CEACAM6 levels were strongly increased in human colorectal cancers from stage II and III patients, compared to matched samples from adjacent normal tissues. Thus, CEACAM6 is a marker of acid-resistant clones in colorectal cancer and a potential motif for targeting therapies to acidic regions within the tumors., Competing Interests: Competing interests statement:The authors declare no competing interest.

The naked mole rat (NMR), Heterocephalus glaber, the longest-living rodent, provides a unique opportunity to explore how evolution has shaped adult stem cell (ASC) activity and tissue function with increasing lifespan. Using cumulative BrdU labelling and a quantitative imaging approach to track intestinal ASCs (Lgr5 + ) in their native in vivo state, we find an expanded pool of Lgr5 + cells in NMRs, and these cells specifically at the crypt base (Lgr5 +CBC ) exhibit slower division rates compared to those in short-lived mice but have a similar turnover as human LGR5 +CBC cells. Instead of entering quiescence (G0), NMR Lgr5 +CBC cells reduce their division rates by prolonging arrest in the G1 and/or G2 phases of the cell cycle. Moreover, we also observe a higher proportion of differentiated cells in NMRs that confer enhanced protection and function to the intestinal mucosa which is able to detect any chemical imbalance in the luminal environment efficiently, triggering a robust pro-apoptotic, anti-proliferative response within the stem/progenitor cell zone., (© 2023. The Author(s).)

Acidic environments reduce the intracellular pH (pHi) of most cells to levels that are sub-optimal for growth and cellular functions. Yet, cancers maintain an alkaline cytoplasm despite low extracellular pH (pHe). Raised pHi is thought to be beneficial for tumor progression and invasiveness. However, the transport mechanisms underpinning this adaptation have not been studied systematically. Here, we characterize the pHe-pHi relationship in 66 colorectal cancer cell lines and identify the acid-loading anion exchanger 2 (AE2, SLC4A2) as a regulator of resting pHi. Cells adapt to chronic extracellular acidosis by degrading AE2 protein, which raises pHi and reduces acid sensitivity of growth. Acidity inhibits mTOR signaling, which stimulates lysosomal function and AE2 degradation, a process reversed by bafilomycin A1. We identify AE2 degradation as a mechanism for maintaining a conducive pHi in tumors. As an adaptive mechanism, inhibiting lysosomal degradation of AE2 is a potential therapeutic target., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Cancer evolution consists of a stepwise acquisition of genetic and epigenetic changes, which alter the gene expression profiles of cells in a particular tissue and result in phenotypic alterations acted upon by natural selection. The recurrent appearance of specific genetic lesions across individual cancers and cancer types suggests the existence of certain "driver mutations," which likely make up the major contribution to tumors' selective advantages over surrounding normal tissue and as such are responsible for the most consequential aspects of the cancer cells' gene expression patterns and phenotypes. We hypothesize that such mutations are likely to cluster with specific dichotomous shifts in the expression of the genes they most closely control, and propose GMMchi, a Python package that leverages Gaussian Mixture Modeling to detect and characterize bimodal gene expression patterns across cancer samples, as a tool to analyze such correlations using 2 × 2 contingency table statistics., Results: Using well-defined simulated data, we were able to confirm the robust performance of GMMchi, reaching 85% accuracy with a sample size of n = 90. We were also able to demonstrate a few examples of the application of GMMchi with respect to its capacity to characterize background florescent signals in microarray data, filter out uninformative background probe sets, as well as uncover novel genetic interrelationships and tumor characteristics. Our approach to analysing gene expression analysis in cancers provides an additional lens to supplement traditional continuous-valued statistical analysis by maximizing the information that can be gathered from bulk gene expression data., Conclusions: We confirm that GMMchi robustly and reliably extracts bimodal patterns from both colorectal cancer (CRC) cell line-derived microarray and tumor-derived RNA-Seq data and verify previously reported gene expression correlates of some well-characterized CRC phenotypes., Availability: The Python package GMMchi and our cell line microarray data used in this paper is available for downloading on GitHub at https://github.com/jeffliu6068/GMMchi ., (© 2022. The Author(s).)

Growth of cancer cells in vitro can be attenuated by genetically inactivating selected metabolic pathways. However, loss-of-function mutations in metabolic pathways are not negatively selected in human cancers, indicating that these genes are not essential in vivo. We hypothesize that spontaneous mutations in 'metabolic genes' will not necessarily produce functional defects because mutation-bearing cells may be rescued by metabolite exchange with neighboring wild-type cells via gap junctions. Using fluorescent substances to probe intercellular diffusion, we show that colorectal cancer (CRC) cells are coupled by gap junctions assembled from connexins, particularly Cx26. Cells with genetically inactivated components of pH regulation ( SLC9A1 ), glycolysis ( ALDOA ), or mitochondrial respiration ( NDUFS1 ) could be rescued through access to functional proteins in co-cultured wild-type cells. The effect of diffusive coupling was also observed in co-culture xenografts. Rescue was largely dependent on solute exchange via Cx26 channels, a uniformly and constitutively expressed isoform in CRCs. Due to diffusive coupling, the emergent phenotype is less heterogenous than its genotype, and thus an individual cell should not be considered as the unit under selection, at least for metabolite-handling processes. Our findings can explain why certain loss-of-function mutations in genes ascribed as 'essential' do not influence the growth of human cancers., Competing Interests: SM, JM, AH, JK, EB, AH, GA, WB, PS No competing interests declared, (© 2022, Monterisi et al.)

Short tandem repeat (STR) polymorphisms are traditionally assessed by measuring allele lengths via capillary electrophoresis (CE). Massively parallel sequencing (MPS) reveals differences among alleles of the same length, thus improving discrimination, but also identifying groups of alleles likely related by descent. These may have relatively restricted geographical distributions and thus MPS could detect population structure more effectively than CE-based analysis. We addressed this question by applying an MPS multiplex, the Promega PowerSeq™ Auto/Mito/Y System prototype, to 362 individuals chosen to represent a wide geographical spread from the People of the British Isles (PoBI) cohort, which represents at least three generations of local rural ancestry. As well as 22 autosomal STRs (aSTRs; equivalent to PowerPlex Fusion loci) the system sequences 23 Y-STRs (the PowerPlexY 23 loci) and the control region (CR) of mitochondrial DNA (mtDNA), allowing population structure to be compared across biparentally and uniparentally inherited segments of the genome. For all loci, F ST -based tests of population structure were done based on historical, linguistic, and geographical partitions, and for aSTRs the clustering algorithm STRUCTURE was also applied. STRs were considered using both length and sequence. Sequencing increased aSTR allele diversity by 87.5% compared to CE-based designations, reducing random match probability to 1.25E-30, compared to a CE-based 6.72E-27. Significant population structure was detectable in just one pairwise comparison (Central/South East England compared to the rest), and for sequence-based alleles only. The 362 samples carried 308 distinct mtDNA CR haplotypes corresponding to 13 broad haplogroups, representing a haplotype diversity of 0.9985 ( ± 0.0005), and a haplotype match probability of 0.0043. No significant population structure was observed. Y-STR haplotypes belonged to ten broad predicted Y-haplogroups. Allele diversity increased by 33% when considered at the sequence rather than length level, although haplotype diversity was unchanged at 0.999969 ( ± 0.000001); haplotype match probability was 2.79E-03. In contrast to the biparentally and maternally inherited loci, Y-STR haplotypes showed significant population structure at several levels, but most markedly in a comparison of regions subject to Anglo-Saxon influence in the east with the rest of the sample. This was evident for both length- and sequence-based allele designations, with no systematic difference between the two. We conclude that MPS analysis of aSTRs or Y-STRs does not generally reveal stronger population structure than length-based analysis, that UK maternal lineages are not significantly structured, and that Y-STR haplotypes reveal significant population structure that may reflect the Anglo-Saxon migrations to Britain in the 6th century., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Unlike most cell types, many cancer cells survive at low extracellular pH (pHe), a chemical signature of tumors. Genes that facilitate survival under acid stress are therefore potential targets for cancer therapies. We performed a genome-wide CRISPR-Cas9 cell viability screen at physiological and acidic conditions to systematically identify gene knockouts associated with pH-related fitness defects in colorectal cancer cells. Knockouts of genes involved in oxidative phosphorylation (NDUFS1) and iron-sulfur cluster biogenesis (IBA57, NFU1) grew well at physiological pHe, but underwent profound cell death under acidic conditions. We identified several small-molecule inhibitors of mitochondrial metabolism that can kill cancer cells at low pHe only. Xenografts established from NDUFS1 -/- cells grew considerably slower than their wild-type controls, but growth could be stimulated with systemic bicarbonate therapy that lessens the tumoral acid stress. These findings raise the possibility of therapeutically targeting mitochondrial metabolism in combination with acid stress as a cancer treatment option., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: The molecular pathogenesis of solid tumour was first assessed in colorectal cancer (CRC). To date, ≤ 100 genes with somatic alterations have been found to inter-connectively promote neoplastic transformation through specific pathways. The process of colorectal carcinogenesis via genome landscape is reviewed on the basis of an adenoma-to-carcinoma sequence, as shown by serial histological and epidemiological observations., Methods: The relevant literatures from PubMed (1980-2021) have been reviewed for this article., Results: The major routes of CRC development, chromosomal instability (CIN), microsatellite instability (MSI), and the serrated route either via CIN or MSI, proceed through the respective molecular pathway of colorectal carcinogenesis. Particular aspects of CRC carcinogenesis can also be determined by evaluating familial CRCs (FCRC) and genotype-phenotype correlations. Specific causative gene alterations still leave to be identified in several FCRCs. Otherwise, recently verified FCRC can be particularly notable, for example, EPCAM-associated Lynch syndrome, polymerase proofreading-associated polyposis, RNF43-associated polyposis syndrome or NTHL1 tumour syndrome, and hereditary mixed polyposis syndrome. The oncogenic landscape is described, including representative pathway genes, the three routes of carcinogenesis, familial CRCs, genotype-phenotype correlations, the identification of causative genes, and consensus molecular subtypes (CMS)., Conclusion: Whole genome research using multi-gene panels (MGPs) has facilitated high through-put detection of previously unidentified genes involved in colorectal carcinogenesis. New approaches designed to identify rare variants are recommended to consider their alterations implicated in the molecular pathogenesis., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Following a brief over view of the contents of Michael Wooldridge's book I give an account of my own background in computing and AI. I then cover a range of topics stimulated by reading the book including machine learning's relationship to AI, applications to medical areas, the need to consider probabilistic effects on decisions, the importance of self-reproduction and whether AI can be made moral. I finish with a discussion of the mind-brain relationship and what makes us human., (© The Author(s) 2022.)

The genomic causes and clinical manifestations of hereditary colorectal cancer (HCRC) might be stratified into 2 groups, namely, familial (FCRC) and a limited sense of HCRC, respectively. Otherwise, FCRC is canonically classified into 2 major categories; Lynch syndrome (LS) or associated spectra and inherited polyposis syndrome. By contrast, despite an increasing body of genotypic and phenotypic traits, some FCRC cannot be clearly differentiated as definitively single type, and the situation has become more complex as additional causative genes have been discovered. This review provides an overview of HCRC, including 6 LS or associated spectra and 8 inherited polyposis syndromes, according to molecular pathogenesis. Variants and newly-identified FCRC are particularly emphasized, including MUTYH (or MYH)-associated polyposis, Muir-Torre syndrome, constitutional mismatch repair deficiency, EPCAM-associated LS, polymerase proofreading-associated polyposis, RNF43- or NTHL1-associated serrated polyposis syndrome, PTEN hamartoma tumor syndrome, and hereditary mixed polyposis syndrome. We also comment on the clinical utility of multigene panel tests, focusing on comprehensive cancer panels that include HCRC. Finally, HCRC surveillance strategies are recommended, based on revised or notable concepts underpinned by competent validation and clinical implications, and favoring major guidelines. As hereditary syndromes are mainly attributable to genomic constitutions of distinctive ancestral groups, an integrative national HCRC registry and guideline is an urgent priority.

Background/aim: Interactions between colorectal cancer (CRC) cells and myofibroblasts govern many processes such as cell growth, migration, invasion and differentiation, and contribute to CRC progression. Robust experimental tests are needed to investigate the nature of these interactions for future anticancer studies. The purpose of the study was to design and validate in vitro assays for studying the communication between myofibroblasts and CRC epithelial cell lines., Materials and Methods: The influence of co-culture of myofibroblasts and CRC cell lines is discussed using various in vitro assays including direct co-culture, transwell assays, Matrigel-based differentiation and cell invasion experiments., Results: The results from these in vitro assays clearly demonstrated various aspects of the crosstalk between myofibroblasts and CRC cell lines, which include cell growth, differentiation, migration and invasion., Conclusion: The reported in vitro assays provide a basis for investigating the factors that control the myofibroblast-epithelial cell interactions in CRC in vivo., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

The reconciliation between Mendelian inheritance of discrete traits and the genetically based correlation between relatives for quantitative traits was Fisher's infinitesimal model of a large number of genetic variants, each with very small effects, whose causal effects could not be individually identified. The development of genome-wide genetic association studies (GWAS) raised the hope that it would be possible to identify single polymorphic variants with identifiable functional effects on complex traits. It soon became clear that, with larger and larger GWAS on more and more complex traits, most of the significant associations had such small effects, that identifying their individual functional effects was essentially hopeless. Polygenic risk scores that provide an overall estimate of the genetic propensity to a trait at the individual level have been developed using GWAS data. These provide useful identification of groups of individuals with substantially increased risks, which can lead to recommendations of medical treatments or behavioral modifications to reduce risks. However, each such claim will require extensive investigation to justify its practical application. The challenge now is to use limited genetic association studies to find individually identifiable variants of significant functional effect that can help to understand the molecular basis of complex diseases and traits, and so lead to improved disease prevention and treatment. This can best be achieved by 1) the study of rare variants, often chosen by careful candidate assessment, and 2) the careful choice of phenotypes, often extremes of a quantitative variable, or traits with relatively high heritability., Competing Interests: The authors declare no competing interest.

Any replicating system in which heritable variants with differing replicative potentials can arise is subject to a Darwinian evolutionary process. The continually replicating adult tissue stem cells that control the integrity of many tissues of long-lived, multicellular, complex vertebrate organisms, including humans, constitute such a replicating system. Our suggestion is that somatic selection for mutations (or stable epigenetic changes) that cause an increased rate of adult tissue stem cell proliferation, and their long-term persistence, at the expense of normal differentiation, is a major key to the ageing process. Once an organism has passed the reproductive age, there is no longer any significant counterselection at the organismal level to this inevitable cellular level Darwinian process., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020. Published by Elsevier Ltd.)

Colonic epithelial cells are highly polarised with a lumen-facing apical membrane, termed the brush border, and a basal membrane in contact with the underlying extracellular matrix (ECM). This polarity is often maintained in cancer tissue in the form of neoplastic glands and has prognostic value. We compared the cellular polarity of several ex vivo spheroid colonic cancer cultures with their parental tumours and found that those grown as non-attached colonies exhibited apical brush border proteins on their outer cellular membranes. Transfer of these cultures to an ECM, such as collagen, re-established the centralised apical polarity observed in vivo. The multidrug resistance protein ABCB1 also became aberrantly polarised to outer colony membranes in suspension cultures, unlike cultures grown in collagen, where it was polarised to central lumens. This polarity switch was dependent on the presence of serum or selected serum components, including epidermal growth factor (EGF), transforming growth factor-β1 (TGF-β1) and insulin-like growth factor-1 (IGF-1). The apical/basal orientation of primary cancer colon cultures cultured in collagen/serum was modulated by α2β1 integrin signalling. The polarisation of ABCB1 in colonies significantly altered drug uptake and sensitivity, as the outward polarisation of ABCB1 in suspension colonies effluxed substrates more effectively than ECM-grown colonies with ABCB1 polarised to central lumens. Thus, serum-free suspension colonies were more resistant to a variety of anti-cancer drugs than ECM-grown colonies. In conclusion, the local stroma, or absence thereof, can have profound effects on the sensitivity of colorectal cultures to drugs that are ABCB1 substrates. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

To elucidate cellular diversity and clonal evolution in tissues and tumors, one must resolve genomic heterogeneity in single cells. To this end, we have developed low-cost, mass-producible micro-/nanofluidic chips for DNA extraction from individual cells. These chips have modules that collect genomic DNA for sequencing or map genomic structure directly, on-chip, with denaturation-renaturation (D-R) optical mapping [Marie R, et al. (2013) Proc Natl Acad Sci USA 110:4893-4898]. Processing of single cells from the LS174T colorectal cancer cell line showed that D-R mapping of single molecules can reveal structural variation (SV) in the genome of single cells. In one experiment, we processed 17 fragments covering 19.8 Mb of the cell's genome. One megabase-large fragment aligned well to chromosome 19 with half its length, while the other half showed variable alignment. Paired-end single-cell sequencing supported this finding, revealing a region of complexity and a 50-kb deletion. Sequencing struggled, however, to detect a 20-kb gap that D-R mapping showed clearly in a megabase fragment that otherwise mapped well to the reference at the pericentromeric region of chromosome 4. Pericentromeric regions are complex and show substantial sequence homology between different chromosomes, making mapping of sequence reads ambiguous. Thus, D-R mapping directly, from a single molecule, revealed characteristics of the single-cell genome that were challenging for short-read sequencing., Competing Interests: Conflict of interest statement: R.M., J.N.P., A.K., and K.U.M. filed patents. K.U.M. declares that XGenomes is developing nucleic acid sequencing technologies.

To discover specific variants with relatively large effects on the human face, we have devised an approach to identifying facial features with high heritability. This is based on using twin data to estimate the additive genetic value of each point on a face, as provided by a 3D camera system. In addition, we have used the ethnic difference between East Asian and European faces as a further source of face genetic variation. We use principal components (PCs) analysis to provide a fine definition of the surface features of human faces around the eyes and of the profile, and chose upper and lower 10% extremes of the most heritable PCs for looking for genetic associations. Using this strategy for the analysis of 3D images of 1,832 unique volunteers from the well-characterized People of the British Isles study and 1,567 unique twin images from the TwinsUK cohort, together with genetic data for 500,000 SNPs, we have identified three specific genetic variants with notable effects on facial profiles and eyes., Competing Interests: The authors declare no conflict of interest.

Proliferation and invasion of cancer cells require favorable pH, yet potentially toxic quantities of acid are produced metabolically. Membrane-bound transporters extrude acid from cancer cells, but little is known about the mechanisms that handle acid once it is released into the poorly perfused extracellular space. Here, we studied acid handling by myofibroblasts (colon cancer-derived Hs675.T, intestinal InMyoFib, embryonic colon-derived CCD-112-CoN), skin fibroblasts (NHDF-Ad), and colorectal cancer (CRC) cells (HCT116, HT29) grown in monoculture or coculture. Expression of the acid-loading transporter anion exchanger 2 (AE2) (SLC4A2 product) was detected in myofibroblasts and fibroblasts, but not in CRC cells. Compared with CRC cells, Hs675.T and InMyoFib myofibroblasts had very high capacity to absorb extracellular acid. Acid uptake into CCD-112-CoN and NHDF-Ad cells was slower and comparable to levels in CRC cells, but increased alongside SLC4A2 expression under stimulation with transforming growth factor β1 (TGFβ1), a cytokine involved in cancer-stroma interplay. Myofibroblasts and fibroblasts are connected by gap junctions formed by proteins such as connexin-43, which allows the absorbed acid load to be transmitted across the stromal syncytium. To match the stimulatory effect on acid uptake, cell-to-cell coupling in NHDF-Ad and CCD-112-CoN cells was strengthened with TGFβ1. In contrast, acid transmission was absent between CRC cells, even after treatment with TGFβ1. Thus, stromal cells have the necessary molecular apparatus for assembling an acid-venting route that can improve the flow of metabolic acid through tumors. Importantly, the activities of stromal AE2 and connexin-43 do not place an energetic burden on cancer cells, allowing resources to be diverted for other activities., Competing Interests: The authors declare no conflict of interest.

Pericryptal myofibroblasts in the colon and rectum play an important role in regulating the normal colorectal stem cell niche and facilitating tumor progression. Myofibroblasts previously have been distinguished from normal fibroblasts mostly by the expression of α smooth muscle actin (αSMA). We now have identified AOC3 (amine oxidase, copper containing 3), a surface monoamine oxidase, as a new marker of myofibroblasts by showing that it is the target protein of the myofibroblast-reacting mAb PR2D3. The normal and tumor tissue distribution and the cell line reactivity of AOC3 match that expected for myofibroblasts. We have shown that the surface expression of AOC3 is sensitive to digestion by trypsin and collagenase and that anti-AOC3 antibodies can be used for FACS sorting of myofibroblasts obtained by nonenzymatic procedures. Whole-genome microarray mRNA-expression profiles of myofibroblasts and skin fibroblasts revealed four additional genes that are significantly differentially expressed in these two cell types: NKX2-3 and LRRC17 in myofibroblasts and SHOX2 and TBX5 in skin fibroblasts. TGFβ substantially down-regulated AOC3 expression in myofibroblasts but in skin fibroblasts it dramatically increased the expression of αSMA. A knockdown of NKX2-3 in myofibroblasts caused a decrease of myofibroblast-related gene expression and increased expression of the fibroblast-associated gene SHOX2, suggesting that NKX2-3 is a key mediator for maintaining myofibroblast characteristics. Our results show that colorectal myofibroblasts, as defined by the expression of AOC3, NKX2-3, and other markers, are a distinctly different cell type from TGFβ-activated fibroblasts.

The transcription factor caudal-type homeobox 1 (CDX1) is a key regulator of differentiation in the normal colon and in colorectal cancer (CRC). CDX1 activates the expression of enterocyte genes, but it is not clear how the concomitant silencing of stem cell genes is achieved. MicroRNAs (miRNAs) are important mediators of gene repression and have been implicated in tumor suppression and carcinogenesis, but the roles of miRNAs in differentiation, particularly in CRC, remain poorly understood. Here, we identified microRNA-215 (miR-215) as a direct transcriptional target of CDX1 by using high-throughput small RNA sequencing to profile miRNA expression in two pairs of CRC cell lines: CDX1-low HCT116 and HCT116 with stable CDX1 overexpression, and CDX1-high LS174T and LS174T with stable CDX1 knockdown. Validation of candidate miRNAs identified by RNA-seq in a larger cell-line panel revealed miR-215 to be most significantly correlated with CDX1 expression. Quantitative ChIP-PCR and promoter luciferase assays confirmed that CDX1 directly activates miR-215 transcription. miR-215 expression is depleted in FACS-enriched cancer stem cells compared with unsorted samples. Overexpression of miR-215 in poorly differentiated cell lines causes a decrease in clonogenicity, whereas miR-215 knockdown increases clonogenicity and impairs differentiation in CDX1-high cell lines. We identified the genome-wide targets of miR-215 and found that miR-215 mediates the repression of cell cycle and stemness genes downstream of CDX1. In particular, the miR-215 target gene BMI1 has been shown to promote stemness and self-renewal and to vary inversely with CDX1. Our work situates miR-215 as a link between CDX1 expression and BMI1 repression that governs differentiation in CRC.

Background: Opportunities provided by rapidly increasing access to educational resources, clinical and epidemiological data, DNA collections, cheaper technology and financial investment, suggest that researchers in sub-Saharan Africa outside South Africa (SSAOSA) could now join the genomics revolution on equal terms with those in the West., Findings: Current evidence, however, suggests that, in some cases, various factors may be compromising this development. One interpretation is that urgent practical problems, which may compromise motivation, aspiration and ambition, are blocking opportunity., Conclusions: Those wishing to help should support the SSAOSA scientists both at the level of extending collaboration networks and in stimulating academic leadership at national and institutional levels to ensure adequate resources are allocated. Members of organisations representing the international community of human geneticists, such as HUGO, have a significant responsibility in supporting such activities.

Drugs that inhibit insulin-like growth factor 1 (IGFI) receptor IGFIR were encouraging in early trials, but predictive biomarkers were lacking and the drugs provided insufficient benefit in unselected patients. In this study, we used genetic screening and downstream validation to identify the WNT pathway element DVL3 as a mediator of resistance to IGFIR inhibition. Sensitivity to IGFIR inhibition was enhanced specifically in vitro and in vivo by genetic or pharmacologic blockade of DVL3. In breast and prostate cancer cells, sensitization tracked with enhanced MEK-ERK activation and relied upon MEK activity and DVL3 expression. Mechanistic investigations showed that DVL3 is present in an adaptor complex that links IGFIR to RAS, which includes Shc, growth factor receptor-bound-2 (Grb2), son-of-sevenless (SOS), and the tumor suppressor DAB2. Dual DVL and DAB2 blockade synergized in activating ERKs and sensitizing cells to IGFIR inhibition, suggesting a nonredundant role for DVL3 in the Shc-Grb2-SOS complex. Clinically, tumors that responded to IGFIR inhibition contained relatively lower levels of DVL3 protein than resistant tumors, and DVL3 levels in tumors correlated inversely with progression-free survival in patients treated with IGFIR antibodies. Because IGFIR does not contain activating mutations analogous to EGFR variants associated with response to EGFR inhibitors, we suggest that IGF signaling achieves an equivalent integration at the postreceptor level through adaptor protein complexes, influencing cellular dependence on the IGF axis and identifying a patient population with potential to benefit from IGFIR inhibition., (©2014 American Association for Cancer Research.)

We have developed a simple procedure for deriving pure cultures of growing cancer cells from colorectal cancers, including material refrigerated overnight, for pathological characterization and cytotoxicity assays. Forty-six cancers were processed and cultures set up under varying culture conditions. Use of a Rho kinase (ROCK1) inhibitor markedly increased culture survival, resulting in 80% of samples growing in culture for at least 1 month and beyond. Overnight refrigeration of samples before culture initiation had little effect on success rates, paving the way for cultures to be established for samples collected over wide geographical areas, such as those for clinical trials. Primary cultures demonstrated good correlation for differentiation markers compared to parent cancers, and were highly dynamic in 3D culture. In Matrigel, many colonies formed central lumens, indicating the presence of stem-like cells. Viable colonies in these cultures recapitulated the in vivo generation of carcinoembryonic antigen (CEA)-positive necrotic/apoptotic debris, much of which was derived from abnormal vacuolated dynamic 'bubble cells' that have not previously been described. Although bubble cells morphologically resembled signet ring cells, a rare cancer subtype, immunostaining suggested that they were most likely derived from terminally differentiated enterocytes. Micro-assays showed that drug toxicity could be measured in these cultures within hours and with sensitivity down to a few hundred cells. Primary cultures derived by our method provide valid in vitro avatars for studying the pathology of cancers in vitro and are amenable to pre-clinical drug testing, paving the way for personalized cancer treatment., (Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Human colorectal cancer cell lines are used widely to investigate tumor biology, experimental therapy, and biomarkers. However, to what extent these established cell lines represent and maintain the genetic diversity of primary cancers is uncertain. In this study, we profiled 70 colorectal cancer cell lines for mutations and DNA copy number by whole-exome sequencing and SNP microarray analyses, respectively. Gene expression was defined using RNA-Seq. Cell line data were compared with those published for primary colorectal cancers in The Cancer Genome Atlas. Notably, we found that exome mutation and DNA copy-number spectra in colorectal cancer cell lines closely resembled those seen in primary colorectal tumors. Similarities included the presence of two hypermutation phenotypes, as defined by signatures for defective DNA mismatch repair and DNA polymerase ε proofreading deficiency, along with concordant mutation profiles in the broadly altered WNT, MAPK, PI3K, TGFβ, and p53 pathways. Furthermore, we documented mutations enriched in genes involved in chromatin remodeling (ARID1A, CHD6, and SRCAP) and histone methylation or acetylation (ASH1L, EP300, EP400, MLL2, MLL3, PRDM2, and TRRAP). Chromosomal instability was prevalent in nonhypermutated cases, with similar patterns of chromosomal gains and losses. Although paired cell lines derived from the same tumor exhibited considerable mutation and DNA copy-number differences, in silico simulations suggest that these differences mainly reflected a preexisting heterogeneity in the tumor cells. In conclusion, our results establish that human colorectal cancer lines are representative of the main subtypes of primary tumors at the genomic level, further validating their utility as tools to investigate colorectal cancer biology and drug responses., (©2014 American Association for Cancer Research.)

Despite the millions of dollars spent on target validation and drug optimization in preclinical models, most therapies still fail in phase III clinical trials. Our current model systems, or the way we interpret data from them, clearly do not have sufficient clinical predictive power. Current opinion suggests that this is because the cell lines and xenografts that are commonly used are inadequate models that do not effectively mimic and predict human responses. This has become such a widespread belief that it approaches dogma in the field of drug discovery and optimization and has spurred a surge in studies devoted to the development of more sophisticated animal models such as orthotopic patient-derived xenografts in an attempt to obtain more accurate estimates of whether particular cancers will respond to given treatments. Here, we explore the evidence that has led to the move away from the use of in vitro cell lines and toward various forms of xenograft models for drug screening and development. We review some of the pros and cons of each model and give an overview of ways in which the use of cell lines could be modified to improve the predictive capacity of this well-defined model., (©2014 AACR.)

Single cancer stem-like cells (CSC) from colorectal cancers can be functionally identified by their ability to form large lumen-containing colonies in three-dimensional Matrigel cultures. These colonies contain the three types of differentiated colorectal epithelial cells, and single cells obtained from them can reproduce themselves and form tumors efficiently in immunodeficient mice. In this study, we show how hypoxia affects these CSC-derived lumens to control differentiation of stem-like cells and enterocytes via the homeobox gene CDX1. Lumens were identified by F-actin staining and they expressed many characteristics associated with normal differentiated intestinal epithelium, including brush border enzymes, polarization, and tight junctions. RNA interference-mediated silencing of CDX1 reduced lumen formation. Inhibitory effects of hypoxia on lumen formation and stem cell differentiation, including suppression of CDX1 expression, could be mimicked by inhibiting prolyl-hydroxylases that activate HIF1, suggesting that HIF1 is a critical mediator of the effects of hypoxia in this setting. Cell line-derived lumens were phenotypically indistinguishable from colorectal tumor glandular structures used by pathologists to grade tumor differentiation. Parallel results to those obtained with established cell lines were seen with primary cultures from fresh tumors. This in vitro approach to functional characterization of CSCs and their differentiation offers a valid model to study colorectal tumor differentiation and differentiation of colorectal CSCs, with additional uses to enable high-throughput screening for novel anticancer compounds., (©2013 AACR.)

Background: Myofibroblasts have an important role in regulating the normal colorectal stem cell niche. While the activation of myofibroblasts in primary colorectal cancers has been previously described, myofibroblast activation in lymph node metastases has not been described before., Methods: Paraffin-embedded lymph node sections from patients with macrometastases, micrometastases and isolated tumour cells were stained to identify myofibroblasts and to characterise the distribution of different cell types in tumour-containing lymph nodes. The extent of myofibroblast presence was quantified and compared with the size of the metastasis and degree of proliferation and differentiation of the cancer cells., Results: We show substantial activation of myofibroblasts in the presence of colorectal metastases in lymph nodes, which is intimately associated with glandular structures, both in micro- and macrometastases. The degree of activation is positively associated with the size of the metastases and the proportion of Ki67+ve cancer cells, and negatively associated with the degree of enterocyte differentiation as measured by CK20 expression., Conclusion: The substantial activation of myofibroblasts in tumour-containing lymph nodes strongly suggests that these metastatic cancer cells are still significantly dependent on their microenvironment. Further understanding of these epithelial-mesenchymal interactions could lead to the development of new therapies in metastatic disease.

A significant proportion of colorectal cancer (CRC) patients are resistant to anti-ERBB1 [avian erythroblastic leukemia viral (v-erb-b) oncogene homolog, receptor for EGF] monoclonal antibodies (Mabs). We evaluated both immune and nonimmune effects of cetuximab (anti-ERBB1 Mab), trastuzumab (anti-ERBB2 Mab), pertuzumab (anti-ERBB2 Mab), and lapatinib (dual ERBB1 and ERBB2 tyrosine kinase inhibitor) in a large well-characterized panel of 64 CRC cell lines to find response predictive tumor characteristics. There was a significant correlation between the direct effects of cetuximab and lapatinib. Both agents were associated (P = 0.0004) with "triple' wild-type status in KRAS, BRAF, and PIK3CA exon 20. Most cell lines were resistant to the direct effects of anti-ERBB2 Mabs, suggesting that the effects of lapatinib might mainly be through ERBB1. Microarray mRNA expression profiles of sensitive and resistant cell lines showed that although ERBB1 receptor or ligand levels did not associate with cetuximab sensitivity, high levels of ERBB2 (P = 0.036) and amphiregulin (P = 0.026) predicted sensitivity to lapatinib. However, higher ERBB1 expression predicted susceptibility to cetuximab-induced antibody-dependent cellular cytotoxicity and occurred independently of KRAS/BRAF/PIK3CA mutations (P = 0.69). Lapatinib may be an effective alternative therapy to cetuximab in triple wild-type tumors. Microarray analysis provides suggestive biomarkers for resistance. ERBB1 levels, independent of mutation status, predict immune killing. Therefore, anti-ERBB1 antibodies may be considered in CRC tumors with higher ERBB1 expression and favorable FcγR polymorphisms.

Some 15-20% of multiple adenomatous polyposis have no genetic explanation and 20-30% of colorectal cancer (CRC) cases are thought to be due to inherited multifactorial causes. Accumulation of deleterious effects of low-frequency dominant and independently acting variants may be a partial explanation for such patients. The aim of this study was to type a selection of rare and low-frequency variants (<5%) to elucidate their role in CRC susceptibility. A total of 1181 subjects were included (866 controls; 315 cases). Cases comprised UK (n=184) and French (n=131) patients with MAP (n=187) or early-onset CRC (n=128). Seventy variants in 17 genes were examined in cases and controls. The effect of the variant effect on protein function was investigated in silico. Out of the 70 variants typed, 36 (51%) were tested for association. Twenty-one variants were rare (minor allele frequency (MAF) <1%). Four rare variants were found to have a significantly higher MAF in cases (EXO1-12, MLH1-1, CTNNB1-1 and BRCA2-37, P<0.05) than in controls. Pooling all rare variants with a MAF <0.5% showed an excess risk in cases (odds ratio=3.2; 95% confidence interval=1.1-9.5; P=0.04). Rare variants are important risk factors in CRC and, as such, should be systematically assayed alongside common variation in the search for the genetic basis of complex diseases., Competing Interests: The authors declare no conflict of interest.

Hypoxia is an important regulator of normal and cancer stem cell (CSC) differentiation. Colorectal CSCs from SW1222, LS180, and CCK81 colorectal cancer-derived cell lines are able to differentiate into complex 3D lumen-containing structures in normoxia, whereas in hypoxia, they form undifferentiated dense colonies that have reduced expression of the enterocyte differentiation marker CDX1, lack goblet cell formation, and have increased expression of BMI1 and activated Notch1. Hypoxia increases the clonogenicity of CSCs, which is cumulative as each round of hypoxia enriches for more CSCs. The hypoxic phenotype is reversible, because cells from hypoxic-dense colonies are able to reform differentiated structures when regrown in normoxia. We show that CDX1 is able to stimulate the generation of lumens even in hypoxia and has a negative feedback on BMI1 expression. Knockdown of CDX1 reduces lumen formation but does not affect goblet cell formation, suggesting that enterocytes and goblet cells form from different progenitor cells. Notch inhibition by dibenzazepine (DBZ) allowed CSCs to form goblet cells in both normoxia and hypoxia. Finally, we show that Hif1α, but not CA9, is an important mediator of the effects of hypoxia on the clonogenicity and differentiation of CSCs. In summary, hypoxia maintains the stem-like phenotype of colorectal cell line-derived CSCs and prevents differentiation of enterocytes and goblet cells by regulating CDX1 and Notch1, suggesting that this regulation is an important component of how hypoxia controls the switch between stemness and differentiation in CSCs.

We examined the influence that rare variants and low-frequency polymorphisms in the cancer candidate gene CCND1 have on the development of multiple intestinal adenomas and the early onset of colorectal cancer. Individuals with <100 multiple polyps and patients with colorectal cancer diagnosed before 50 years of age were recruited in UK, and screened for sequence changes in the coding and regulatory regions of CCND1. A set of about 800 UK control individuals was genotyped for the variants discovered in the cases. Variants in the promoter, intron-exon boundaries and untranslated regions of the CCND1 gene had higher frequencies in cases than in controls. Five of these variants were typed in a set of French multiple adenoma and early-onset patients, who also showed higher allele frequencies than UK controls. When pooled together, variants with frequencies lower than 1% conferred an increased risk of disease that was significant in the multiple adenoma group (odds ratio (OR) 2.2; 95% confidence interval, 1.1-4.4; P = 0.03). Most variants had a putative functional effect when assessed in silico. We conclude that rare variants of CCND1 are risk factors for colorectal cancer, with considerably larger effects than common polymorphisms, and as such should be systematically evaluated in susceptibility studies.

Replication error deficient (RER+) colorectal cancers are a distinct subset of colorectal cancers, characterized by inactivation of the DNA mismatch repair system. These cancers are typically pseudodiploid, accumulate mutations in repetitive sequences as a result of their mismatch repair deficiency, and have distinct pathologies. Regulatory sequences controlling all aspects of mRNA processing, especially including message stability, are found in the 3'UTR sequence of most genes. The relevant sequences are typically A/U-rich elements or U repeats. Microarray analysis of 14 RER+ (deficient) and 16 RER- (proficient) colorectal cancer cell lines confirms a striking difference in expression profiles. Analysis of the incidence of mononucleotide repeat sequences in the 3'UTRs, 5'UTRs, and coding sequences of those genes most differentially expressed in RER+ versus RER- cell lines has shown that much of this differential expression can be explained by the occurrence of a massive enrichment of genes with 3'UTR T repeats longer than 11 base pairs in the most differentially expressed genes. This enrichment was confirmed by analysis of two published consensus sets of RER differentially expressed probesets for a large number of primary colorectal cancers. Sequence analysis of the 3'UTRs of a selection of the most differentially expressed genes shows that they all contain deletions in these repeats in all RER+ cell lines studied. These data strongly imply that deregulation of mRNA stability through accumulation of mutations in repetitive regulatory 3'UTR sequences underlies the striking difference in expression profiles between RER+ and RER- colorectal cancers.

It is now generally accepted that cancers contain a sub-population, the cancer stem cells (CSCs), which initiate and drive a tumour's growth. At least until recently it has been widely assumed that only a small proportion of the cells in a tumour are CSCs. Here we use a mathematical model, supported by experimental evidence, to show that such an assumption is unwarranted. We show that CSCs may comprise any possible proportion of the tumour, and that the higher the proportion the more aggressive the tumour is likely to be., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The HLA region on chromosome 6 is gene-rich and under selective pressure because of the high proportion of immunity-related genes. Linkage disequilibrium (LD) patterns and allele frequencies in this region are highly differentiated across broad geographical populations, making it a region of interest for population genetics and immunity-related disease studies. We examined LD in this important region of the genome in six European populations using 166 putatively neutral SNPs and the classical HLA-A, -B and -C gene alleles. We found that the pattern of association between classic HLA gene alleles and SNPs implied that most of the SNPs predated the origin of classic HLA gene alleles. The SNPs most strongly associated with HLA gene alleles were in some cases highly predictive of the HLA allele carrier status (misclassification rates ranged from <1 to 27%) in independent populations using five or fewer SNPs, a much smaller number than tagSNP panels previously proposed and often with similar accuracy, showing that our approach may be a viable solution to designing new HLA prediction panels. To describe the LD within this region, we developed a new haplotype clustering method/software based on r(2), which may be more appropriate for use within regions of strong LD. Haplotype blocks created using this proposed method, as well as classic HLA gene alleles and SNPs, were predictive of a northern versus southern European population membership (misclassification error rates ranged from 0 to 23%, depending on which independent population was used for prediction), indicating that this region may be a rich source of ancestry informative markers.

Background: Colorectal cancer is (CRC) one of the commonest cancers and its therapy is still based on few drugs. Currently, no biological criteria are used to choose the most effective of the established drugs for treatment., Methods: A panel of 77 CRC cell lines was tested for sensitivity to 5-fluorouracil (5FU) using the SRB assay. The responses were grouped into three categories and correlated with genetic changes in the cell lines., Results: The strongest and most clearcut correlation was between 5-fluorouracil response and replication error status (mismatch repair deficiency). All the other significant correlations (loss of heterozygosity for DCC and mutations in TGFbIIR) are secondary to the association with replication error status., Interpretation and Conclusion: Our findings validate previous analyses based mainly on clinical data, and indicate that replication error status could be a useful guide to 5-fluorouracil-based CRC therapy. Essentially, all previously described correlations with 5FU response are secondary to the association with replication error status.

The WHO Nomenclature Committee for Factors of the HLA System met during the 15th International Histocompatibility and Immunogenetics Workshop in Buzios, Brazil in September 2008. This update is an extract of the main report that documents the additions and revisions to the nomenclature of human leukocyte antigen (HLA) specificities following the principles established in previous reports.

The role of transforming growth factor beta receptor type 1 (TGFBR1) polymorphisms, particularly a coding CGC insertion (rs11466445, TGFBR1*6A/9A) in exon 1, has been extensively investigated in regard to colorectal cancer (CRC) risk. These investigations have generated conflicting results. More recently, allele-specific expression (ASE) of TGFBR1 mRNA has been suggested as predisposing to CRC, with a relative risk of nearly 10-fold and a population attributable risk of approximately 10%. Owing to the potential importance of TGFBR1 variants in CRC, we performed a comprehensive examination of tagging SNPs at and around the gene in 3,101 CRC cases and 3,334 controls of northern European ancestry. To test whether rare or subpolymorphic TGFBR1 variants were associated with CRC risk, we sequenced the gene's exons in a subset of patients. We also evaluated TGFBR1 ASE in a panel of CRC cases and controls. Overall, we found no association between TGFBR1 polymorphisms and CRC risk. The rare variant screen did not identify any changes of potentially pathogenic effects. No evidence of greater ASE in cases than controls was detected, and no haplotype around TGFBR1 could account for the ASE reported in other studies. We conclude that neither genetic variation nor ASE at TGFBR1 is likely to be a major CRC risk factor.

Cancer stem cells (CSCs) are the subpopulation of cells within a tumor that can self-renew, differentiate into multiple lineages, and drive tumor growth. Here we describe a two-pronged approach for the identification and characterization of CSCs from colorectal cancer cell lines, using a Matrigel-based differentiation assay, and cell surface markers CD44 and CD24. About 20 to 30% of cells from the SW1222 cell line form megacolonies in Matrigel that have complex 3D structures resembling colonic crypts. The megacolonies' capacity to self-renew in vitro is direct evidence that they contain the CSCs. Furthermore, just 200 cells from SW1222 megacolonies initiate tumors in NOD/SCID mice. We also showed that CD44(+)CD24(+) cells enriched for colorectal CSCs in the HT29 and SW1222 cell lines, which can self-renew and reform all four CD44/CD24 subpopulations, are the most clonogenic in vitro and can initiate tumors in vivo. A single SW1222 CD44(+)CD24(+) CSC, when grown in Matrigel, can form large megacolonies that differentiate into enterocyte, enteroendocrine, and goblet cell lineages. The HCT116 line does not differentiate or express CDX1, nor does it contain subpopulations of cells with greater tumor-forming capacity, suggesting that HCT116 contains mainly CSCs. However, forced expression of CDX1 in HCT116 leads to reduced clonogenicity and production of differentiating crypt-containing colonies, which can explain the selection for reduced CDX1 expression in many colorectal cancers. In summary, colorectal cancer cell lines contain subpopulations of CSCs, characterized by their cell surface markers and colony morphology, which can self-renew and differentiate into multiple lineages.

Background: The effect of glycoengineering a membrane specific anti-carcinoembryonic antigen (CEA) (this paper uses the original term CEA for the formally designated CEACAM5) antibody (PR1A3) on its ability to enhance killing of colorectal cancer (CRC) cell lines by human immune effector cells was assessed. In vivo efficacy of the antibody was also tested., Methods: The antibody was modified using EBNA cells cotransfected with beta-1,4-N-acetylglucosaminyltransferase III and the humanised hPR1A3 antibody genes., Results: The resulting alteration of the Fc segment glycosylation pattern enhances the antibody's binding affinity to the FcgammaRIIIa receptor on human immune effector cells but does not alter the antibody's binding capacity. Antibody-dependent cellular cytotoxicity (ADCC) is inhibited in the presence of anti-FcgammaRIII blocking antibodies. This glycovariant of hPR1A3 enhances ADCC 10-fold relative to the parent unmodified antibody using either unfractionated peripheral blood mononuclear or natural killer (NK) cells and CEA-positive CRC cells as targets. NK cells are far more potent in eliciting ADCC than either freshly isolated monocytes or granulocytes. Flow cytometry and automated fluorescent microscopy have been used to show that both versions of hPR1A3 can induce antibody-dependent cellular phagocytosis (ADCP) by monocyte-derived macrophages. However, the glycovariant antibody did not mediate enhanced ADCP. This may be explained by the relatively low expression of FcgammaRIIIa on cultured macrophages. In vivo studies show the efficacy of glycoengineered humanised IgG1 PR1A3 in significantly improving survival in a CRC metastatic murine model., Conclusion: The greatly enhanced in vitro ADCC activity of the glycoengineered version of hPR1A3 is likely to be clinically beneficial.

Background: The Wnt signalling pathway directs aspects of embryogenesis and is thought to contribute to maintenance of certain stem cell populations. Disruption of the pathway has been observed in many different tumour types. In bowel, stomach, and endometrial cancer, this is usually due to mutation of genes encoding Wnt pathway components APC or beta-catenin. Such mutations are rare in hepatocellular carcinomas and medulloblastomas with Wnt pathway dysfunction, and there, mutation in genes for other Wnt molecules, such as Axin, is more frequently found., Objective: Although evidence of abnormal activation of the Wnt pathway in prostate cancer has been demonstrated by several groups, APC and beta-catenin mutations are infrequent. We sought mutations in genes encoding Wnt pathway participants in a panel of prostate cancer clinical specimens and cell lines., Design, Setting, and Participants: DNA was obtained from 49 advanced prostate cancer specimens using laser microdissection followed by whole genome amplification and 8 prostate cancer cell lines., Measurements: The DNA samples were screened for mutations in the genes encoding APC, beta-catenin, and Axin. The subcellular distribution of beta-catenin expression was assessed in the clinical specimens using immunohistochemistry., Results and Limitations: Abnormal patterns of beta-catenin expression, suggesting Wnt pathway dysregulation, were observed in 71% of specimens. One APC mutation, two beta-catenin gene mutations, and 7 DNA sequence variations in the Axin gene were detected. Four different Axin polymorphisms were also found in the cell lines. The study does not provide definite evidence that the observed sequence changes alter protein function, promoting neoplasia, but the potential functional relevance of these variants is discussed., Conclusions: These data contribute to our understanding of the role of Wnt dysregulation in prostatic tumourigenesis and support the current interest in the pathway as a therapeutic target. Of particular interest, we identified three new potentially functionally relevant AXIN1 mutations.

CDX1 is a transcription factor that plays a key role in intestinal development and differentiation. However, the downstream targets of CDX1 are less well defined than those of its close homologue, CDX2. We report here the identification of downstream targets of CDX1 using microarray gene-expression analysis and other approaches. Keratin 20 (KRT20), a member of the intermediate filament and a well-known marker of intestinal differentiation, was initially identified as one of the genes likely to be directly regulated by CDX1. CDX1 and KRT20 mRNA expression were significantly correlated in a panel of 38 colorectal cancer cell lines. Deletion and mutation analysis of the KRT20 promoter showed that the minimum regulatory region for the control of KRT20 expression by CDX1 is within 246 bp upstream of the KRT20 transcription start site. ChIP analysis confirmed that CDX1 binds to the predicted CDX elements in this region of the KRT20 promoter in vivo. In addition, immunohistochemistry showed expression of CDX1 parallels that of KRT20 in the normal crypt, which further supports their close relationship. In summary, our observations strongly imply that KRT20 is directly regulated by CDX1, and therefore suggest a role for CDX1 in maintaining differentiation in intestinal epithelial cells. Because a key feature of the development of a cancer is an unbalanced program of proliferation and differentiation, dysregulation of CDX1 may be an advantage for the development of a colorectal carcinoma. This could, therefore, explain the relatively frequent down regulation of CDX1 in colorectal carcinomas by hypermethylation.

We have developed an automated, highly sensitive and specific method for identifying and enumerating circulating tumour cells (CTCs) in the blood. Blood samples from 10 prostate, 25 colorectal and 4 ovarian cancer patients were analysed. Eleven healthy donors and seven men with elevated serum prostate-specific antigen (PSA) levels but no evidence of malignancy served as controls. Spiking experiments with cancer cell lines were performed to estimate recovery yield. Isolation was performed either by density gradient centrifugation or by filtration, and the CTCs were labelled with monoclonal antibodies against cytokeratins 7/8 and either AUA1 (against EpCam) or anti-PSA. The slides were analysed with the Ikoniscope robotic fluorescence microscope imaging system. Spiking experiments showed that less than one epithelial cell per millilitre of blood could be detected, and that fluorescence in situ hybridisation (FISH) could identify chromosomal abnormalities in these cells. No positive cells were detected in the 11 healthy control samples. Circulating tumour cells were detected in 23 out of 25 colorectal, 10 out of 10 prostate and 4 out of 4 ovarian cancer patients. Five samples (three colorectal and two ovarian) were analysed by FISH for chromosomes 7 and 8 combined and all had significantly more than four dots per cell. We have demonstrated an Ikoniscope based relatively simple and rapid procedure for the clear-cut identification of CTCs. The method has considerable promise for screening, early detection of recurrence and evaluation of treatment response for a wide variety of carcinomas.

The distribution of carcinoembryonic antigen (CEA) in colorectal cancer (CRC) differs from that in normal colorectal tissue, being found on all borders of the cell membrane and hence enabling access to intravenous antibody, making CEA a good target for antibody-based therapy. The distinctive anti-CEA antibody, PR1A3, binds only membrane-bound CEA. Humanised PR1A3 (hPR1A3) was assessed both in vitro cytotoxicity and binding assays with colorectal cancer cell lines expressing varying levels of CEA. Human peripheral blood mononuclear cells (PBMCs) and purified natural killer (NK) cells were used as effectors. The in vitro assays demonstrated hPR1A3 CEA-specific binding and antibody-dependent and CEA-specific killing of human colorectal cancer cell lines by human PBMCs. The effect increased with increasing concentration of antibody and surface CEA, and was lost by using the parent murine IgG1 PR1A3. Killing was also blocked by antibody to the Fc-gammaIIIA receptor. Purified human NK cells were effective at much lower effector:target ratios than unfractionated PBMCs, indicating that NK cells were the main mediators of hPR1A3-based CEA-specific killing. The results support the development of hPR1A3 for therapy of colorectal cancer.

The heterogeneity of cancer cell signaling is a significant obstacle for the effective development and clinical use of molecularly targeted therapies. As a contribution to a better understanding of the diversity of signaling activities in colorectal cancers (CRCs), we have analyzed the activity of Src family kinases (SFKs), which are implicated in human cancer development, in 64 CRC cell lines. A striking diversity of SFK activity was observed within this panel. Importantly, all CRC lines tested depend on SFK activity for their growth. In addition, SFK activity levels strongly correlated with global levels of tyrosine-phosphorylated (pTyr) proteins in CRC lines. SFK inhibition substantially reduced these pTyr levels, suggesting that SFKs may function as signal integration points and master controllers for the pTyr protein status in CRC lines. The majority of analyzed CRC lines with high-SFK activity express activated c-Met (pYpY1234/1235), a receptor tyrosine kinase contributing to the regulation of cell proliferation, migration, and invasion. Inhibition of SFKs reduced c-Met phosphorylation in most cases, indicating a reversed signal flow from SFK to c-Met. We conclude that SFK activity is important for the growth of CRC lines, although only low activity levels are required. If this also is true for CRC patients, tumors with low-SFK activity may be particularly sensitive to SFK inhibitors, and such patients should be targeted in clinical trials testing SFK inhibitors.

Mathematical modeling is being increasingly recognized within the biomedical sciences as an important tool that can aid the understanding of biological systems. The heavily regulated cell renewal cycle in the colonic crypt provides a good example of how modeling can be used to find out key features of the system kinetics, and help to explain both the breakdown of homeostasis and the initiation of tumorigenesis. We use the cell population model by Johnston et al. to illustrate the power of mathematical modeling by considering two key questions about the cell population dynamics in the colonic crypt. We ask: how can a model describe both homeostasis and unregulated growth in tumorigenesis; and to which parameters in the system is the model most sensitive? In order to address these questions, we discuss what type of modeling approach is most appropriate in the crypt. We use the model to argue why tumorigenesis is observed to occur in stages with long lag phases between periods of rapid growth, and we identify the key parameters.

We have developed a procedure for massively parallel resequencing of multiple human genes by combining a highly multiplexed and target-specific amplification process with a high-throughput parallel sequencing technology. The amplification process is based on oligonucleotide constructs, called selectors, that guide the circularization of specific DNA target regions. Subsequently, the circularized target sequences are amplified in multiplex and analyzed by using a highly parallel sequencing-by-synthesis technology. As a proof-of-concept study, we demonstrate parallel resequencing of 10 cancer genes covering 177 exons with average sequence coverage per sample of 93%. Seven cancer cell lines and one normal genomic DNA sample were studied with multiple mutations and polymorphisms identified among the 10 genes. Mutations and polymorphisms in the TP53 gene were confirmed by traditional sequencing.