Jane A. Wakeman

BRACHYURY confers cancer stem cell characteristics on colorectal cancer cells

Cancer stem cells (CSCs) are initiating cells in colorectal cancer (CRC). Colorectal tumours undergo epithelial to mesenchymal transition (EMT)‐like processes at the invasive front, enabling invasion and metastasis, and recent studies have linked this process to the acquisition of stem cell‐like properties. It is of fundamental importance to understand the molecular events leading to the establishment of cancer initiating cells and how these mechanisms relate to cellular transitions during tumourigenesis. We use an in vitro system to recapitulate changes in CRC cells at the invasive front (mesenchymal‐like cells) and central mass (epithelial‐like cells) of tumours. We show that the mesoderm inducer BRACHYURY is expressed in a subpopulation of CRC cells that resemble invasive front mesenchymal‐like cells, where it acts to impose characteristics of CSCs in a fully reversible manner, suggesting reversible formation and modulation of such cells. BRACHYURY, itself regulated by the oncogene β‐catenin, influences NANOG and other ‘stemness’ markers including a panel of markers defining CRC‐CSC whose presence has been linked to poor patient prognosis. Similar regulation of NANOG through BRACHYURY was observed in other cells lines, suggesting this might be a pathway common to cancer cells undergoing mesenchymal transition. We suggest that BRACHYURY may regulate NANOG in mesenchymal‐like CRC cells to impose a ‘plastic‐state’, allowing competence of cells to respond to signals prompting invasion or metastasis.

Human Wnt-13 is developmentally regulated during the differentiation of NTERA-2 pluripotent human embryonal carcinoma cells

The Wnt gene family encodes a series of conserved glycoproteins that regulate pattern formation during embryogenesis, in a variety of tissues including the nervous system. As with other genes that control embryonic cell differentiation, members of the Wnt family have also been implicated in tumourigenesis. To search for Wnt genes involved in human teratocarcinomas, with a possible role in human embryogenesis, we used RT–PCR primed with degenerate oligonucleotides to analyse mRNA from differentiating cultures of the pluripotent human embryonal carcinoma (EC) cell line NTERA-2. NTERA-2 EC cells differentiate into neurons and other cell types when induced with retinoic acid. Wnt gene expression was not detected in the undifferentiated EC cells, but Wnt-related PCR fragments were amplified from differentiating cultures, 4–14 days after induction with retinoic acid. The RT–PCR products were composed primarily of DNA fragments corresponding to the recently identified human Wnt-13 gene. No other Wnt-related genes were identified. Northern analysis confirmed induction of Wnt-13 as a 2.4 kb mRNA during the early phases of retinoic acid-induced differentiation, and during differentiation along a non-neural pathway induced by hexamethylene bisacetamide (HMBA), but not in the terminally differentiated neurons. Wnt-13 remained expressed in non-neural differentiated NTERA-2 cells, even several weeks after the induction of differentiation. The time course of induction, its induction by HMBA, and its persistence in differentiated cells indicate that Wnt-13 expression is not dependent upon direct activation by retinoic acid. Wnt-13 was not detected, or only detected at low levels, in other human EC cells. However, it was found to be expressed at a high level in one malignant teratoma cell line, 577MF, that does not exhibit an EC phenotype although it was derived from a testicular teratocarcinoma. At least two members of the human frizzled gene family, thought to encode receptors for Wnt proteins, were also expressed in the NTERA-2 cells, suggesting the presence of a mechanism by which endogenously expressed Wnt-13 could modulate the histogenesis of teratocarcinomas by mediating interactions between sub-populations of differentiating EC cells. We note that Wnt-13 maps to chromosome 1p13, a region reported to be subject to relatively frequent loss of heterozygosity in germ cell tumours. Further analysis indicated that 465 bp of the published Wnt-13 sequence, within the predicted 5′ UTR, is incorrect and is possibly derived from a human mitochondrial DNA sequence.

Density-dependent location and interactions of truncated APC and beta-catenin.

Adenomatous polyposis coli (APC) is a multifunctional tumour suppressor protein, central to development and the mature organism. It is mutated in most cases of colorectal cancer, rendering it ineffective in mediating β-catenin degradation. We show that localization of full-length APC in colon carcinoma and noncancer cell lines is independent of cell density. However, the location of truncated APC is a function of cell density and in high-density cells truncated APC is predominantly not nuclear. Although the distribution of truncated APC and β-catenin is closely linked in subconfluent SW480 cells, at high cell density they are not colocalized. We postulated that in this cell line this could be due to an increase in β-catenin bound to E-cadherin with formation of adherens junctions at high cell density. However, while in coimmunoprecipitation assays we observe an increase in binding between β-catenin and E-cadherin and a corresponding decrease in binding between β-catenin and APC at high cell density, we did not observe a strict colocalization of β-catenin and E-cadherin at the membrane of all cells.

Interaction between Ku80 protein and a widely used antibody to adenomatous polyposis coli

The adenomatous polyposis coli (APC) gene and its expressed product are highly studied because of its role as a tumour-suppressor protein. Inherited mutations in APC lead to the condition known as familial adenomatous polyposis (FAP), which predisposes the affected individuals to colorectal cancer. Furthermore, mutations in APC are found in the majority of sporadic cases of colon cancer. There have been many published studies concerning the cellular localisation of APC, this being fundamental to our understanding of its function, but there has also been much concern over the specificity of certain commercially available antibodies to APC. Here we report that the widely used antibody APC(N15) demonstrates a strong interaction with the Ku80 subunit of the Ku heterodimer under defined experimental conditions. Based on the data presented here, we suggest that APC(N15) is not suitable for many applications used for the study of APC.

Analysis of a panel of antibodies to APC reveals consistent activity towards an unidentified protein

Acquisition of truncating mutations in the adenomatous polyposis coli (APC) protein underlies the progression of the majority of sporadic and familial colorectal cancers. As such, the localisation patterns and interacting partners of APC have been extensively studied in a range of systems, relying on the use of a broad panel of antibodies. Until recently, antibodies to APC have been used largely unchecked. However, several recent reports have been invaluable in clarifying the use of a number of antibodies commonly used to detect APC. Here, we analyse the specificity of a further subset of antibodies to APC. We used a panel of six commercially available antibodies (directed to the amino and carboxy termini of APC) and confirm the detection of full-length APC by immunoblotting. We demonstrate that a 150 kDa protein, also reproducibly detected by this panel of antibodies, is unlikely to be APC. We present data for the immunological staining patterns of the APC antibodies and validate the results through RNAi. Using this approach, we confirm that the apical staining pattern, observed by immunofluorescence and previously reported in cell systems, is unlikely to be APC. Finally, we present our data as a summary of APC-antibody specificities for APC.

Translin and Trax differentially regulate telomere-associated transcript homeostasis

Translin and Trax proteins are highly conserved nucleic acid binding proteins that have been implicated in RNA regulation in a range of biological processes including tRNA processing, RNA interference, microRNA degradation during oncogenesis, spermatogenesis and neuronal regulation. Here, we explore the function of this paralogue pair of proteins in the fission yeast. Using transcript analysis we demonstrate a reciprocal mechanism for control of telomere-associated transcripts. Mutation of tfx1+ (Trax) elevates transcript levels from silenced sub-telomeric regions of the genome, but not other silenced regions, such as the peri-centromeric heterochromatin. In the case of some sub-telomeric transcripts, but not all, this elevation is dependent on the Trax paralogue, Tsn1 (Translin). In a reciprocal fashion, Tsn1 (Translin) serves to repress levels of transcripts (TERRAs) from the telomeric repeats, whereas Tfx1 serves to maintain these elevated levels. This reveals a novel mechanism for the regulation of telomeric transcripts. We extend this to demonstrate that human Translin and Trax also control telomere-associated transcript levels in human cells in a telomere-specific fashion.

Brachyury identifies a class of enteroendocrine cells in normal human intestinal crypts and colorectal cancer

Normal homeostasis of adult intestinal epithelium and repair following tissue damage is maintained by a balance of stem and differentiated cells, many of which are still only poorly characterised. Enteroendocrine cells of the gut are a small population of differentiated, secretory cells that are critical for integrating nutrient sensing with metabolic responses, dispersed amongst other epithelial cells. Recent evidence suggests that sub-sets of secretory enteroendocrine cells can act as reserve stem cells. Given the link between cells with stem-like properties and cancer, it is important that we identify factors that might provide a bridge between the two. Here, we identify a sub-set of chromogranin A-positive enteroendocrine cells that are positive for the developmental and cancer-associated transcription factor Brachyury in normal human small intestinal and colonic crypts. Whilst chromogranin A-positive enteroendocrine cells are also Brachyury-positive in colorectal tumours, expression of Brachyury becomes more diffuse in these samples, suggesting a more widespread function in cancer. The finding of the developmental transcription factor Brachyury in normal adult human intestinal crypts may extend the functional complexity of enteroendocrine cells and serves as a platform for assessment of the molecular processes of intestinal homeostasis that underpins our understanding of human health, cancer and aging.

Human germ/stem cell-specific gene TEX19 influences cancer cell proliferation and cancer prognosis

BackgroundCancer/testis (CT) genes have expression normally restricted to the testis, but become activated during oncogenesis, so they have excellent potential as cancer-specific biomarkers. Evidence is starting to emerge to indicate that they also provide function(s) in the oncogenic programme. Human TEX19 is a recently identified CT gene, but a functional role for TEX19 in cancer has not yet been defined.MethodssiRNA was used to deplete TEX19 levels in various cancer cell lines. This was extended using shRNA to deplete TEX19 in vivo. Western blotting, fluorescence activated cell sorting and immunofluorescence were used to study the effect of TEX19 depletion in cancer cells and to localize TEX19 in normal testis and cancer cells/tissues. RT-qPCR and RNA sequencing were employed to determine the changes to the transcriptome of cancer cells depleted for TEX19 and Kaplan-Meier plots were generated to explore the relationship between TEX19 expression and prognosis for a range of cancer types.ResultsDepletion of TEX19 levels in a range of cancer cell lines in vitro and in vivo restricts cellular proliferation/self-renewal/reduces tumour volume, indicating TEX19 is required for cancer cell proliferative/self-renewal potential. Analysis of cells depleted for TEX19 indicates they enter a quiescent-like state and have subtle defects in S-phase progression. TEX19 is present in both the nucleus and cytoplasm in both cancerous cells and normal testis. In cancer cells, localization switches in a context-dependent fashion. Transcriptome analysis of TEX19 depleted cells reveals altered transcript levels of a number of cancer-/proliferation-associated genes, suggesting that TEX19 could control oncogenic proliferation via a transcript/transcription regulation pathway. Finally, overall survival analysis of high verses low TEX19 expressing tumours indicates that TEX19 expression is linked to prognostic outcomes in different tumour types.ConclusionsTEX19 is required to drive cell proliferation in a range of cancer cell types, possibly mediated via an oncogenic transcript regulation mechanism. TEX19 expression is linked to a poor prognosis for some cancers and collectively these findings indicate that not only can TEX19 expression serve as a novel cancer biomarker, but may also offer a cancer-specific therapeutic target with broad spectrum potential.

Meiosis-like Functions in Oncogenesis: A New View of Cancer

Cancer cells have many abnormal characteristics enabling tumors to grow, spread, and avoid immunologic and therapeutic destruction. Central to this is the innate ability of populations of cancer cells to rapidly evolve. One feature of many cancers is that they activate genes that are normally associated with distinct developmental states, including germ cell-specific genes. This has historically led to the proposal that tumors take on embryonal characteristics, the so called embryonal theory of cancer. However, one group of germline genes, not directly associated with embryonic somatic tissue genesis, is the one that encodes the specific factors to drive the unique reductional chromosome segregation of meiosis I, which also results in chromosomal exchanges. Here, we propose that meiosis I-specific modulators of reductional segregation can contribute to oncogenic chromosome dynamics and that the embryonal theory for cancer cell growth/proliferation is overly simplistic, as meiotic factors are not a feature of most embryonic tissue development. We postulate that some meiotic chromosome-regulatory functions contribute to a soma-to-germline model for cancer, in which activation of germline (including meiosis) functions drive oncogenesis, and we extend this to propose that meiotic factors could be powerful sources of targets for therapeutics and biomonitoring in oncology. Cancer Res; 77(21); 5712-6. ©2017 AACR.

The immortal strand hypothesis: still non-randomly segregating opinions

Abstract Cairns first suggested a mechanism for protecting the genomes of stem cells (SCs) from replicative errors some 40 years ago when he proposed the immortal strand hypothesis, which argued for the inheritance of a so-called immortal strand by an SC following asymmetric SC divisions. To date, the existence of immortal strands remains contentious with published evidence arguing in favour of and against the retention of an immortal strand by asymmetrically dividing SCs. The conflicting evidence is derived from a diverse array of studies on adult SC types and is predominantly based on following the fate of labelled DNA strands during asymmetric cell division events. Here, we review current data, highlighting limitations of such labelling techniques, and suggest how interpretation of such data may be improved in the future.