Norman J. Maitland

Mutations of the BRAF gene in human cancer

Cancers arise owing to the accumulation of mutations in critical genes that alter normal programmes of cell proliferation, differentiation and death. As the first stage of a systematic genome-wide screen for these genes, we have prioritized for analysis signalling pathways in which at least one gene is mutated in human cancer. The RAS–RAF–MEK–ERK–MAP kinase pathway mediates cellular responses to growth signals. RAS is mutated to an oncogenic form in about 15% of human cancer. The three RAF genes code for cytoplasmic serine/threonine kinases that are regulated by binding RAS. Here we report BRAF somatic missense mutations in 66% of malignant melanomas and at lower frequency in a wide range of human cancers. All mutations are within the kinase domain, with a single substitution (V599E) accounting for 80%. Mutated BRAF proteins have elevated kinase activity and are transforming in NIH3T3 cells. Furthermore, RAS function is not required for the growth of cancer cell lines with the V599E mutation. As BRAF is a serine/threonine kinase that is commonly activated by somatic point mutation in human cancer, it may provide new therapeutic opportunities in malignant melanoma.

Prospective Identification of Tumorigenic Prostate Cancer Stem Cells

Existing therapies for prostate cancer eradicates the bulk of cells within a tumor. However, most patients go on to develop androgen-independent disease that remains incurable by current treatment strategies. There is now increasing evidence in some malignancies that the tumor cells are organized as a hierarchy originating from rare stem cells that are responsible for maintaining the tumor. We report here the identification and characterization of a cancer stem cell population from human prostate tumors, which possess a significant capacity for self-renewal. These cells are also able to regenerate the phenotypically mixed populations of nonclonogenic cells, which express differentiated cell products, such as androgen receptor and prostatic acid phosphatase. The cancer stem cells have a CD44+/alpha2beta1hi/CD133+ phenotype, and we have exploited these markers to isolate cells from a series of prostate tumors with differing Gleason grade and metastatic states. Approximately 0.1% of cells in any tumor expressed this phenotype, and there was no correlation between the number of CD44+/alpha2beta1hi/CD133+ cells and tumor grade. The identification of a prostate cancer stem cell provides a powerful tool to investigate the tumorigenic process and to develop therapies targeted to the stem cell.

CD133, a novel marker for human prostatic epithelial stem cells

Stem cells are clonogenic cells with self-renewal and differentiation properties, which may represent a major target for genetic damage leading to prostate cancer and benign prostatic hyperplasia. Stem cells remain poorly characterised because of the absence of specific molecular markers that permit us to distinguish them from their progeny, the transit amplifying cells, which have a more restricted proliferative potential. Human CD133 antigen, also known as AC133, was recently identified as a haematopoietic stem cell marker. Here we show that a small population (approximately 1%) of human prostate basal cells express the cell surface marker CD133 and are restricted to the α2β1hi population, previously shown to be a marker of stem cells in prostate epithelia (Collins, A. T., Habib, F. K., Maitland, N. J. and Neal, D. E. (2001). J. Cell Sci. 114, 3865-3872). α2β1hi/CD133+ cells exhibit two important attributes of epithelial stem cells: they possess a high in vitro proliferative potential and can reconstitute prostatic-like acini in immunocompromised male nude mice.

Predisposing Gene for Early-Onset Prostate Cancer, Localized on Chromosome 1q42.2-43

There is genetic predisposition associated with >=10% of all cancer of the prostate (CaP). By means of a genomewide search on a selection of 47 French and German families, parametric and nonparametric linkage (NPL) analysis allowed identification of a locus, on chromosome 1q42.2-43, carrying a putative predisposing gene for CaP (PCaP). The primary localization was confirmed with several markers, by use of three different genetic models. We obtained a maximum two-point LOD score of 2.7 with marker D1S2785. Multipoint parametric and NPL analysis yielded maximum HLOD and NPL scores of 2.2 and 3.1, respectively, with an associated P value of . 001. Homogeneity analysis with multipoint LOD scores gave an estimate of the proportion of families with linkage to this locus of 50%, with a likelihood ratio of 157/1 in favor of heterogeneity. Furthermore, the 9/47 families with early-onset CaP at age <60 years gave multipoint LOD and NPL scores of 3.31 and 3.32, respectively, with P = .001.

Prostate Cancer Stem Cells: A New Target for Therapy

The existence of prostate cancer stem cells offers a theoretical explanation for many of the enduring uncertainties surrounding the etiology and treatment of the most commonly diagnosed tumor in US males. The study of cancer stem cells in prostate, as in other complex tissues, is critically dependent on the availability of pure cell populations, a situation complicated by the heterogeneity of prostate tumors. However, selection of cells with a CD133(+)/alpha 2 beta 1 integrin/ CD44(+) phenotype enriches for a tumor-initiating population from human prostate cancers. Among the most pressing needs is for enduring therapy in patients who have experienced failure of hormonal treatments. Because the putative cancer stem cell does not express androgen receptor, it is likely to be immune from most androgen-based therapies, and an inherent genetic instability would enable the tumor to develop the new variants present in hormone-refractory disease. Prostate cancer stem cells have a unique gene expression signature that can also be related to Gleason grade and patient outcome. The scarcity of cancer stem cells in a prostate tumor will probably limit their usefulness in cancer diagnosis and prognosis. However, the emergence of new stem-cell therapeutic targets not only will require new assays for efficacy (because of their relatively quiescent nature), but also holds real promise of more lasting treatments to augment those currently directed against the remaining tumor cells, which comprise 99.9% of tumor mass, but paradoxically have a poor tumor-initiating capacity.

Gene expression profiling of human prostate cancer stem cells reveals a pro-inflammatory phenotype and the importance of extracellular matrix interactions.

BackgroundThe tumor-initiating capacity of many cancers is considered to reside in a small subpopulation of cells (cancer stem cells). We have previously shown that rare prostate epithelial cells with a CD133+/α2β1hi phenotype have the properties of prostate cancer stem cells. We have compared gene expression in these cells relative to their normal and differentiated (CD133-/α2β1low) counterparts, resulting in an informative cancer stem cell gene-expression signature.ResultsCell cultures were generated from specimens of human prostate cancers (n = 12) and non-malignant control tissues (n = 7). Affymetrix gene-expression arrays were used to analyze total cell RNA from sorted cell populations, and expression changes were selectively validated by quantitative RT-PCR, flow cytometry and immunocytochemistry. Differential expression of multiple genes associated with inflammation, cellular adhesion, and metastasis was observed. Functional studies, using an inhibitor of nuclear factor κB (NF-κB), revealed preferential targeting of the cancer stem cell and progenitor population for apoptosis whilst sparing normal stem cells. NF-κB is a major factor controlling the ability of tumor cells to resist apoptosis and provides an attractive target for new chemopreventative and chemotherapeutic approaches.ConclusionWe describe an expression signature of 581 genes whose levels are significantly different in prostate cancer stem cells. Functional annotation of this signature identified the JAK-STAT pathway and focal adhesion signaling as key processes in the biology of cancer stem cells.

Identification of degradome components associated with prostate cancer progression by expression analysis of human prostatic tissues

Extracellular proteases of the matrix metalloproteinase (MMP) and serine protease families participate in many aspects of tumour growth and metastasis. Using quantitative real-time RT–PCR analysis, we have undertaken a comprehensive survey of the expression of these enzymes and of their natural inhibitors in 44 cases of human prostate cancer and 23 benign prostate specimens. We found increased expression of MMP10, 15, 24, 25 and 26, urokinase plasminogen activator-receptor (uPAR) and plasminogen activator inhibitor-1 (PAI1), and the newly characterised serine proteases hepsin and matriptase-1 (MTSP1) in malignant tissue compared to benign prostate tissue. In contrast, there was significantly decreased expression of MMP2 and MMP23, maspin, and the protease inhibitors tissue inhibitor of metalloproteinase 3 (TIMP3), TIMP4 and RECK (reversion-inducing cysteine-rich protein with Kazal motifs) in the cancer specimens. The expression of MMP15 and MMP26 correlated positively with Gleason score, whereas TIMP3, TIMP4 and RECK expression correlated negatively with Gleason score. The cellular localisation of the expression of the deregulated genes was evaluated using primary malignant epithelial and stromal cell cultures derived from radical prostatectomy specimens. MMP10 and 25, hepsin, MTSP1 and maspin showed predominantly epithelial expression, whereas TIMP 3 and 4, RECK, MMP2 and 23, uPAR and PAI1 were produced primarily by stromal cells. These data provide the first comprehensive and quantitative analysis of the expression and localisation of MMPs and their inhibitors in human prostate cancer, leading to the identification of several genes involved in proteolysis as potential prognostic indicators, in particular hepsin, MTSP1, MMP26, PAI1, uPAR, MMP15, TIMP3, TIMP4, maspin and RECK.

Macrophages in gene therapy: cellular delivery vehicles and in vivo targets

The appearance and activation of macrophages are thought to be rapid events in the development of many pathological lesions, including malignant tumors, atherosclerotic plaques, and arthritic joints. This has prompted recent attempts to use macrophages as novel cellular vehicles for gene therapy, in which macrophages are genetically modified ex vivo and then reintroduced into the body with the hope that a proportion will then home to the diseased site. Here, we critically review the efficacy of various gene transfer methods (viral, bacterial, protozoan, and various chemical and physical methods in transfecting macrophages in vitro, and the results obtained when transfected macrophages are used as gene delivery vehicles. Finally, we discuss the use of various viral and nonviral methods to transfer genes to macrophages in vivo. As will be seen, definitive evidence for the use of macrophages as gene transfer vehicles has yet to be provided and awaits detailed trafficking studies in vivo. Moreover, although methods for transfecting macrophages have improved considerably in efficiency in recent years, targeting of gene transfer specifically to macrophages in vivo remains a problem. However, possible solutions to this include placing transgenes under the control of macrophage‐specific promoters to limit expression to macrophages or stably transfecting CD34+ precursors of monocytes/macrophages and then differentiating these cells into monocytes/macrophages ex vivo. The latter approach could conceivably lead to the bone marrow precursor cells of patients with inherited genetic disorders being permanently fortified or even replaced with genetically modified cells.

Structure of the intact transactivation domain of the human papillomavirus E2 protein.

Papillomaviruses cause warts and proliferative lesions in skin and other epithelia. In a minority of papillomavirus types (‘high risk’, including human papillomaviruses 16, 18, 31, 33, 45 and 56), further transformation of the wart lesions can produce tumours. The papillomavirus E2 protein controls primary transcription and replication of the viral genome. Both activities are governed by a ∼200 amino-acid amino-terminal module (E2NT) which is connected to a DNA-binding carboxy-terminal module by a flexible linker. Here we describe the crystal structure of the complete E2NT module from human papillomavirus 16. The E2NT module forms a dimer both in the crystal and in solution. Amino acids that are necessary for transactivation are located at the dimer interface, indicating that the dimer structure may be important in the interactions of E2NT with viral and cellular transcription factors. We propose that dimer formation may contribute to the stabilization of DNA loops which may serve to relocate distal DNA-binding transcription factors to the site of human papillomavirus transcription initiation.

Prostate epithelial cell lines form spheroids with evidence of glandular differentiation in three-dimensional Matrigel cultures

Normal (PNT2-C2) and metastatic (PC-3) prostate cell lines were grown in Matrigel to observe the effects on morphology and phenotype in comparison to monolayer culture. In monolayer cultures, PNT2-C2 showed typical round/cuboidal epithelial morphology, with tight cell associations, whereas in Matrigel they formed smooth spheroids, tightly packed with cells. In both monolayer and Matrigel, PNT2-C2 had a differentiated luminal epithelial phenotype with high expression of cytokeratin 8, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), E-cadherin and desmoglein. In contrast, PC-3 cells possessed an epithelial/mesenchyme morphology in monolayer with loose cell to cell contact and pseudopodial extensions. Immunohistochemical phenotyping indicated the cells were undifferentiated, expressing high levels of vimentin, β1 integrin, CD44 and low expression of cytokeratin 8. In Matrigel they formed smooth and irregular spheroids, which had a lumen surrounded by a single cell layer. Matrigel also influenced the expression of PSA, PSMA and CD44. These results indicate that Matrigel culture can induce morphological differentiation of prostate cancer cells which initially had a basal phenotype.