Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells

Transformed, oncogenic precursors, possessing both defining neural-stem-cell properties and the ability to initiate intracerebral tumours, have been identified in human brain cancers. Here we report that bone morphogenetic proteins (BMPs), amongst which BMP4 elicits the strongest effect, trigger a significant reduction in the stem-like, tumour-initiating precursors of human glioblastomas (GBMs). Transient in vitro exposure to BMP4 abolishes the capacity of transplanted GBM cells to establish intracerebral GBMs. Most importantly, in vivo delivery of BMP4 effectively blocks the tumour growth and associated mortality that occur in 100% of mice after intracerebral grafting of human GBM cells. We demonstrate that BMPs activate their cognate receptors (BMPRs) and trigger the Smad signalling cascade in cells isolated from human glioblastomas (GBMs). This is followed by a reduction in proliferation, and increased expression of markers of neural differentiation, with no effect on cell viability. The concomitant reduction in clonogenic ability, in the size of the CD133+ population and in the growth kinetics of GBM cells indicates that BMP4 reduces the tumour-initiating cell pool of GBMs. These findings show that the BMP-BMPR signalling system--which controls the activity of normal brain stem cells--may also act as a key inhibitory regulator of tumour-initiating, stem-like cells from GBMs and the results also identify BMP4 as a novel, non-cytotoxic therapeutic effector, which may be used to prevent growth and recurrence of GBMs in humans.     

Identification and expansion of human colon-cancer-initiating cells

Colon carcinoma is the second most common cause of death from cancer. The isolation and characterization of tumorigenic colon cancer cells may help to devise novel diagnostic and therapeutic procedures. Although there is increasing evidence that a rare population of undifferentiated cells is responsible for tumour formation and maintenance, this has not been explored for colorectal cancer. Here, we show that tumorigenic cells in colon cancer are included in the high-density CD133+ population, which accounts for about 2.5% of the tumour cells. Subcutaneous injection of colon cancer CD133+ cells readily reproduced the original tumour in immunodeficient mice, whereas CD133- cells did not form tumours. Such tumours were serially transplanted for several generations, in each of which we observed progressively faster tumour growth without significant phenotypic alterations. Unlike CD133- cells, CD133+ colon cancer cells grew exponentially for more than one year in vitro as undifferentiated tumour spheres in serum-free medium, maintaining the ability to engraft and reproduce the same morphological and antigenic pattern of the original tumour. We conclude that colorectal cancer is created and propagated by a small number of undifferentiated tumorigenic CD133+ cells, which should therefore be the target of future therapies.     

Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9

Leukaemias and other cancers possess a rare population of cells capable of the limitless self-renewal necessary for cancer initiation and maintenance. Eradication of these cancer stem cells is probably a critical part of any successful anti-cancer therapy, and may explain why conventional cancer therapies are often effective in reducing tumour burden, but are only rarely curative. Given that both normal and cancer stem cells are capable of self-renewal, the extent to which cancer stem cells resemble normal tissue stem cells is a critical issue if targeted therapies are to be developed. However, it remains unclear whether cancer stem cells must be phenotypically similar to normal tissue stem cells or whether they can retain the identity of committed progenitors. Here we show that leukaemia stem cells (LSC) can maintain the global identity of the progenitor from which they arose while activating a limited stem-cell- or self-renewal-associated programme. We isolated LSC from leukaemias initiated in committed granulocyte macrophage progenitors through introduction of the MLL-AF9 fusion protein encoded by the t(9;11)(p22;q23). The LSC were capable of transferring leukaemia to secondary recipient mice when only four cells were transferred, and possessed an immunophenotype and global gene expression profile very similar to that of normal granulocyte macrophage progenitors. However, a subset of genes highly expressed in normal haematopoietic stem cells was re-activated in LSC. LSC can thus be generated from committed progenitors without widespread reprogramming of gene expression, and a leukaemia self-renewal-associated signature is activated in the process. Our findings define progression from normal progenitor to cancer stem cell, and suggest that targeting a self-renewal programme expressed in an abnormal context may be possible.     

Mediators of vascular remodelling co-opted for sequential steps in lung metastasis

Metastasis entails numerous biological functions that collectively enable cancerous cells from a primary site to disseminate and overtake distant organs. Using genetic and pharmacological approaches, we show that the epidermal growth factor receptor ligand epiregulin, the cyclooxygenase COX2, and the matrix metalloproteinases 1 and 2, when expressed in human breast cancer cells, collectively facilitate the assembly of new tumour blood vessels, the release of tumour cells into the circulation, and the breaching of lung capillaries by circulating tumour cells to seed pulmonary metastasis. These findings reveal how aggressive primary tumorigenic functions can be mechanistically coupled to greater lung metastatic potential, and how such biological activities may be therapeutically targeted with specific drug combinations.     

Identification of human brain tumour initiating cells

The cancer stem cell (CSC) hypothesis suggests that neoplastic clones are maintained exclusively by a rare fraction of cells with stem cell properties. Although the existence of CSCs in human leukaemia is established, little evidence exists for CSCs in solid tumours, except for breast cancer. Recently, we prospectively isolated a CD133+ cell subpopulation from human brain tumours that exhibited stem cell properties in vitro. However, the true measures of CSCs are their capacity for self renewal and exact recapitulation of the original tumour. Here we report the development of a xenograft assay that identified human brain tumour initiating cells that initiate tumours in vivo. Only the CD133+ brain tumour fraction contains cells that are capable of tumour initiation in NOD-SCID (non-obese diabetic, severe combined immunodeficient) mouse brains. Injection of as few as 100 CD133+ cells produced a tumour that could be serially transplanted and was a phenocopy of the patient's original tumour, whereas injection of 10(5) CD133- cells engrafted but did not cause a tumour. Thus, the identification of brain tumour initiating cells provides insights into human brain tumour pathogenesis, giving strong support for the CSC hypothesis as the basis for many solid tumours, and establishes a previously unidentified cellular target for more effective cancer therapies.     

Regulation of cancer cell migration and bone metastasis by RANKL

Bone metastases are a frequent complication of many cancers that result in severe disease burden and pain. Since the late nineteenth century, it has been thought that the microenvironment of the local host tissue actively participates in the propensity of certain cancers to metastasize to specific organs, and that bone provides an especially fertile 'soil'. In the case of breast cancers, the local chemokine milieu is now emerging as an explanation for why these tumours preferentially metastasize to certain organs. However, as the inhibition of chemokine receptors in vivo only partially blocks metastatic behaviour, other factors must exist that regulate the preferential metastasis of breast cancer cells. Here we show that the cytokine RANKL (receptor activator of NF-kappaB ligand) triggers migration of human epithelial cancer cells and melanoma cells that express the receptor RANK. RANK is expressed on cancer cell lines and breast cancer cells in patients. In a mouse model of melanoma metastasis, in vivo neutralization of RANKL by osteoprotegerin results in complete protection from paralysis and a marked reduction in tumour burden in bones but not in other organs. Our data show that local differentiation factors such as RANKL have an important role in cell migration and the tissue-specific metastatic behaviour of cancer cells.     

Somatic support cells restrict germline stem cell self-renewal and promote differentiation

Stem cells maintain populations of highly differentiated, short-lived cell-types, including blood, skin and sperm, throughout adult life. Understanding the mechanisms that regulate stem cell behaviour is crucial for realizing their potential in regenerative medicine. A fundamental characteristic of stem cells is their capacity for asymmetric division: daughter cells either retain stem cell identity or initiate differentiation. However, stem cells are also capable of symmetric division where both daughters remain stem cells, indicating that mechanisms must exist to balance self-renewal capacity with differentiation. Here we present evidence that support cells surrounding the stem cells restrict self-renewal and control stem cell number by ensuring asymmetric division. Loss of function of the Drosophila Epidermal growth factor receptor in somatic cells disrupted the balance of self-renewal versus differentiation in the male germline, increasing the number of germline stem cells. We propose that activation of this receptor specifies normal behaviour of somatic support cells; in turn, the somatic cells play a guardian role, providing information that prevents self-renewal of stem cell identity by the germ cell they enclose.     

Transforming ras genes from human melanoma: a manifestation of tumour heterogeneity?

Variability in the phenotype of cells comprising individual tumours is a striking feature of animal and human cancer and is generally referred to as tumour heterogeneity. Studies of clonally derived cell populations from tumours that originated presumably from a single transformed cell have shown that tumours are made up of cells that differ in a variety of traits, including drug resistance, antigen expression and metastatic potential. The origin and maintenance of tumour heterogeneity are unclear, but mutational and epigenetic mechanisms are thought to be involved. Here we report the results of a search for transforming genes in human melanoma which have raised the possibility that ras gene activation follows the same variable pattern as other traits involved in tumour heterogeneity. DNA from 4 of 30 melanoma cell lines yielded transforming ras genes in the NIH/3T3 assay. Of five cell lines originating from separate metastatic deposits of a single patient, only one contained activated ras, indicating heterogeneity in ras activation in this case and suggesting that ras activation was not involved in tumour initiation or maintenance in this patient.     

NUMB controls p53 tumour suppressor activity

NUMB is a cell fate determinant, which, by asymmetrically partitioning at mitosis, controls cell fate choices by antagonising the activity of the plasma membrane receptor of the NOTCH family. NUMB is also an endocytic protein, and the NOTCH-NUMB counteraction has been linked to this function. There might be, however, additional functions of NUMB, as witnessed by its proposed role as a tumour suppressor in breast cancer. Here we describe a previously unknown function for human NUMB as a regulator of tumour protein p53 (also known as TP53). NUMB enters in a tricomplex with p53 and the E3 ubiquitin ligase HDM2 (also known as MDM2), thereby preventing ubiquitination and degradation of p53. This results in increased p53 protein levels and activity, and in regulation of p53-dependent phenotypes. In breast cancers there is frequent loss of NUMB expression. We show that, in primary breast tumour cells, this event causes decreased p53 levels and increased chemoresistance. In breast cancers, loss of NUMB expression causes increased activity of the receptor NOTCH. Thus, in these cancers, a single event-loss of NUMB expression-determines activation of an oncogene (NOTCH) and attenuation of the p53 tumour suppressor pathway. Biologically, this results in an aggressive tumour phenotype, as witnessed by findings that NUMB-defective breast tumours display poor prognosis. Our results uncover a previously unknown tumour suppressor circuitry.     

Paraneoplastic myasthenic syndrome IgG inhibits 45Ca2+ flux in a human small cell carcinoma line

Certain cancers exert unexplained remote effects on the nervous system. Small cell carcinoma (SCC) of the lung, a tumour capable of spike electrogenesis and which is of possible neural crest origin, is present in approximately 70% of patients with the Lambert-Eaton myasthenic syndrome (LEMS), a disorder characterized by fatigable muscle weakness. Patients with this syndrome have a defect in the (Ca2+-dependent) quantal release of acetylcholine from motor nerve terminals evoked by a nerve impulse or by high K+ (ref.5), and a decreased number of presynaptic active zone particles. The physiological and morphological features of the syndrome can be transferred to mice by the patients' IgG, consistent with an autoantibody interfering with the function of voltage-dependent Ca2+ channels. Here we demonstrate that K+-induced 45Ca2+ flux in a cultured human SCC line is significantly reduced by LEMS IgG, suggesting that in SCC-LEMS an autoantibody to tumour Ca2+-channel determinants is triggered; its cross-reaction with similar determinants at the motor nerve terminal could lead to the remote neurological syndrome.     

Glioblastoma stem-like cells give rise to tumour endothelium

Glioblastoma (GBM) is among the most aggressive of human cancers. A key feature of GBMs is the extensive network of abnormal vasculature characterized by glomeruloid structures and endothelial hyperplasia. Yet the mechanisms of angiogenesis and the origin of tumour endothelial cells remain poorly defined. Here we demonstrate that a subpopulation of endothelial cells within glioblastomas harbour the same somatic mutations identified within tumour cells, such as amplification of EGFR and chromosome 7. We additionally demonstrate that the stem-cell-like CD133(+) fraction includes a subset of vascular endothelial-cadherin (CD144)-expressing cells that show characteristics of endothelial progenitors capable of maturation into endothelial cells. Extensive in vitro and in vivo lineage analyses, including single cell clonal studies, further show that a subpopulation of the CD133(+) stem-like cell fraction is multipotent and capable of differentiation along tumour and endothelial lineages, possibly via an intermediate CD133(+)/CD144(+) progenitor cell. The findings are supported by genetic studies of specific exons selected from The Cancer Genome Atlas, quantitative FISH and comparative genomic hybridization data that demonstrate identical genomic profiles in the CD133(+) tumour cells, their endothelial progenitor derivatives and mature endothelium. Exposure to the clinical anti-angiogenesis agent bevacizumab or to a γ-secretase inhibitor as well as knockdown shRNA studies demonstrate that blocking VEGF or silencing VEGFR2 inhibits the maturation of tumour endothelial progenitors into endothelium but not the differentiation of CD133(+) cells into endothelial progenitors, whereas γ-secretase inhibition or NOTCH1 silencing blocks the transition into endothelial progenitors. These data may provide new perspectives on the mechanisms of failure of anti-angiogenesis inhibitors currently in use. The lineage plasticity and capacity to generate tumour vasculature of the putative cancer stem cells within glioblastoma are novel findings that provide new insight into the biology of gliomas and the definition of cancer stemness, as well as the mechanisms of tumour neo-angiogenesis.     

Creation of human tumour cells with defined genetic elements.

During malignant transformation, cancer cells acquire genetic mutations that override the normal mechanisms controlling cellular proliferation. Primary rodent cells are efficiently converted into tumorigenic cells by the coexpression of cooperating oncogenes. However, similar experiments with human cells have consistently failed to yield tumorigenic transformants, indicating a fundamental difference in the biology of human and rodent cells. The few reported successes in the creation of human tumour cells have depended on the use of chemical or physical agents to achieve immortalization, the selection of rare, spontaneously arising immortalized cells, or the use of an entire viral genome. We show here that the ectopic expression of the telomerase catalytic subunit (hTERT) in combination with two oncogenes (the simian virus 40 large-T oncoprotein and an oncogenic allele of H-ras) results in direct tumorigenic conversion of normal human epithelial and fibroblast cells. These results demonstrate that disruption of the intracellular pathways regulated by large-T, oncogenic ras and telomerase suffices to create a human tumor cell.     

A human colon cancer cell capable of initiating tumour growth in immunodeficient mice

Colon cancer is one of the best-understood neoplasms from a genetic perspective, yet it remains the second most common cause of cancer-related death, indicating that some of its cancer cells are not eradicated by current therapies. What has yet to be established is whether every colon cancer cell possesses the potential to initiate and sustain tumour growth, or whether the tumour is hierarchically organized so that only a subset of cells--cancer stem cells--possess such potential. Here we use renal capsule transplantation in immunodeficient NOD/SCID mice to identify a human colon cancer-initiating cell (CC-IC). Purification experiments established that all CC-ICs were CD133+; the CD133- cells that comprised the majority of the tumour were unable to initiate tumour growth. We calculated by limiting dilution analysis that there was one CC-IC in 5.7 x 10(4) unfractionated tumour cells, whereas there was one CC-IC in 262 CD133+ cells, representing >200-fold enrichment. CC-ICs within the CD133+ population were able to maintain themselves as well as differentiate and re-establish tumour heterogeneity upon serial transplantation. The identification of colon cancer stem cells that are distinct from the bulk tumour cells provides strong support for the hierarchical organization of human colon cancer, and their existence suggests that for therapeutic strategies to be effective, they must target the cancer stem cells.     

Increased pH and tumorigenicity of fibroblasts expressing a yeast proton pump

A common early response of eukaryotic cells to stimuli which activate their proliferation is an increase in intracellular pH (ref. 1). In animal cells this is caused by the activation of an Na+/H+ exchange system; in fungi and plants an H+-pumping ATPase is involved. The critical question is whether this intracellular alkalinization is merely coincident with the activation of cell proliferation or whether it is a regulatory signal. To increase intracellular pH bypassing the usual physiological stimuli (growth factors, hormones etc.) alkaline media or ammonia have been used in the past. Both approaches suffer from long-term toxicity effects and cannot be used in tumorigenic assays with whole organisms. We introduce here a more specific approach which involves expressing the gene for the yeast plasma membrane H+-ATPase in fibroblasts. The resulting cells have an elevated intracellular pH and acquire tumorigenic properties, suggesting that the yeast ATPase gene behaves as an oncogene in mammalian cells. These experiments support a crucial role of intracellular pH in the growth control of animal cells.     

A restricted cell population propagates glioblastoma growth after chemotherapy

Glioblastoma multiforme is the most common primary malignant brain tumour, with a median survival of about one year. This poor prognosis is due to therapeutic resistance and tumour recurrence after surgical removal. Precisely how recurrence occurs is unknown. Using a genetically engineered mouse model of glioma, here we identify a subset of endogenous tumour cells that are the source of new tumour cells after the drug temozolomide (TMZ) is administered to transiently arrest tumour growth. A nestin-ΔTK-IRES-GFP (Nes-ΔTK-GFP) transgene that labels quiescent subventricular zone adult neural stem cells also labels a subset of endogenous glioma tumour cells. On arrest of tumour cell proliferation with TMZ, pulse-chase experiments demonstrate a tumour re-growth cell hierarchy originating with the Nes-ΔTK-GFP transgene subpopulation. Ablation of the GFP+ cells with chronic ganciclovir administration significantly arrested tumour growth, and combined TMZ and ganciclovir treatment impeded tumour development. Thus, a relatively quiescent subset of endogenous glioma cells, with properties similar to those proposed for cancer stem cells, is responsible for sustaining long-term tumour growth through the production of transient populations of highly proliferative cells.