A vascular niche and a VEGF-Nrp1 loop regulate the initiation and stemness of skin tumours

Angiogenesis is critical during tumour initiation and malignant progression. Different strategies aimed at blocking vascular endothelial growth factor (VEGF) and its receptors have been developed to inhibit angiogenesis in cancer patients. It has become increasingly clear that in addition to its effect on angiogenesis, other mechanisms including a direct effect of VEGF on tumour cells may account for the efficiency of VEGF-blockade therapies. Cancer stem cells (CSCs) have been described in various cancers including squamous tumours of the skin. Here we use a mouse model of skin tumours to investigate the impact of the vascular niche and VEGF signalling on controlling the stemness (the ability to self renew and differentiate) of squamous skin tumours during the early stages of tumour progression. We show that CSCs of skin papillomas are localized in a perivascular niche, in the immediate vicinity of endothelial cells. Furthermore, blocking VEGFR2 caused tumour regression not only by decreasing the microvascular density, but also by reducing CSC pool size and impairing CSC renewal properties. Conditional deletion of Vegfa in tumour epithelial cells caused tumours to regress, whereas VEGF overexpression by tumour epithelial cells accelerated tumour growth. In addition to its well-known effect on angiogenesis, VEGF affected skin tumour growth by promoting cancer stemness and symmetric CSC division, leading to CSC expansion. Moreover, deletion of neuropilin-1 (Nrp1), a VEGF co-receptor expressed in cutaneous CSCs, blocked VEGF's ability to promote cancer stemness and renewal. Our results identify a dual role for tumour-cell-derived VEGF in promoting cancer stemness: by stimulating angiogenesis in a paracrine manner, VEGF creates a perivascular niche for CSCs, and by directly affecting CSCs through Nrp1 in an autocrine loop, VEGF stimulates cancer stemness and renewal. Finally, deletion of Nrp1 in normal epidermis prevents skin tumour initiation. These results may have important implications for the prevention and treatment of skin cancers.     

Defining the mode of tumour growth by clonal analysis

Recent studies using the isolation of a subpopulation of tumour cells followed by their transplantation into immunodeficient mice provide evidence that certain tumours, including squamous skin tumours, contain cells with high clonogenic potential that have been referred to as cancer stem cells (CSCs). Until now, CSC properties have only been investigated by transplantation assays, and their existence in unperturbed tumour growth is unproven. Here we make use of clonal analysis of squamous skin tumours using genetic lineage tracing to unravel the mode of tumour growth in vivo in its native environment. To this end, we used a genetic labelling strategy that allows individual tumour cells to be marked and traced over time at different stages of tumour progression. Surprisingly, we found that the majority of labelled tumour cells in benign papilloma have only limited proliferative potential, whereas a fraction has the capacity to persist long term, giving rise to progeny that occupy a significant part of the tumour. As well as confirming the presence of two distinct proliferative cell compartments within the papilloma, mirroring the composition, hierarchy and fate behaviour of normal tissue, quantitative analysis of clonal fate data indicates that the more persistent population has stem-cell-like characteristics and cycles twice per day, whereas the second represents a slower cycling transient population that gives rise to terminally differentiated tumour cells. Such behaviour is shown to be consistent with double-labelling experiments and detailed clonal fate characteristics. By contrast, measurements of clone size and proliferative potential in invasive squamous cell carcinoma show a different pattern of behaviour, consistent with geometric expansion of a single CSC population with limited potential for terminal differentiation. This study presents the first experimental evidence for the existence of CSCs during unperturbed solid tumour growth.     

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.     

NF-kappaB blockade and oncogenic Ras trigger invasive human epidermal neoplasia

The nuclear factor NF-kappaB and oncogenic Ras can alter proliferation in epidermis, the most common site of human cancer. These proteins are implicated in epidermal squamous cell carcinoma in mice, however, the potential effects of altering their function are uncertain. Whereas inhibition of NF-kappaB enhances apoptosis in certain tumours, blockade of NF-kappaB predisposes murine skin to squamous cell carcinoma. Because therapeutics inhibiting Ras and NF-kappaB pathways are being developed to treat human cancer, it is essential to assess the effects of altering these regulators. The medical relevance of murine studies is limited, however, by differences between mouse and human skin, and by the greater ease of transforming murine cells. Here we show that in normal human epidermal cells both NF-kappaB and oncogenic Ras trigger cell-cycle arrest. Growth arrest triggered by oncogenic Ras can be bypassed by IkappaBalpha-mediated blockade of NF-kappaB, generating malignant human epidermal tissue resembling squamous cell carcinoma. Human cell tumorigenesis is dependent on laminin 5 and alpha6beta4 integrin. Thus, IkappaBalpha circumvents restraints on growth promotion induced by oncogenic Ras and can act with Ras to induce invasive human tissue neoplasia.     

Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration

In the age of stem cell engineering it is critical to understand how stem cell activity is regulated during regeneration. Hairs are mini-organs that undergo cyclic regeneration throughout adult life, and are an important model for organ regeneration. Hair stem cells located in the follicle bulge are regulated by the surrounding microenvironment, or niche. The activation of such stem cells is cyclic, involving periodic beta-catenin activity. In the adult mouse, regeneration occurs in waves in a follicle population, implying coordination among adjacent follicles and the extrafollicular environment. Here we show that unexpected periodic expression of bone morphogenetic protein 2 (Bmp2) and Bmp4 in the dermis regulates this process. This BMP cycle is out of phase with the WNT/beta-catenin cycle, thus dividing the conventional telogen into new functional phases: one refractory and the other competent for hair regeneration, characterized by high and low BMP signalling, respectively. Overexpression of noggin, a BMP antagonist, in mouse skin resulted in a markedly shortened refractory phase and faster propagation of the regenerative wave. Transplantation of skin from this mutant onto a wild-type host showed that follicles in donor and host can affect their cycling behaviours mutually, with the outcome depending on the equilibrium of BMP activity in the dermis. Administration of BMP4 protein caused the competent region to become refractory. These results show that BMPs may be the long-sought 'chalone' inhibitors of hair growth postulated by classical experiments. Taken together, results presented in this study provide an example of hierarchical regulation of local organ stem cell homeostasis by the inter-organ macroenvironment. The expression of Bmp2 in subcutaneous adipocytes indicates physiological integration between these two thermo-regulatory organs. Our findings have practical importance for studies using mouse skin as a model for carcinogenesis, intra-cutaneous drug delivery and stem cell engineering studies, because they highlight the acute need to differentiate supportive versus inhibitory regions in the host skin.     

Multi-genetic events collaboratively contribute to Pten-null leukaemia stem-cell formation

Cancer stem cells, which share many common properties and regulatory machineries with normal stem cells, have recently been proposed to be responsible for tumorigenesis and to contribute to cancer resistance. The main challenges in cancer biology are to identify cancer stem cells and to define the molecular events required for transforming normal cells to cancer stem cells. Here we show that Pten deletion in mouse haematopoietic stem cells leads to a myeloproliferative disorder, followed by acute T-lymphoblastic leukaemia (T-ALL). Self-renewable leukaemia stem cells (LSCs) are enriched in the c-Kit(mid)CD3(+)Lin(-) compartment, where unphosphorylated beta-catenin is significantly increased. Conditional ablation of one allele of the beta-catenin gene substantially decreases the incidence and delays the occurrence of T-ALL caused by Pten loss, indicating that activation of the beta-catenin pathway may contribute to the formation or expansion of the LSC population. Moreover, a recurring chromosomal translocation, T(14;15), results in aberrant overexpression of the c-myc oncogene in c-Kit(mid)CD3(+)Lin(-) LSCs and CD3(+) leukaemic blasts, recapitulating a subset of human T-ALL. No alterations in Notch1 signalling are detected in this model, suggesting that Pten inactivation and c-myc overexpression may substitute functionally for Notch1 abnormalities, leading to T-ALL development. Our study indicates that multiple genetic or molecular alterations contribute cooperatively to LSC transformation.     

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.     

The circadian molecular clock creates epidermal stem cell heterogeneity

Murine epidermal stem cells undergo alternate cycles of dormancy and activation, fuelling tissue renewal. However, only a subset of stem cells becomes active during each round of morphogenesis, indicating that stem cells coexist in heterogeneous responsive states. Using a circadian-clock reporter-mouse model, here we show that the dormant hair-follicle stem cell niche contains coexisting populations of cells at opposite phases of the clock, which are differentially predisposed to respond to homeostatic cues. The core clock protein Bmal1 modulates the expression of stem cell regulatory genes in an oscillatory manner, to create populations that are either predisposed, or less prone, to activation. Disrupting this clock equilibrium, through deletion of Bmal1 (also known as Arntl) or Per1/2, resulted in a progressive accumulation or depletion of dormant stem cells, respectively. Stem cell arrhythmia also led to premature epidermal ageing, and a reduction in the development of squamous tumours. Our results indicate that the circadian clock fine-tunes the temporal behaviour of epidermal stem cells, and that its perturbation affects homeostasis and the predisposition to tumorigenesis.     

Promotion of Hras-induced squamous carcinomas by a polymorphic variant of the Patched gene in FVB mice

Mice of the C57BL/6 strain are resistant to the development of skin squamous carcinomas (SCCs) induced by an activated Ras oncogene, whereas FVB/N mice are highly susceptible. The genetic basis of this difference in phenotype is unknown. Here we show that susceptibility to SCC is under the control of a carboxy-terminal polymorphism in the mouse Ptch gene. F1 hybrids between C57BL/6 and FVB/N strains ((B6FVB)F1) are resistant to Ras-induced SCCs, but resistance can be overcome either by elimination of the C57BL/6 Ptch allele (Ptch(B6)) or by overexpression of the FVB/N Ptch allele (Ptch(FVB)) in the epidermis of K5Hras-transgenic (B6FVB)F1 hybrid mice. The human Patched (PTCH) gene is a classical tumour suppressor gene for basal cell carcinomas and medulloblastomas, the loss of which causes increased signalling through the Sonic Hedgehog (SHH) pathway. SCCs that develop in PtchB6+/- mice do not lose the wild-type Ptch gene or show evidence of increased SHH signalling. Although Ptch(FVB) overexpression can promote SCC formation, continued expression is not required for tumour maintenance, suggesting a role at an early stage of tumour cell lineage commitment. The Ptch polymorphism affects Hras-induced apoptosis, and binding to Tid1, the mouse homologue of the Drosophila l(2)tid tumour suppressor gene. We propose that Ptch occupies a critical niche in determining basal or squamous cell lineage, and that both tumour types can arise from the same target cell depending on carcinogen exposure and host genetic background.     

Stem cells, cancer, and cancer stem cells.

Stem cell biology has come of age. Unequivocal proof that stem cells exist in the haematopoietic system has given way to the prospective isolation of several tissue-specific stem and progenitor cells, the initial delineation of their properties and expressed genetic programmes, and the beginnings of their utility in regenerative medicine. Perhaps the most important and useful property of stem cells is that of self-renewal. Through this property, striking parallels can be found between stem cells and cancer cells: tumours may often originate from the transformation of normal stem cells, similar signalling pathways may regulate self-renewal in stem cells and cancer cells, and cancer cells may include 'cancer stem cells' - rare cells with indefinite potential for self-renewal that drive tumorigenesis.     

Mesenchymal stem cells within tumour stroma promote breast cancer metastasis

Mesenchymal stem cells have been recently described to localize to breast carcinomas, where they integrate into the tumour-associated stroma. However, the involvement of mesenchymal stem cells (or their derivatives) in tumour pathophysiology has not been addressed. Here, we demonstrate that bone-marrow-derived human mesenchymal stem cells, when mixed with otherwise weakly metastatic human breast carcinoma cells, cause the cancer cells to increase their metastatic potency greatly when this cell mixture is introduced into a subcutaneous site and allowed to form a tumour xenograft. The breast cancer cells stimulate de novo secretion of the chemokine CCL5 (also called RANTES) from mesenchymal stem cells, which then acts in a paracrine fashion on the cancer cells to enhance their motility, invasion and metastasis. This enhanced metastatic ability is reversible and is dependent on CCL5 signalling through the chemokine receptor CCR5. Collectively, these data demonstrate that the tumour microenvironment facilitates metastatic spread by eliciting reversible changes in the phenotype of cancer cells.     

Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding

The mammalian hair follicle is a complex 'mini-organ' thought to form only during development; loss of an adult follicle is considered permanent. However, the possibility that hair follicles develop de novo following wounding was raised in studies on rabbits, mice and even humans fifty years ago. Subsequently, these observations were generally discounted because definitive evidence for follicular neogenesis was not presented. Here we show that, after wounding, hair follicles form de novo in genetically normal adult mice. The regenerated hair follicles establish a stem cell population, express known molecular markers of follicle differentiation, produce a hair shaft and progress through all stages of the hair follicle cycle. Lineage analysis demonstrated that the nascent follicles arise from epithelial cells outside of the hair follicle stem cell niche, suggesting that epidermal cells in the wound assume a hair follicle stem cell phenotype. Inhibition of Wnt signalling after re-epithelialization completely abrogates this wounding-induced folliculogenesis, whereas overexpression of Wnt ligand in the epidermis increases the number of regenerated hair follicles. These remarkable regenerative capabilities of the adult support the notion that wounding induces an embryonic phenotype in skin, and that this provides a window for manipulation of hair follicle neogenesis by Wnt proteins. These findings suggest treatments for wounds, hair loss and other degenerative skin disorders.     

Inhibition of Dll4 signalling inhibits tumour growth by deregulating angiogenesis

Haploinsufficiency of Dll4, a vascular-specific Notch ligand, has shown that it is essential for embryonic vascular development and arteriogenesis. Mechanistically, it is unclear how the Dll4-mediated Notch pathway contributes to complex vascular processes that demand meticulous coordination of multiple signalling pathways. Here we show that Dll4-mediated Notch signalling has a unique role in regulating endothelial cell proliferation and differentiation. Neutralizing Dll4 with a Dll4-selective antibody rendered endothelial cells hyperproliferative, and caused defective cell fate specification or differentiation both in vitro and in vivo. In addition, blocking Dll4 inhibited tumour growth in several tumour models. Remarkably, antibodies against Dll4 and antibodies against vascular endothelial growth factor (VEGF) had paradoxically distinct effects on tumour vasculature. Our data also indicate that Dll4-mediated Notch signalling is crucial during active vascularization, but less important for normal vessel maintenance. Furthermore, unlike blocking Notch signalling globally, neutralizing Dll4 had no discernable impact on intestinal goblet cell differentiation, supporting the idea that Dll4-mediated Notch signalling is largely restricted to the vascular compartment. Therefore, targeting Dll4 might represent a broadly efficacious and well-tolerated approach for the treatment of solid tumours.     

Wnt signalling regulates adult hippocampal neurogenesis

The generation of new neurons from neural stem cells is restricted to two regions of the adult mammalian central nervous system: the subventricular zone of the lateral ventricle, and the subgranular zone of the hippocampal dentate gyrus. In both regions, signals provided by the microenvironment regulate the maintenance, proliferation and neuronal fate commitment of the local stem cell population. The identity of these signals is largely unknown. Here we show that adult hippocampal stem/progenitor cells (AHPs) express receptors and signalling components for Wnt proteins, which are key regulators of neural stem cell behaviour in embryonic development. We also show that the Wnt/beta-catenin pathway is active and that Wnt3 is expressed in the hippocampal neurogenic niche. Overexpression of Wnt3 is sufficient to increase neurogenesis from AHPs in vitro and in vivo. By contrast, blockade of Wnt signalling reduces neurogenesis from AHPs in vitro and abolishes neurogenesis almost completely in vivo. Our data show that Wnt signalling is a principal regulator of adult hippocampal neurogenesis and provide evidence that Wnt proteins have a role in adult hippocampal function.     

Mouse skin carcinomas induced in vivo by chemical carcinogens have a transforming Harvey-ras oncogene

Several groups have shown that the malignant phenotype can be transferred to NIH/3T3 fibroblasts by incorporation of DNA isolated from tumour cell lines. These studies have demonstrated that the transforming activity of DNA isolated from human bladder, lung and colon carcinoma cell lines is related to an alteration of the cellular homologues of the ras genes of Harvey or Kirsten murine sarcoma viruses. It is, however, unclear what relevance these observations have to the multi-stage nature of tumorigenesis in vivo, in which several independent events are required in both humans and experimental animals. The activation of a cellular oncogene in a defined experimental system for the progressive induction of solid tumours has not yet been demonstrated. We report here that high molecular weight DNA from transplanted squamous cell carcinomas induced by sequential treatment of mouse skin with initiators and promoters of carcinogenesis causes morphological transformation of NIH/3T3 fibroblasts at high frequency. The transforming properties are due to the transfer of an activated cellular homologue of the Harvey-ras (rasH) oncogene.     

Deletion of vascular endothelial growth factor in myeloid cells accelerates tumorigenesis

Angiogenesis and the development of a vascular network are required for tumour progression, and they involve the release of angiogenic factors, including vascular endothelial growth factor (VEGF-A), from both malignant and stromal cell types. Infiltration by cells of the myeloid lineage is a hallmark of many tumours, and in many cases the macrophages in these infiltrates express VEGF-A. Here we show that the deletion of inflammatory-cell-derived VEGF-A attenuates the formation of a typical high-density vessel network, thus blocking the angiogenic switch in solid tumours in mice. Vasculature in tumours lacking myeloid-cell-derived VEGF-A was less tortuous, with increased pericyte coverage and decreased vessel length, indicating vascular normalization. In addition, loss of myeloid-derived VEGF-A decreases the phosphorylation of VEGF receptor 2 (VEGFR2) in tumours, even though overall VEGF-A levels in the tumours are unaffected. However, deletion of myeloid-cell VEGF-A resulted in an accelerated tumour progression in multiple subcutaneous isograft models and an autochthonous transgenic model of mammary tumorigenesis, with less overall tumour cell death and decreased tumour hypoxia. Furthermore, loss of myeloid-cell VEGF-A increased the susceptibility of tumours to chemotherapeutic cytotoxicity. This shows that myeloid-derived VEGF-A is essential for the tumorigenic alteration of vasculature and signalling to VEGFR2, and that these changes act to retard, not promote, tumour progression.     

The Wnt/beta-catenin pathway regulates cardiac valve formation

Truncation of the tumour suppressor adenomatous polyposis coli (Apc) constitutively activates the Wnt/beta-catenin signalling pathway. Apc has a role in development: for example, embryos of mice with truncated Apc do not complete gastrulation. To understand this role more fully, we examined the effect of truncated Apc on zebrafish development. Here we show that, in contrast to mice, zebrafish do complete gastrulation. However, mutant hearts fail to loop and form excessive endocardial cushions. Conversely, overexpression of Apc or Dickkopf 1 (Dkk1), a secreted Wnt inhibitor, blocks cushion formation. In wild-type hearts, nuclear beta-catenin, the hallmark of activated canonical Wnt signalling, accumulates only in valve-forming cells, where it can activate a Tcf reporter. In mutant hearts, all cells display nuclear beta-catenin and Tcf reporter activity, while valve markers are markedly upregulated. Concomitantly, proliferation and epithelial-mesenchymal transition, normally restricted to endocardial cushions, occur throughout the endocardium. Our findings identify a novel role for Wnt/beta-catenin signalling in determining endocardial cell fate.