Gaëlle Lapouge

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.

Mesp1 Acts as a Master Regulator of Multipotent Cardiovascular Progenitor Specification

During embryonic development, multipotent cardiovascular progenitor cells are specified from early mesoderm. Using mouse ESCs in which gene expression can be temporally regulated, we have found that transient expression of Mesp1 dramatically accelerates and enhances multipotent cardiovascular progenitor specification through an intrinsic and cell autonomous mechanism. Genome-wide transcriptional analysis indicates that Mesp1 rapidly activates and represses a discrete set of genes, and chromatin immunoprecipitation shows that Mesp1 directly binds to regulatory DNA sequences located in the promoter of many key genes in the core cardiac transcriptional machinery, resulting in their rapid upregulation. Mesp1 also directly represses the expression of key genes regulating other early mesoderm and endoderm cell fates. Our results demonstrate that Mesp1 acts as a key regulatory switch during cardiovascular specification, residing at the top of the hierarchy of the gene network responsible for cardiovascular cell-fate determination.

SOX2 controls tumour initiation and cancer stem-cell functions in squamous-cell carcinoma

Cancer stem cells (CSCs) have been reported in various cancers, including in skin squamous-cell carcinoma (SCC). The molecular mechanisms regulating tumour initiation and stemness are still poorly characterized. Here we find that Sox2, a transcription factor expressed in various types of embryonic and adult stem cells, was the most upregulated transcription factor in the CSCs of squamous skin tumours in mice. SOX2 is absent in normal epidermis but begins to be expressed in the vast majority of mouse and human pre-neoplastic skin tumours, and continues to be expressed in a heterogeneous manner in invasive mouse and human SCCs. In contrast to other SCCs, in which SOX2 is frequently genetically amplified, the expression of SOX2 in mouse and human skin SCCs is transcriptionally regulated. Conditional deletion of Sox2 in the mouse epidermis markedly decreases skin tumour formation after chemical-induced carcinogenesis. Using green fluorescent protein (GFP) as a reporter of Sox2 transcriptional expression (SOX2–GFP knock-in mice), we showed that SOX2-expressing cells in invasive SCC are greatly enriched in tumour-propagating cells, which further increase upon serial transplantations. Lineage ablation of SOX2-expressing cells within primary benign and malignant SCCs leads to tumour regression, consistent with the critical role of SOX2-expressing cells in tumour maintenance. Conditional Sox2 deletion in pre-existing skin papilloma and SCC leads to tumour regression and decreases the ability of cancer cells to be propagated upon transplantation into immunodeficient mice, supporting the essential role of SOX2 in regulating CSC functions. Transcriptional profiling of SOX2–GFP-expressing CSCs and of tumour epithelial cells upon Sox2 deletion uncovered a gene network regulated by SOX2 in primary tumour cells in vivo. Chromatin immunoprecipitation identified several direct SOX2 target genes controlling tumour stemness, survival, proliferation, adhesion, invasion and paraneoplastic syndrome. We demonstrate that SOX2, by marking and regulating the functions of skin tumour-initiating cells and CSCs, establishes a continuum between tumour initiation and progression in primary skin tumours.

Identifying the cellular origin of squamous skin tumors

Squamous cell carcinoma (SCC) is the second most frequent skin cancer. The cellular origin of SCC remains controversial. Here, we used mouse genetics to determine the epidermal cell lineages at the origin of SCC. Using mice conditionally expressing a constitutively active KRas mutant (G12D) and an inducible CRE recombinase in different epidermal lineages, we activated Ras signaling in different cellular compartments of the skin epidermis and determined from which epidermal compartments Ras activation induces squamous tumor formation. Expression of mutant KRas in hair follicle bulge stem cells (SCs) and their immediate progeny (hair germ and outer root sheath), but not in their transient amplifying matrix cells, led to benign squamous skin tumor (papilloma). Expression of KRasG12D in interfollicular epidermis also led to papilloma formation, demonstrating that squamous tumor initiation is not restricted to the hair follicle lineages. Whereas no malignant tumor was observed after KRasG12D expression alone, expression of KRasG12D combined with the loss of p53 induced invasive SCC. Our studies demonstrate that different epidermal lineages including bulge SC are competent to initiate papilloma formation and that multiple genetic hits in the context of oncogenic KRas are required for the development of invasive SCC.

Bcl-2 and accelerated DNA repair mediates resistance of hair follicle bulge stem cells to DNA-damage-induced cell death

Adult stem cells (SCs) are at high risk of accumulating deleterious mutations because they reside and self-renew in adult tissues for extended periods. Little is known about how adult SCs sense and respond to DNA damage within their natural niche. Here, using mouse epidermis as a model, we define the functional consequences and the molecular mechanisms by which adult SCs respond to DNA damage. We show that multipotent hair-follicle-bulge SCs have two important mechanisms for increasing their resistance to DNA-damage-induced cell death: higher expression of the anti-apoptotic gene Bcl-2 and transient stabilization of p53 after DNA damage in bulge SCs. The attenuated p53 activation is the consequence of a faster DNA repair activity, mediated by a higher non-homologous end joining (NHEJ) activity, induced by the key protein DNA-PK. Because NHEJ is an error-prone mechanism, this novel characteristic of adult SCs may have important implications in cancer development and ageing.

p53 induces formation of NEAT1 lncRNA-containing paraspeckles that modulate replication stress response and chemosensitivity

In a search for mediators of the p53 tumor suppressor pathway, which induces pleiotropic and often antagonistic cellular responses, we identified the long noncoding RNA (lncRNA) NEAT1. NEAT1 is an essential architectural component of paraspeckle nuclear bodies, whose pathophysiological relevance remains unclear. Activation of p53, pharmacologically or by oncogene-induced replication stress, stimulated the formation of paraspeckles in mouse and human cells. Silencing Neat1 expression in mice, which prevents paraspeckle formation, sensitized preneoplastic cells to DNA-damage-induced cell death and impaired skin tumorigenesis. We provide mechanistic evidence that NEAT1 promotes ATR signaling in response to replication stress and is thereby engaged in a negative feedback loop that attenuates oncogene-dependent activation of p53. NEAT1 targeting in established human cancer cell lines induced synthetic lethality with genotoxic chemotherapeutics, including PARP inhibitors, and nongenotoxic activation of p53. This study establishes a key genetic link between NEAT1 paraspeckles, p53 biology and tumorigenesis and identifies NEAT1 as a promising target to enhance sensitivity of cancer cells to both chemotherapy and p53 reactivation therapy.

Adult interfollicular tumour-initiating cells are reprogrammed into an embryonic hair follicle progenitor-like fate during basal cell carcinoma initiation.

Basal cell carcinoma, the most frequent human skin cancer, arises from activating hedgehog (HH) pathway mutations; however, little is known about the temporal changes that occur in tumour-initiating cells from the first oncogenic hit to the development of invasive cancer. Using an inducible mouse model enabling the expression of a constitutively active Smoothened mutant (SmoM2) in the adult epidermis, we carried out transcriptional profiling of SmoM2-expressing cells at different times during cancer initiation. We found that tumour-initiating cells are massively reprogrammed into a fate resembling that of embryonic hair follicle progenitors (EHFPs). Wnt/ β-catenin signalling was very rapidly activated following SmoM2 expression in adult epidermis and coincided with the expression of EHFP markers. Deletion of β-catenin in adult SmoM2-expressing cells prevents EHFP reprogramming and tumour initiation. Finally, human basal cell carcinomas also express genes of the Wnt signalling and EHFP signatures.

Different Levels of Twist1 Regulate Skin Tumor Initiation, Stemness, and Progression

Twist1 promotes epithelial-to-mesenchymal transition (EMT), invasion, metastasis, and cancer stem cell (CSC) properties. However, it remains unclear whether Twist1 is also required for tumor initiation and whether Twist1-induced cancer stemness and EMT are functionally linked. Using a conditional deletion of Twist1 at different stages of skin carcinogenesis, we show that Twist1 is required for skin tumor initiation and progression in a gene-dosage-dependent manner. Moreover, conditional ablation of Twist1 in benign tumors leads to increased apoptosis, reduced cell proliferation, and defective tumor maintenance and propagation independently of its EMT-inducing abilities. Concomitant deletion of Twist1 and p53 rescues the apoptotic response, but not the cell proliferation and propagation defects. These results reveal that Twist1 is required for tumor initiation and maintenance in a p53-dependent and -independent manner. Importantly, our findings also indicate that tumor stemness and EMT can be regulated by distinct mechanisms.

Identification of novel truncated androgen receptor (AR) mutants including unreported pre-mRNA splicing variants in the 22Rv1 hormone-refractory prostate cancer (PCa) cell line†

Advanced prostate cancer (PCa) has emerged as a public health concern due to population aging. Although androgen deprivation has proven efficacy in this condition, most advanced PCa patients will have to face failure of androgen deprivation as a treatment. Mutations in the androgen receptor (AR) from tumor cells have been shown to induce androgen independency both in PCa cell lines and in the clinic. We have investigated the molecular events leading to androgen independency in the 22Rv1 cell line, a commonly used preclinical model of PCa. Besides AR mutants that have been described so far, including nonsense mutations, recent data have focused on AR pre‐mRNA aberrant splicing as a new mechanism leading to constitutively active truncated AR variants. In this article, we describe two novel variants arising from aberrant splicing of AR pre‐mRNA, characterized by long mRNA transcripts that encode truncated, constitutively active proteins. We also describe several new nonsense mutants that share ligand independency and transcriptional activity. Finally, we show that alongside these mutants, 22Rv1 cells also express a mutant AR lacking exon 3 tandem duplication, a major feature of this cell line. By describing unreported AR mutants in the 22Rv1 cell line, our data emphasize the complexity and heterogeneity of molecular events that occur in preclinical models, and supposedly in the clinic. Future work on the 22Rv1 cell line should take into account the concomitant expression of various AR mutants. Hum Mutat 31:74–80, 2010.

Skin squamous cell carcinoma propagating cells increase with tumour progression and invasiveness.

Cancer stem cells have been described in various cancers including squamous tumours of the skin by their ability to reform secondary tumours upon transplantation into immunodeficient mice. Here, we used transplantation of limiting dilution of different populations of FACS‐isolated tumour cells from four distinct mouse models of squamous skin tumours to investigate the frequency of tumour propagating cells (TPCs) at different stages of tumour progression. We found that benign papillomas, despite growing rapidly in vivo and being clonogenic in vitro, reformed secondary tumours upon transplantation at very low frequency and only when tumour cells were co‐transplanted together with tumour‐associated fibroblasts or endothelial cells. In two models of skin squamous cell carcinoma (SCC), TPCs increased with tumour invasiveness. Interestingly, the frequency of TPCs increased in CD34HI but not in CD34LO SCC cells with serial transplantations, while the two populations initially gave rise to secondary tumours with the same frequency. Our results illustrate the progressive increase of squamous skin TPCs with tumour progression and invasiveness and reveal that serial transplantation may be required to define the long‐term renewal potential of TPCs.