Gian Paolo Dotto, M.D., Ph.D.
Biologist (Dermatology)
Massachusetts General Hospital
Massachusetts General Hospital
Cutaneous Biology Research Center
Building 149, 13th Street
Charlestown MA 02129
Tel: (617) 724-9538
Fax: (617) 726-9572
Email: gdotto@partners.org
Human epithelial stem cell signaling and carcinogenesis.
Self-renewal of human epithelial stem cells versus their commitment to differentiation and/or cancer development are closely linked. Understanding this process is of great potential impact for new therapeutic approaches to human tumors, which are mostly of epithelial origin.
Keratinocytes provide an attractive experimental system to study the connection between growth/differentiation potential of epithelial cells and transformation. Over the last few years we have been focusing on the role of the Notch signaling pathway in keratinocyte growth/differentiation control and tumor development 1.
Notch signalling is an important form of intercellular communication and plays a key role in cell-fate determination and differentiation. The biological function of this pathway is critically dependent on context-specific interactions with other signalling pathways. In many mammalian systems, Notch signaling enhances stem cell potential and suppresses differentiation, while in others, notably keratinocytes, we showed that it exerts an opposite function 1.
Our main working hypothesis is that the pro-differentiation function of Notch in keratinocytes depends on integration with other cell-type-specific regulatory pathways. In our previous work, with mouse primary keratinocytes and skin, we have established important interconnections between Notch signaling and other pathways with a significant role in this cell type, shown that these interactions occur in a keratinocyte-specific manner and provided underlying biochemical mechanisms 2-6. Our ongoing work is based on the insights and information that we have gained so far, with a shift of focus from mouse to human keratinocytes. In fact, significant differences are known to exist between growth control of cells of mouse versus human origin, including, as we have found in keratinocytes, their response to Notch activation 6.
Over the last couple of years, our laboratory has been responsible for two major findings :
1) Cross-regulation between Notch and p63 in keratinocyte commitment to differentiation 6.
While Notch signaling promotes commitment of keratinocytes to differentiation, p63, a transcription factor of the p53 family, has been implicated in establishment of the keratinocyte cell fate and/or maintenance of epithelial self renewal. Furthermore, this gene is over-expressed in a variety of epithelial tumors including oral and skin squamous cell carcinomas. p63 is expressed in proliferating keratinocytes of the basal layer of the epidermis and hair follicles, and in the basal layers of the mammary gland and the prostate, while it is strongly down modulated with differentiation. The molecular basis for control of p63 expression is not known. Similarly, while elevated p63 expression can suppress differentiation, the underlying mechanisms have not been defined.
In our work we found that p63 expression is under Notch control in both mouse and human keratinocytes 6 . Specifically, p63 expression is negatively regulated by Notch activation through a mechanism independent of cell cycle withdrawal and involving modulation of genes in the interferon response pathway. In turn, p63 counteracts the ability of Notch1 to promote the irreversible versus reversible commitment to differentiation, through selective modulation of Notch-dependent transcription. Thus, as illustrated in Fig. 1, a cross-talk between Notch and p63 is involved in the balance between keratinocyte self renewal and differentiation.
Figure 1. Dynamic cross-regulation of Notch with other key signaling pathways in keratinocyte stem self renewal, differentiation and/or tumorigenesis. An inverse gradient of p63 expression versus Notch activity exists in the lower versus upper epidermal layers that results from their reciprocal negative regulation. p63 exerts a dual function in suppressing Notch signaling in epidermal cells with high self-renewal potential, while synergizing with other aspects of Notch function in early stages of differentiation. Notch1 and p21 WAF1/Cip1, a “canonical” Notch target in keratinocytes, suppress Wnt ligand expression and signaling, and function as negative regulator of stem cell potential and tumorigenesis. In fact, UV light exposure is a major ethiological agent of human skin cancer, and the Notch1 gene is a p53 target with a key role in human keratinocyte tumor suppression.
2) Notch1 is a p53 target gene involved in human keratinocyte tumor suppression 7 .
An inverse relationship exists between induction of differentiation and tumor development. Consistent with its role in maintaining lymphocyte stem cell populations, the role of Notch signaling in promoting human T cell leukemia is by now well demonstrated. A proto-oncogenic function of this pathway in human breast and ovarian carcinogenesis and in melanoma progression is also emerging. By contrast, the possibility that Notch signaling plays an alternative tumor suppressing function in human cancers of other type, where it can promote differentiation, has only been suggested on the basis of activated Notch1 over-expression studies. In our recent work, we have shown that Notch1 gene expression and activity are substantially down-modulated in keratinocyte cancer cell lines and tumors, with expression of this gene being under positive control of the p53 tumor suppressor in these cells. Genetic suppression of Notch signaling in primary human keratinocytes is sufficient, together with an activated ras oncogene, to cause aggressive squamous cell carcinoma formation (Fig. 2). Similar tumor promoting effects are also caused by in vivo treatment of mice, grafted with keratinocytes expressing oncogenic ras alone, with a pharmacological inhibitor of endogenous Notch signaling. These effects are linked with a lesser commitment of keratinocytes to differentiation, an expansion of stem cell populations and a mechanism involving effectors of small Rho GTPases, which were previously implicated in neoplastic progression.
Figure 2. Malignant tumorigenic conversion of human primary keratinocytes by suppression of Notch signaling. To evaluate the functional consequences of decreased Notch signaling, primary human keratinocytes were infected with a retroviral vector expressing a 51 amino acid peptide (MAM51) which is widely used as a specific tool to block the “canonical” Notch-pathway. After sub-cutaneous injection into SCID mice , keratinocytes expressing oncogenic ras together with MAM51gave rise to consistent tumor formation, while control cells expressing oncogenic ras alone produced either no tumors or only small nodules (upper panel). The nodules formed by control keratinocytes expressing oncogenic ras alone had histological features of well differentiated squamous cell carcinomas or benign keratinized cysts. By contrast, tumors formed by ras and MAM51 expressing keratinocytes were moderately to poorly differentiated carcinomas, with areas of spindle cell transformation, as identified by positive vimentin expression (lower panels).
Ongoing Research efforts
We are currently addressing three main topics :
- Cross-talk between Notch signaling and other pathways involved in control of human keratinocyte stem cell potential and carcinogenesis;
- Cell-type specific regulation of Notch1 gene expression;
- Identification of positive regulators of the Notch pathway by Chemical Genetics (in collaboration with the Broad Institute, Harvard and MIT).
Work is focused on primary keratinocytes of human origin, as well as on human squamous cancer cell lines and primary tumors. We are taking advantage of recent advances in genetic manipulations of these cells and of their use in a 3D organotypic culture system that recapitulates the program of vertical differentiation and epithelial-mesenchymal interactions that occur in vivo. In addition, we have developed organ culture conditions for maintenance of intact human skin as well as freshly excised skin tumors up to 1 week, for genetic and pharmacological manipulations. These in vitro approaches are complemented in vivo by skin reconstitution grafting assays, to assess long term stem cell potential and tumorigenesis (Fig. 3).
Figure 3. The keratinocyte grafting technique and resulting formation of epidermal proliferative units (EPU) by genetically labeled keratinocytes. Cultured keratinocytes are trypsinized and injected, either alone or admixed with dermal fibroblasts, into open-bottom silicon transplantation chambers that have been placed onto the back of the mouse. Grafted cells readily attach to the underlying subcutaneous tissue and form a well differentiated epidermis by 1-2 weeks of grafting 8 . Grafting of unlabeled and genetically labeled keratinocytes (in this case infected with an alkaline-phosphatase expressing adenovirus) allows detection of well delimited vertical epidermal proliferative units (EPUs), which are each the predicted product of individual progenitors or stem cells 9 .
Referred Publications
1. Lefort, K. & Dotto, G.P. Notch signaling in the integrated control of keratinocyte growth/differentiation and tumor suppression. Semin Cancer Biol14, 374-86 (2004).
2. Rangarajan, A. et al. Notch signaling is a direct determinant of keratinocyte growth arrest and entry into differentiation. Embo J20, 3427-36 (2001). Link to Article
3. Okuyama, R. et al. High commitment of embryonic keratinocytes to terminal differentiation through a Notch1-caspase 3 regulatory mechanism. Dev Cell6, 551-62 (2004).
4. Mammucari, C. et al. Integration of Notch 1 and calcineurin/NFAT signaling pathways in keratinocyte growth and differentiation control. Dev Cell8, 665-76 (2005).
5. Devgan, V., Mammucari, C., Millar, S.E., Brisken, C. & Dotto, G.P. p21WAF1/Cip1 is a negative transcriptional regulator of Wnt4 expression downstream of Notch1 activation. Genes Dev19, 1485-95 (2005). Link to Article
6. Nguyen, B.C. et al. Cross-regulation between Notch and p63 in keratinocyte commitment to differentiation. Genes Dev20, 1028-42 (2006). Link to Article
7. Lefort, K. et al. Notch1 is a p53 target gene involved in human keratinocyte tumor suppression through negative regulation of ROCK1/2 and MRCK{alpha} kinases. Genes Dev21, 562-77 (2007).Link to Article
8. Dotto, G.P., Moellmann, G., Ghosh, S., Edwards, M. & Halaban, R. Transformation of murine melanocytes by basic fibroblast growth factor cDNA and oncogenes and selective suppression of the transformed phenotype in a reconstituted cutaneous environment. J Cell Biol109, 3115-28 (1989).
9. Kamimura, J., Lee, D., Baden, H., Brissette, J. & Dotto, G.P. Primary mouse keratinocyte cultures contain hair follicle progenitor cells with multiple differentiation potential. J Invest Derm109, 534-540 (1997).
The gene expression database that has resulted in our listed publications is being made available upon request or can be retrieved from the “Gene Expression Omnibus” initiative of the NCBI (http://www.ncbi.nlm.nih.gov/geo/). Email address: gdotto@partners.org
G. Paolo Dotto, MD, PhD
Professor
Cutaneous Biology Research Center
Massachusetts General Hospital
Building 149 - 13th Street
Charlestown, MA 02129
Tel. (617) 724 9538
Fax (617) 724 9572
Cell (857) 544 3020.