Prof Colin R Goding
| Research Area: | Developmental and Stem Cell Biology |
|---|---|
| Technology Exchange: | Immunohistochemistry, In situ hybridisation and Transcript profiling |
| Scientific Themes: | Cancer Biology and Physiology, Cellular & Molecular Biology |
| Keywords: | melanocytes, melanoma, stem cells, transcription, Signalling and cancer |
| Web Links: |
Heterogeneity of melanoma cells within a tumour stained with anti-Brn2 (red) and anti-Mitf (green)
Tbx2 associated with small subnuclear heterochromatin foci stained with anti-Tbx2 (green) and ...
Left: melanoma cell; right: invasive melanoma stem cell-like cell stained for Diaph1 (green) and ...
The interface between signal transduction pathways and transcription regulation acts to coordinate the programme of gene expression underlying the genesis of specific cell lineages and cellular proliferation and differentiation during development. Deregulation of transcription is a key factor in cancer. Our laboratory is interested in determining how a precise programme of transcription regulation is achieved, particularly in the transition between normal and cancer stem cells and their proliferating progeny, and hte parallels with normal stem cell populations.
Over recent years we have made use of two model systems: understanding the controls operating in melanoma that arise through deregulation of the normal program of transcription in pigment cells; and the molecular mechanisms underlying regulation of gene expression and in particular transcription memory and the role of non-coding RNAs.
Melanocytes offer an interesting developmental system as mutations affecting the lineage result in an obvious coat colour phenotype, yet do not affect the viability of the animal. Moreover, melanoma, originating in the skin, offers a unique opportunity to examine a cancer at all stages of its development from benign naevi/moles to highly aggressive metastatic tumours.
We have recently proposed a new model for melanoma metastasis that reflects a dynamic and reversible epigenetic mechanism rather than irreversible genetic lesions, and have identified the transcription factor Mitf as a master regulator of melanoma proliferation, differentiation and invasiveness. We are currently analysing how Mitf integrates a wide range of signals to give a variable biological output and how this relates to melanoma stem cells that are proposed to provide a therapeutically resistant sub-population of cancer cells. Moreover, our most recent work is using 3C technology to detect differences chromatin architecture in cancer cell sub-populations. This technology has the potential to provide and antibody independent means to detect and monitor changes cancer subpopulations in response to therapy or disease progression.
In addition, our knowledge of melanoma subpopulations has enabled us to identify, and develop novel fluorescent reporters for melanoma stem cells that will be invaluable in their characterisation and ultimately screens for drugs that eradicate them.
We have also shown the potential of pro-senescence therapy for this disease based on inhibition of the T-box transcription factors Tbx2 and Tbx3. Understanding how these factors inhibit senescence and how we can inhibit their function in melanoma is also a key aim and we are currently working towards developing pro-senescence drugs.
Finally our mechanistic studies on gene regulation have revealed a novel class of transcription factor we term 'promoter education factors' that act in a temporally distinct fashion from classical transcription activators to bring about the correct kinetics of gene expression in response to extracellular signals. We are currently exploring how such factors may contribute to priming stem cells for activation.
| Name | Department | Institution | Country |
|---|---|---|---|
| Lionel Larue | Institute Curie | France | |
| Eirkur Steingrimsson | University of Iceland | Iceland | |
| Luisa Lanfrancone | Euorpean Instute of Oncology, Milan | Italy | |
| Svenja Meierjohann | University of Würzburg | Germany | |
| Dr Jose Neptuno Rodriguez Lopez | University of Murcia | Spain | |
| Dr Irwin Davidson | IGBMC | France |
2007. Beta-catenin induces immortalization of melanocytes by suppressing p16INK4a expression and cooperates with N-Ras in melanoma development. Genes Dev, 21 (22), pp. 2923-2935. Read abstract | Read more
Tumor progression is a multistep process in which proproliferation mutations must be accompanied by suppression of senescence. In melanoma, proproliferative signals are provided by activating mutations in NRAS and BRAF, whereas senescence is bypassed by inactivation of the p16(Ink4a) gene. Melanomas also frequently exhibit constitutive activation of the Wnt/beta-catenin pathway that is presumed to induce proliferation, as it does in carcinomas. We show here that, contrary to expectations, stabilized beta-catenin reduces the number of melanoblasts in vivo and immortalizes primary skin melanocytes by silencing the p16(Ink4a) promoter. Significantly, in a novel mouse model for melanoma, stabilized beta-catenin bypasses the requirement for p16(Ink4a) mutations and, together with an activated N-Ras oncogene, leads to melanoma with high penetrance and short latency. The results reveal that synergy between the Wnt and mitogen-activated protein (MAP) kinase pathways may represent an important mechanism underpinning the genesis of melanoma, a highly aggressive and increasingly common disease. Hide abstract
2006. Mitf regulation of Dia1 controls melanoma proliferation and invasiveness. Genes Dev, 20 (24), pp. 3426-3439. Read abstract | Read more
It is widely held that cells with metastatic properties such as invasiveness and expression of matrix metalloproteinases arise through the stepwise accumulation of genetic lesions arising from genetic instability and "clonal evolution." By contrast, we show here that in melanomas invasiveness can be regulated epigenetically by the microphthalmia-associated transcription factor, Mitf, via regulation of the DIAPH1 gene encoding the diaphanous-related formin Dia1 that promotes actin polymerization and coordinates the actin cytoskeleton and microtubule networks at the cell periphery. Low Mitf levels lead to down-regulation of Dia1, reorganization of the actin cytoskeleton, and increased ROCK-dependent invasiveness, whereas increased Mitf expression leads to decreased invasiveness. Significantly the regulation of Dia1 by Mitf also controls p27(Kip1)-degradation such that reduced Mitf levels lead to a p27(Kip1)-dependent G1 arrest. Thus Mitf, via regulation of Dia1, can both inhibit invasiveness and promote proliferation. The results imply variations in the repertoire of environmental cues that determine Mitf activity will dictate the differentiation, proliferative, and invasive/migratory potential of melanoma cells through a dynamic epigenetic mechanism. Hide abstract
2005. Tbx2 is overexpressed and plays an important role in maintaining proliferation and suppression of senescence in melanomas. Cancer Res, 65 (6), pp. 2260-2268. Read abstract | Read more
The INK4a and ARF genes found at the CDKN2A locus are key effectors of cellular senescence that is believed to act as a powerful anticancer mechanism. Accordingly, mutations in these genes are present in a wide variety of spontaneous human cancers and CDKN2A germ line mutations are found in familial melanoma. The TBX2 gene encoding a key developmental transcription factor is amplified in pancreatic cancer cell lines and preferentially amplified and overexpressed in BRCA1 and BRCA2 mutated breast tumors. Overexpression of Tbx2 and the related factor Tbx3, which is also overexpressed in breast cancer and melanomas, can suppress senescence in defined experimental systems through repression of ARF expression. However, it is not known how Tbx2 mediates its repressive effect nor whether endogenous Tbx2 or Tbx3 perform a similar antisenescence function in transformed cells. This is a particularly important question because the loss of CDKN2A in many human cancers would, in principle, bypass the requirement for Tbx2/3-mediated repression of ARF in suppressing senescence. We show here that Tbx2 is overexpressed in melanoma cell lines and that Tbx2 targets histone deacetylase 1 to the p21Cip1 (CDKN1A) initiator. Strikingly, expression of an inducible dominant-negative Tbx2 (dnTbx2) leads to displacement of histone deacetylase 1, up-regulation of p21(Cip1) expression, and the induction of replicative senescence in CDKN2A-null B16 melanoma cells. In human melanoma cells, expression of dnTbx2 leads to severely reduced growth and induction of senescence-associated heterochromatin foci. The results suggest that the activity of endogenous Tbx2 is critically required to maintain proliferation and suppress senescence in melanomas. Hide abstract
2005. Mitf cooperates with Rb1 and activates p21Cip1 expression to regulate cell cycle progression. Nature, 433 (7027), pp. 764-769. Read abstract | Read more
The controls that enable melanoblasts and melanoma cells to proliferate are likely to be related, but so far no key regulator of cell cycle progression specific to the melanocyte lineage has been identified. The microphthalmia-associated transcription factor Mitf has a crucial but poorly defined role in melanoblast and melanocyte survival and in differentiation. Here we show that Mitf can act as a novel anti-proliferative transcription factor able to induce a G1 cell-cycle arrest that is dependent on Mitf-mediated activation of the p21(Cip1) (CDKN1A) cyclin-dependent kinase inhibitor gene. Moreover, cooperation between Mitf and the retinoblastoma protein Rb1 potentiates the ability of Mitf to activate transcription. The results indicate that Mitf-mediated activation of p21Cip1 expression and consequent hypophosphorylation of Rb1 will contribute to cell cycle exit and activation of the differentiation programme. The mutation of genes associated with melanoma, such as INK4a or BRAF that would affect either Mitf cooperation with Rb1 or Mitf stability respectively, would impair Mitf-mediated cell cycle control. Hide abstract
2004. Precise nucleosome positioning and the TATA box dictate requirements for the histone H4 tail and the bromodomain factor Bdf1. Mol Cell, 15 (1), pp. 69-81. Read abstract | Read more
Acetylation of histone tails plays a key role in chromatin dynamics and is associated with the potential for gene expression. We show here that a 2-3 bp mispositioning of the nucleosome covering the TATA box at PHO5 induces a dependency on the acetylatable lysine residues of the histone H4 N-terminal region and on the TFIID-associated bromodomain factor Bdf1. This dependency arises either through fusion of the PHO5 promoter to a lacZ reporter or by mutation of the TATA box in the natural gene. The results suggest that promoters in which the TATA box is either absent or poorly accessible on the surface of a nucleosome may compensate by using Bdf1 bromodomains and acetylated H4 tails to anchor TFIID to the promoter during the initial stages of transcription activation. We propose that nucleosome positioning at the nucleotide level provides a subtle, but highly effective, mechanism for gene regulation. Hide abstract
2004. The Brn-2 transcription factor links activated BRAF to melanoma proliferation. Mol Cell Biol, 24 (7), pp. 2923-2931. Read abstract | Read more
Malignant melanoma, an aggressive and increasingly common cancer, is characterized by a strikingly high rate (70%) of mutations in BRAF, a key component of the mitogen-activated protein (MAP) kinase signaling pathway. How signaling events downstream from BRAF affect the underlying program of gene expression is poorly understood. We show that the Brn-2 POU domain transcription factor is highly expressed in melanoma cell lines but not in melanocytes or melanoblasts and that overexpression of Brn-2 in melanocytes results in increased proliferation. Expression of Brn-2 is strongly upregulated by Ras and MAP kinase signaling. Importantly, the Brn-2 promoter is stimulated by kinase-activating BRAF mutants and endogenous Brn-2 expression is inhibited by RNA interference-mediated downregulation of BRAF. Moreover, silent interfering RNA-mediated depletion of Brn-2 in melanoma cells expressing activated BRAF leads to decreased proliferation. The results suggest that the high levels of Brn-2 expression observed in melanomas link BRAF signaling to increased proliferation. Hide abstract
2003. Regulated displacement of TBP from the PHO8 promoter in vivo requires Cbf1 and the Isw1 chromatin remodeling complex. Mol Cell, 11 (6), pp. 1609-1620. Read abstract | Read more
Regulated binding of TBP to a promoter is a key event in transcriptional regulation. We show here that on glucose depletion, the S. cerevisiae Isw1 chromatin remodeling complex is required for the displacement of TBP from the PHO8 promoter. Displacement of TBP also requires the sequence-specific bHLH-LZ factor Cbf1p that targets Isw1p to the PHO8 UAS. Cbf1p- and Isw1p-dependent displacement of TBP is also observed at the PHO84 promoter, but not at the ADH1 promoter, where loss of TBP is Cbf1p- and Isw1p independent. The results point to a promoter-specific Isw1p-dependent mechanism for targeted regulation of basal transcription by displacement of TBP from a promoter. Hide abstract
2001. The Usf-1 transcription factor is a novel target for the stress-responsive p38 kinase and mediates UV-induced Tyrosinase expression. EMBO J, 20 (17), pp. 5022-5031. Read abstract | Read more
The stress-activated signalling cascade leading to phosphorylation of the p38 family of kinases plays a crucial role during development and in the cellular response to a wide variety of stress-inducing agents. Although alterations in gene expression characteristic of the stress response require the regulation of key transcription factors by the p38 family, few downstream targets for this signalling pathway have been identified. By examining the ability of pigment cells to respond to UV irradiation as part of the UV-induced tanning response, we show that while the microphthalmia-associated transcription factor Mitf regulates basal Tyrosinase expression, it is the ubiquitous basic helix-loop-helix-leucine zipper transcription factor Usf-1 that is required for the UV activation of the Tyrosinase promoter. Consistent with this we demonstrate that Usf-1 is phosphorylated and activated by the stress-responsive p38 kinase. The results suggest that activation of Usf-1 by p38 at a wide variety of viral and cellular promoters will provide a link between stimuli as diverse as UV irradiation, glucose, viral infection and pro-inflammatory cytokines, and the changes in gene expression associated with the stress response. Hide abstract
Signalling epigenetic switches in cancer stem cells
A major challenge to effective anti-cancer therapy is tumour cell heterogeneity. Within cancers, multiple sub-populations of cancer cells may co-exist, each with differing biological properties. Some may exhibit features of differentiation, others proliferate, while some possess stem cell-like properties, able to initiate new tumours and provide a pool of therapeutically resistant cells. Understanding the origins of cancer cell heterogeneity and how it can be managed to provide more effective ...
Targetting cancer stem cells using small-molecule drug screens
A major challenge to effective anti-cancer therapy is tumour cell heterogeneity. Within cancers, multiple sub-populations of cancer cells may co-exist, each with differing biological properties. Some may exhibit features of differentiation, others proliferate, while some possess stem cell-like properties, able to initiate new tumours and provide a pool of therapeutically resistant cells. Understanding the origins of cancer cell heterogeneity and how it can be managed to provide more effective ...
Regulation of tumour-specific vascular genes
The formation of new blood vessels is an essential requirement for the development of solid tumours. Consequently, understanding the regulatory cascade involved in vessel recruitment during tumour growth is necessary for the development of therapies to prevent or modulate this process. In recent years, a number of tumour endothelial markers (TEM) have been described. These are genes which are highly expressed in human tumor vasculature compared with vasculature in normal tissue. This DPhil ...
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