News and Views surprising. Testing the consequences of the KITLG p53-RE in a cellular mechanism deregulated in cancer, the authors investigated its effect on proliferation in the developing pigmentary system in mice. They observed an increase in epidermal melanocyte count and KITLG expression in pups upon UV irradiation; this response was severely decreased in p53 null mice. Furthermore, melanocyte proliferation was increased in wildtype mice compared with p53 null, based on immunostaining with TYRP1 and the proliferative marker Ki67. The anticancer role of p53 has been well established, and somatic mutations have been found in 50% of human cancers, making it the most commonly mutated cancer gene. However, testicular cancers are an exception with a low rate of p53 mutations (Peng et al., 1993). The normal p53 activity in this type of cancer may be explained by p53-KITLG-driven male germ cell proliferation despite the presence of DNA damage, contributing to tumorigenesis. This mechanism would be more prevalent in Caucasians than Africans, explain-
ing why this type of cancer is 4–5-fold more common in Caucasians compared with Africans (Holmes et al., 2008). It is a difficult task to interpret the functional consequences of SNPs that reside in non-coding DNA. ZeronMedina et al. have paved the way for systematic functional identification and, importantly, validation of these variants. Published whole-genome data, such as that provided by the ENCODE project, hold vast information on diverse properties of non-coding sequences in the human genome. It will now be important to make use of the available data combined with functional studies to shed light on cellular mechanisms involving non-coding DNA sequences. This approach will without doubt continue to reveal interesting and surprising findings regarding pigmentation.
References
response and pathologic hyperpigmentation. Cell 128, 853–864. Holmes, L. Jr., Escalante, C., Garrison, O., Foldi, B.X., Ogungbade, G.O., Essien, E.J., and Ward, D. (2008). Testicular cancer incidence trends in the USA (1975–2004): plateau or shifting racial paradigm? Public Health 122, 862–872. Lennartsson, J., and Ronnstrand, L. (2012). Stem cell factor receptor/c-Kit: from basic science to clinical implications. Physiol. Rev. 92, 1619–1649. Murase, D., Hachiya, A., Amano, Y., Ohuchi, A., Kitahara, T., and Takema, Y. (2009). The essential role of p53 in hyperpigmentation of the skin via regulation of paracrine melanogenic cytokine receptor signaling. J. Biol. Chem. 284, 4343–4353. Peng, H.Q., Hogg, D., Malkin, D., Bailey, D., Gallie, B.L., Bulbul, M., Jewett, M., Buchanan, J., and Goss, P.E (1993). Mutations of the p53 gene do not occur in testis cancer. Cancer Res. 53, 3574–3578.
Cui, R., Widlund, H.R., Feige, E. et al. (2007). Central role of p53 in the suntan
Fishing for melanoma drivers Adam Hurlstone e-mail: [email protected]
As the first melanoma genome was sequenced, the mutational complexity of this cancer type has been apparent: almost 300 protein altering mutations were found. Almost twice as many synonymous mutations were also
Coverage on: Yen, J., White, R.M., Wedge, D.C., Van Loo, P., de Ridder, J., Capper, A., Richardson, J., Jones, D., Raine, K., Watson, I.R., Wu, C.J., Cheng, J., Martincorena, I., Nik-Zainal, S., Mudie, L., Moreau, Y., Marshall, J., Ramakrishna, M., Tarpey, P., Shlien, A., Whitmore, I., Gamble, S., Latimer, C., Langdon, E., Kaufman, C., Dovey, M., Taylor, A., Menzies, A., McLaren, S.O., Meara, S., Butler, A., Teague, J., Lister, J., Chin, L., Campbell, P., Adams, D.J., Zon, L.I., Patton, E.E., Stemple, D.L., Futreal, P.A. (2013). The genetic heterogeneity and mutational burden of engineered melanomas in zebrafish models. Genome Biol. Oct 23;14(10):R113. [Epub ahead of print]. doi: 10.1111/pcmr.12214
detected, indicating that the majority of mutations were likely to be mere ‘passengers’ – irrelevant to the transformed phenotype (Pleasance et al., 2010). The challenge prompted by this study was to devise a strategy that could pinpoint mutations responsible for the transformed phenotype, so-called driver mutations. Proteins with driver mutations, and in particular acquiring a gainof-function, are high-value targets, as antagonists of such drivers have proven particularly potent at shrinking tumours. Identifying drivers in melanoma is made more challenging by the very high mutation frequencies resulting from the aetiological agent UV radiation (UVR). (A UVR mutation signature is apparent in the genome of melanoma from sunexposed sights comprising frequent C > T transitions occurring especially within CC dinucleotides.) One solution has been to analyse a greater number of genomes and identify non-random recurrent mutations. Throughput has been augmented by either zooming in on a subset of the genome or scrutinising only the protein coding fraction (the
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
exome) [for a recent example see (Krauthammer et al., 2012)]. Substitutions and indels have been supplemented by copy number gains and losses and rearrangements. Collectively, these studies have confirmed the supremacy of BRAF and NRAS gain-offunction and PTEN and CDKN2A lossof-function and revealed that CCND1 amplification and RAC1 gain-of-function and ARID2, TP53 and PPP6C loss-offunction are close runners-up. Other contenders have also been advanced, including ERBB4, GRIN2A, GRM3 and -8, MAPK3K5 and -9, MEK1 and -2, MITF, PREX2 and TRRAP, although with seemingly little cross-study agreement, possibly reflecting methodological differences. Independently, very frequent mutations have been detected in the promoter of the telomerase reverse transcriptase gene TERT resulting in its increased activity in melanoma. The heterogeneity of melanoma genomes confounds a comprehensive and coherent model of deregulated signalling emerging from the above studies. Nonetheless, it can now be
155
News and Views stated with a high degree of confidence that melanocyte transformation requires deregulation of MAPK/ERK and PI3K/ AKT signalling, and abrogation of cell-cycle, senescence and crisis checkpoints, consistent with a classical/ pregenomic understanding that has guided drug discovery programmes over the past two decades. Comparing cancer genomes across animal taxa is an alternative method for highlighting driver mutations. The comparison made has typically been between mice and humans, but had not yet been made for melanoma. Now, the groups of Andrew Futreal (The University of Texas MD Anderson Cancer Center) and Derek Stemple (the Sanger Insititute) in collaboration with teams lead by Liz Patton (MRC Human Genetics Unit and Edinburgh Cancer Research Centre) and Len Zon (Harvard Medical School, Boston) used targeted exome capture and Illumina sequencing of zebrafish melanoma and normal DNA to scrutinise all the protein coding genes present in the Zv9 genome release for sequence and copy number changes. The zebrafish melanoma models utilised in the study by Yen et al. featured either exogenous BRAFV600E or Q61K NRAS driven by the zebrafish mitf gene promoter mostly in combination with loss-of-function of p53, and sometimes additionally of mitf or pten. In total, 38 exomes were sequenced from animals expressing BRAFV600E and fifteen from animals expressing NRASQ61K. On average, 21.8 Gb of sequence was generated per sample giving >3009 coverage The median number of coding mutations per sample was four, significantly fewer than from sun-exposed human melanomas but comparable to the rate found in human melanoma from sun-shielded sites (Krauthammer et al., 2012), in keeping with the absence of UVR as an aetiological agent in zebrafish melanoma. More substitutions were found when fewer initiating events were frontloaded that is the highest rate of mutation was detected in a melanoma with only BRAFV600E engineered, while exomes from melanoma containing two or more additional initiating drivers often had no de novo substitutions detected. Owing to the low substitution rate, no recurrent mutations were detected in known melanoma genes or genes in the Cancer Gene Census.
156
While additional substitutions may not be required for melanoma development in zebrafish already harbouring BRAF – or NRAS – and p53 mutations, Yen et al. detected 991 amplified segments, with 85% of samples harbouring at least one amplification, revealing significant aneuploidy in zebrafish melanoma also. One known cancer gene recurrently amplified in zebrafish melanoma was TERT, which is amplified in human melanoma and as mentioned harbours frequent promoter mutations. Intriguingly, a recurrent amplification of the gene encoding the catalytic domain of PKA (prkacaa) was detected in half of a subset of melanomas expressing BRAFV600E, in which melanoma cells were constrained to also express MITF. In this scenario, increased PKA activity could stimulate MITF expression (through activating the transcription factor CREB), comparable to MITF amplification that has been detected in a subset of human melanoma. 436 segments of homozygous deletions were also detected, but arising in only 30% of samples. Few genes were recurrently deleted and none of these had previously been associated with cancer. A notable recurrent deletion (three samples belonging to both BRAF and NRAS mutant lines) ‘when it comes contained multiple nitr genes, to protein which encode drivers which novel immune could make for type receptors therapeutic found in teleotargets, the lowsts. This indilying fruit have cates a potential mostly already requirement to been picked.’ avoid immune surveillance for melanoma development in zebrafish, comparable to mammals. The scarcity of de novo recurrent mutations in zebrafish melanoma engineered with mutant BRAF – or NRAS – and p53 implies that only a few mutations are needed to establish malignancy. Yen et al. conclude that genetically engineered animal cancer models can be used to ‘bound and estimate the number of events in human cancers’. As already stated, human melanoma from sun-shielded sites also typically harbour only a handful of mutations, few of which are recurrent (Krauthammer et al., 2012). In addition to mutation, epigenetic
change, encompassing both altered chromatin structure, transcription factor activity, and signalling activity, is also important in allowing cancer cells to acquire malignant hallmarks. A zebrafish melanoma model was used to demonstrate that the histone methylase SETDB1, which in humans is encoded by a gene located in a recurrent amplicon, is an oncogene (Ceol et al., 2011). There is significant overlap in the programme of gene expression in zebrafish and human melanoma (Dovey et al., 2009), and studies have also shown that increased activity of both PI3K and Rac promotes melanoma progression in zebrafish and man (Dalton et al., 2013). Thus, several key molecular aberrations are conserved in the transformation of fish and mammalian melanocytes, which may be achieved through mutation or epigenetic factors. Perhaps, it would be fruitful to start exploring the therapeutic potential of epigenetic factors and misexpressed gene products. Model organisms like zebrafish could prove invaluable in identifying and validating such targets.
References Ceol, C.J., Houvras, Y., Jane-Valbuena, J. et al. (2011). The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset. Nature 471, 513–517. Dalton, L.E., Kamarashev, J., BarinagaRementeria Ramirez, I., White, G., Malliri, A., and Hurlstone, A. (2013). Constitutive RAC activation is not sufficient to initiate melanocyte neoplasia but accelerates malignant progression. J. Invest. Dermatol. 133, 1572–1581. Dovey, M., White, R.M., and Zon, L.I. (2009). Oncogenic NRAS cooperates with p53 loss to generate melanoma in zebrafish. Zebrafish 6, 397–404. Krauthammer, M., Kong, Y., Ha, B.H. et al. (2012). Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma. Nat. Genet. 44, 1006– 1014. Pleasance, E.D., Cheetham, R.K., Stephens, P.J. et al. (2010). A comprehensive catalogue of somatic mutations from a human cancer genome. Nature 463, 191–196.
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
ing why this type of cancer is 4–5-fold more common in Caucasians compared with Africans (Holmes et al., 2008). It is a difficult task to interpret the functional consequences of SNPs that reside in non-coding DNA. ZeronMedina et al. have paved the way for systematic functional identification and, importantly, validation of these variants. Published whole-genome data, such as that provided by the ENCODE project, hold vast information on diverse properties of non-coding sequences in the human genome. It will now be important to make use of the available data combined with functional studies to shed light on cellular mechanisms involving non-coding DNA sequences. This approach will without doubt continue to reveal interesting and surprising findings regarding pigmentation.
References
response and pathologic hyperpigmentation. Cell 128, 853–864. Holmes, L. Jr., Escalante, C., Garrison, O., Foldi, B.X., Ogungbade, G.O., Essien, E.J., and Ward, D. (2008). Testicular cancer incidence trends in the USA (1975–2004): plateau or shifting racial paradigm? Public Health 122, 862–872. Lennartsson, J., and Ronnstrand, L. (2012). Stem cell factor receptor/c-Kit: from basic science to clinical implications. Physiol. Rev. 92, 1619–1649. Murase, D., Hachiya, A., Amano, Y., Ohuchi, A., Kitahara, T., and Takema, Y. (2009). The essential role of p53 in hyperpigmentation of the skin via regulation of paracrine melanogenic cytokine receptor signaling. J. Biol. Chem. 284, 4343–4353. Peng, H.Q., Hogg, D., Malkin, D., Bailey, D., Gallie, B.L., Bulbul, M., Jewett, M., Buchanan, J., and Goss, P.E (1993). Mutations of the p53 gene do not occur in testis cancer. Cancer Res. 53, 3574–3578.
Cui, R., Widlund, H.R., Feige, E. et al. (2007). Central role of p53 in the suntan
Fishing for melanoma drivers Adam Hurlstone e-mail: [email protected]
As the first melanoma genome was sequenced, the mutational complexity of this cancer type has been apparent: almost 300 protein altering mutations were found. Almost twice as many synonymous mutations were also
Coverage on: Yen, J., White, R.M., Wedge, D.C., Van Loo, P., de Ridder, J., Capper, A., Richardson, J., Jones, D., Raine, K., Watson, I.R., Wu, C.J., Cheng, J., Martincorena, I., Nik-Zainal, S., Mudie, L., Moreau, Y., Marshall, J., Ramakrishna, M., Tarpey, P., Shlien, A., Whitmore, I., Gamble, S., Latimer, C., Langdon, E., Kaufman, C., Dovey, M., Taylor, A., Menzies, A., McLaren, S.O., Meara, S., Butler, A., Teague, J., Lister, J., Chin, L., Campbell, P., Adams, D.J., Zon, L.I., Patton, E.E., Stemple, D.L., Futreal, P.A. (2013). The genetic heterogeneity and mutational burden of engineered melanomas in zebrafish models. Genome Biol. Oct 23;14(10):R113. [Epub ahead of print]. doi: 10.1111/pcmr.12214
detected, indicating that the majority of mutations were likely to be mere ‘passengers’ – irrelevant to the transformed phenotype (Pleasance et al., 2010). The challenge prompted by this study was to devise a strategy that could pinpoint mutations responsible for the transformed phenotype, so-called driver mutations. Proteins with driver mutations, and in particular acquiring a gainof-function, are high-value targets, as antagonists of such drivers have proven particularly potent at shrinking tumours. Identifying drivers in melanoma is made more challenging by the very high mutation frequencies resulting from the aetiological agent UV radiation (UVR). (A UVR mutation signature is apparent in the genome of melanoma from sunexposed sights comprising frequent C > T transitions occurring especially within CC dinucleotides.) One solution has been to analyse a greater number of genomes and identify non-random recurrent mutations. Throughput has been augmented by either zooming in on a subset of the genome or scrutinising only the protein coding fraction (the
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
exome) [for a recent example see (Krauthammer et al., 2012)]. Substitutions and indels have been supplemented by copy number gains and losses and rearrangements. Collectively, these studies have confirmed the supremacy of BRAF and NRAS gain-offunction and PTEN and CDKN2A lossof-function and revealed that CCND1 amplification and RAC1 gain-of-function and ARID2, TP53 and PPP6C loss-offunction are close runners-up. Other contenders have also been advanced, including ERBB4, GRIN2A, GRM3 and -8, MAPK3K5 and -9, MEK1 and -2, MITF, PREX2 and TRRAP, although with seemingly little cross-study agreement, possibly reflecting methodological differences. Independently, very frequent mutations have been detected in the promoter of the telomerase reverse transcriptase gene TERT resulting in its increased activity in melanoma. The heterogeneity of melanoma genomes confounds a comprehensive and coherent model of deregulated signalling emerging from the above studies. Nonetheless, it can now be
155
News and Views stated with a high degree of confidence that melanocyte transformation requires deregulation of MAPK/ERK and PI3K/ AKT signalling, and abrogation of cell-cycle, senescence and crisis checkpoints, consistent with a classical/ pregenomic understanding that has guided drug discovery programmes over the past two decades. Comparing cancer genomes across animal taxa is an alternative method for highlighting driver mutations. The comparison made has typically been between mice and humans, but had not yet been made for melanoma. Now, the groups of Andrew Futreal (The University of Texas MD Anderson Cancer Center) and Derek Stemple (the Sanger Insititute) in collaboration with teams lead by Liz Patton (MRC Human Genetics Unit and Edinburgh Cancer Research Centre) and Len Zon (Harvard Medical School, Boston) used targeted exome capture and Illumina sequencing of zebrafish melanoma and normal DNA to scrutinise all the protein coding genes present in the Zv9 genome release for sequence and copy number changes. The zebrafish melanoma models utilised in the study by Yen et al. featured either exogenous BRAFV600E or Q61K NRAS driven by the zebrafish mitf gene promoter mostly in combination with loss-of-function of p53, and sometimes additionally of mitf or pten. In total, 38 exomes were sequenced from animals expressing BRAFV600E and fifteen from animals expressing NRASQ61K. On average, 21.8 Gb of sequence was generated per sample giving >3009 coverage The median number of coding mutations per sample was four, significantly fewer than from sun-exposed human melanomas but comparable to the rate found in human melanoma from sun-shielded sites (Krauthammer et al., 2012), in keeping with the absence of UVR as an aetiological agent in zebrafish melanoma. More substitutions were found when fewer initiating events were frontloaded that is the highest rate of mutation was detected in a melanoma with only BRAFV600E engineered, while exomes from melanoma containing two or more additional initiating drivers often had no de novo substitutions detected. Owing to the low substitution rate, no recurrent mutations were detected in known melanoma genes or genes in the Cancer Gene Census.
156
While additional substitutions may not be required for melanoma development in zebrafish already harbouring BRAF – or NRAS – and p53 mutations, Yen et al. detected 991 amplified segments, with 85% of samples harbouring at least one amplification, revealing significant aneuploidy in zebrafish melanoma also. One known cancer gene recurrently amplified in zebrafish melanoma was TERT, which is amplified in human melanoma and as mentioned harbours frequent promoter mutations. Intriguingly, a recurrent amplification of the gene encoding the catalytic domain of PKA (prkacaa) was detected in half of a subset of melanomas expressing BRAFV600E, in which melanoma cells were constrained to also express MITF. In this scenario, increased PKA activity could stimulate MITF expression (through activating the transcription factor CREB), comparable to MITF amplification that has been detected in a subset of human melanoma. 436 segments of homozygous deletions were also detected, but arising in only 30% of samples. Few genes were recurrently deleted and none of these had previously been associated with cancer. A notable recurrent deletion (three samples belonging to both BRAF and NRAS mutant lines) ‘when it comes contained multiple nitr genes, to protein which encode drivers which novel immune could make for type receptors therapeutic found in teleotargets, the lowsts. This indilying fruit have cates a potential mostly already requirement to been picked.’ avoid immune surveillance for melanoma development in zebrafish, comparable to mammals. The scarcity of de novo recurrent mutations in zebrafish melanoma engineered with mutant BRAF – or NRAS – and p53 implies that only a few mutations are needed to establish malignancy. Yen et al. conclude that genetically engineered animal cancer models can be used to ‘bound and estimate the number of events in human cancers’. As already stated, human melanoma from sun-shielded sites also typically harbour only a handful of mutations, few of which are recurrent (Krauthammer et al., 2012). In addition to mutation, epigenetic
change, encompassing both altered chromatin structure, transcription factor activity, and signalling activity, is also important in allowing cancer cells to acquire malignant hallmarks. A zebrafish melanoma model was used to demonstrate that the histone methylase SETDB1, which in humans is encoded by a gene located in a recurrent amplicon, is an oncogene (Ceol et al., 2011). There is significant overlap in the programme of gene expression in zebrafish and human melanoma (Dovey et al., 2009), and studies have also shown that increased activity of both PI3K and Rac promotes melanoma progression in zebrafish and man (Dalton et al., 2013). Thus, several key molecular aberrations are conserved in the transformation of fish and mammalian melanocytes, which may be achieved through mutation or epigenetic factors. Perhaps, it would be fruitful to start exploring the therapeutic potential of epigenetic factors and misexpressed gene products. Model organisms like zebrafish could prove invaluable in identifying and validating such targets.
References Ceol, C.J., Houvras, Y., Jane-Valbuena, J. et al. (2011). The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset. Nature 471, 513–517. Dalton, L.E., Kamarashev, J., BarinagaRementeria Ramirez, I., White, G., Malliri, A., and Hurlstone, A. (2013). Constitutive RAC activation is not sufficient to initiate melanocyte neoplasia but accelerates malignant progression. J. Invest. Dermatol. 133, 1572–1581. Dovey, M., White, R.M., and Zon, L.I. (2009). Oncogenic NRAS cooperates with p53 loss to generate melanoma in zebrafish. Zebrafish 6, 397–404. Krauthammer, M., Kong, Y., Ha, B.H. et al. (2012). Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma. Nat. Genet. 44, 1006– 1014. Pleasance, E.D., Cheetham, R.K., Stephens, P.J. et al. (2010). A comprehensive catalogue of somatic mutations from a human cancer genome. Nature 463, 191–196.
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
