Team effort
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© 2001 Macmillan Magazines Ltd Invasive melanoma is notoriously difficult to treat, but the generation of mouse models of the disease would facilitate the development of vac- cines and expedite screening for new drugs. Sotillo et al., reporting in the 6 November issue of Proceedings of the National Academy of Sciences, describe such a mouse model. Most human tumours have deregulated CDK4 activity, usually due to cyclin D1 overex- pression or inactivation of the INK4 cyclin- dependent kinase inhibitors, but a point muta- tion in CDK4 has also been identified in patients with both spontaneous and familial melanoma. Mutation of this residue (R24C) prevents CDK4 from binding to the INK4 proteins. To investigate the role of R24C in melanoma susceptibility, the authors generat- ed knock-in mice that express Cdk4 R24C. They develop multiple tumours, but do not show melanocyte hyperproliferation (see pic- ture). However, on treatment with the skin carcinogens 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol- 13-acetate (TPA), they develop more, and larger, skin papillomas than wild-type mice, which frequently progress to invasive carcino- mas. Nevi (moles), which are precursors of melanoma, are also more common in the carcinogen-treated Cdk4 R24C mice, and 70% of these mice developed melanomas. Interestingly, although the draining and regional lymph nodes were enlarged and pig- mented, distant metastases were not found. This could be because the mice die before distant metastasis occurs, or because they lack distant metastatic potential. So, is loss of the Ink4 inhibitors at the Cdkn2a and Cdkn2b loci also necessary for the generation of melanoma? Sotillo et al. suggest not, as Southern blotting of the melanomas from Cdk4 R24C mice did not reveal deletion or rearrange- ment of the loci, or promoter methylation in the the genes encoding Ink4a, Ink4b and Arf. In addi- tion to this, immunostaining of Ink4a showed that it was present in all tumours analysed. Cdk4 R24C mice are insensitive to all mem- bers of the Ink4 family, so which confer suscepti- bility to melanoma? The authors investigated tumour incidence following carcinogen treat- ment in mice deficient in Ink4b and Ink4c, as well as in Ink4a∆2,3 mice — which produce neither Ink4a nor Arf. Ink4a∆2,3 mice have an increased susceptibility to lymphomas, so all die by 10–12 weeks — before the incidence of melanoma could be evaluated — and Ink4b-deficient mice do not exhibit a higher rate of melanomas than wild-type mice. Ink4c-deficient mice, however, have an increased incidence of melanomas, but not of papillomas. The incidence of melanoma is still lower than in carcinogen-treated Cdk4 R24C mice, indicating that their susceptibility to melanoma is probably also due to loss of interac- tion with Ink4a, which is supported by results from human tumours and other melanoma mouse models (see further reading). Now, with several melanoma mouse models available, it is time to use them to generate new treatment opportunities for melanoma. Emma Greenwood References and links ORIGINAL RESEARCH PAPER Sotillo, R. et al. Invasive melanoma in Cdk4-targeted mice. Proc. Natl Acad. Sci. USA 98, 13312–13317 (2001) FURTHER READING Krimpenfort, P. et al. Loss of p16 Ink4a confers susceptibility to metastatic melanoma in mice. Nature 413, 83–86 (2001) | Sharpless, N. E. et al. Loss of p16 Ink4a with retention of p19 ARF predisposes mice to tumorigenesis. Nature 413, 86–91 (2001) | Sotillo, R. et al. Wide spectrum of tumors in knock-in mice carrying a Cdk4 protein insensitive to INK4 inhibitors. EMBO J. (in the press) WEB SITE Encyclopedia of Life Sciences: www.els.net melanoma Of mice and moles MOUSE MODELS Melanocytes are barely visible in wild-type cells (left), but hyperproliferate in the Cdk4 R24C mice (right; see green stain). Reproduced with permission © (2001) Proc. Natl Acad. Sci. USA. NATURE REVIEWS | CANCER VOLUME 1 | DECEMBER 2001 | 177 Why has it been so difficult to model hereditary breast cancer in mice? Homozygous loss of either of the known breast-cancer susceptibility genes, Brca1 and Brca2, is not compatible with life, whereas mice bearing truncation mutations die or develop lymphomas. Jos Jonkers and colleagues now describe how they have overcome these problems by developing a conditional knockout of Brca2, using the cre–lox system. The authors targeted exon 11 of Brca2, which encodes its Rad51-binding BRC repeats. Binding to Rad51 is necessary for Brca2 to carry out its poorly understood function in DNA repair. To generate a tissue- specific conditional mutant, the authors put the cre gene under the control of the keratin 14 (K14) promoter, which is active in stratified epithelia such as those in mammary gland and skin. By crossing K14cre mice with mice carrying the Brca2 ∆11 allele, mice were bred in which both Brca2 alleles were mutated in tissues where the K14 promoter is active. Surpsisingly, these mice did not develop tumours any more frequently than wild-type mice. So what else is needed to tip their apparently normal mammary tissue towards tumour formation? The tumours of women with BRCA2 mutations often show loss of TP53; could this situation be reproduced by crossing Brca2 mutant mice with conditional Trp53 knockout mice? The conditional Trp53 knockouts generated by the authors were tumour prone but didn’t develop any mammary tumours. In stark contrast, mice that were homozygous for both mutations in epithelia developed tumours of the mammary gland and skin. Mice with just one functional copy of either Brca2 or Trp53 in epithelium also developed tumours, but with a longer latency. In all the mice lacking one Trp53 allele, the second allele was lost in the tumours, but in those lacking two Trp53 alleles, only about half lost the second Brca2 allele. Loss of both copies of Trp53, then, is necessary for tumour formation in mice lacking Brca2. Another intriguing finding is that there is nothing unique about mammary tissue, as the double knockouts developed tumours in skin. Further experiments will uncover how these two important tumour suppressors normally collaborate to protect against cancer. Cath Brooksbank References and links ORIGINAL RESEARCH PAPER Jonkers, J. et al. Synergistic tumour suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer. Nature Genet. 29, 418–425 (2001) FURTHER READING Scully, R. & Livingston, D. In search of the tumour suppressor functions of BRCA1 and BRCA2. Nature 408, 429–432 (2000) Team effort BREAST CANCER
Transcript of Team effort
© 2001 Macmillan Magazines Ltd
Invasive melanoma is notoriously difficult totreat, but the generation of mouse models of thedisease would facilitate the development of vac-cines and expedite screening for new drugs.Sotillo et al., reporting in the 6 November issueof Proceedings of the National Academy ofSciences, describe such a mouse model.
Most human tumours have deregulatedCDK4 activity, usually due to cyclin D1 overex-pression or inactivation of the INK4 cyclin-dependent kinase inhibitors, but a point muta-tion in CDK4 has also been identified in patientswith both spontaneous and familial melanoma.Mutation of this residue (R24C) prevents CDK4from binding to the INK4 proteins.
To investigate the role of R24C inmelanoma susceptibility, the authors generat-ed knock-in mice that express Cdk4 R24C.They develop multiple tumours, but do notshow melanocyte hyperproliferation (see pic-ture). However, on treatment with the skincarcinogens 7,12-dimethylbenz[a]anthracene(DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA), they develop more, andlarger, skin papillomas than wild-type mice,which frequently progress to invasive carcino-mas. Nevi (moles), which are precursors ofmelanoma, are also more common in the
carcinogen-treated Cdk4 R24C mice, and70% of these mice developed melanomas.
Interestingly, although the draining andregional lymph nodes were enlarged and pig-mented, distant metastases were not found. Thiscould be because the mice die before distantmetastasis occurs, or because they lack distantmetastatic potential.
So, is loss of the Ink4 inhibitors at the Cdkn2aand Cdkn2b loci also necessary for the generationof melanoma? Sotillo et al. suggest not, asSouthern blotting of the melanomas from Cdk4R24C mice did not reveal deletion or rearrange-ment of the loci, or promoter methylation in thethe genes encoding Ink4a, Ink4b and Arf. In addi-tion to this, immunostaining of Ink4a showedthat it was present in all tumours analysed.
Cdk4 R24C mice are insensitive to all mem-bers of the Ink4 family, so which confer suscepti-bility to melanoma? The authors investigatedtumour incidence following carcinogen treat-ment in mice deficient in Ink4b and Ink4c,as wellas in Ink4a∆2,3 mice — which produce neitherInk4a nor Arf. Ink4a∆2,3 mice have an increasedsusceptibility to lymphomas, so all die by 10–12weeks — before the incidence of melanoma
could be evaluated — and Ink4b-deficient micedo not exhibit a higher rate of melanomas thanwild-type mice. Ink4c-deficient mice, however,have an increased incidence of melanomas, butnot of papillomas. The incidence of melanoma isstill lower than in carcinogen-treated Cdk4 R24Cmice, indicating that their susceptibility tomelanoma is probably also due to loss of interac-tion with Ink4a, which is supported by resultsfrom human tumours and other melanomamouse models (see further reading).
Now, with several melanoma mouse modelsavailable, it is time to use them to generate newtreatment opportunities for melanoma.
Emma Greenwood
References and linksORIGINAL RESEARCH PAPER Sotillo, R. et al. Invasivemelanoma in Cdk4-targeted mice. Proc. Natl Acad. Sci. USA98, 13312–13317 (2001) FURTHER READING Krimpenfort, P. et al. Loss of p16Ink4a
confers susceptibility to metastatic melanoma in mice. Nature413, 83–86 (2001) | Sharpless, N. E. et al. Loss of p16Ink4a withretention of p19ARF predisposes mice to tumorigenesis. Nature413, 86–91 (2001) | Sotillo, R. et al. Wide spectrum of tumors inknock-in mice carrying a Cdk4 protein insensitive to INK4inhibitors. EMBO J. (in the press)WEB SITEEncyclopedia of Life Sciences: www.els.netmelanoma
Of mice and moles
M O U S E M O D E L S
Melanocytes are barely visible in wild-type cells (left), but hyperproliferate in the Cdk4 R24C mice (right; see green stain). Reproducedwith permission © (2001) Proc. Natl Acad. Sci. USA.
NATURE REVIEWS | CANCER VOLUME 1 | DECEMBER 2001 | 177
Why has it been so difficult to modelhereditary breast cancer in mice?Homozygous loss of either of the knownbreast-cancer susceptibility genes,Brca1 and Brca2, is not compatible with life,whereas mice bearing truncation mutationsdie or develop lymphomas. Jos Jonkers andcolleagues now describe how they haveovercome these problems by developing aconditional knockout of Brca2, using thecre–lox system.
The authors targeted exon 11 of Brca2,which encodes its Rad51-binding BRCrepeats. Binding to Rad51 is necessary forBrca2 to carry out its poorly understoodfunction in DNA repair. To generate a tissue-specific conditional mutant, the authors putthe cre gene under the control of the keratin14 (K14) promoter, which is active instratified epithelia such as those inmammary gland and skin. By crossingK14cre mice with mice carrying the Brca2∆11
allele, mice were bred in which both Brca2alleles were mutated in tissues where the K14promoter is active. Surpsisingly, these micedid not develop tumours any morefrequently than wild-type mice.
So what else is needed to tip their apparentlynormal mammary tissue towards tumourformation? The tumours of women withBRCA2 mutations often show loss of TP53;could this situation be reproduced by crossingBrca2 mutant mice with conditional Trp53knockout mice? The conditional Trp53knockouts generated by the authors weretumour prone but didn’t develop anymammary tumours. In stark contrast, mice
that were homozygous for both mutations inepithelia developed tumours of the mammarygland and skin. Mice with just one functionalcopy of either Brca2 or Trp53 in epitheliumalso developed tumours, but with a longerlatency. In all the mice lacking one Trp53 allele,the second allele was lost in the tumours, butin those lacking two Trp53 alleles, only abouthalf lost the second Brca2 allele.
Loss of both copies of Trp53, then, isnecessary for tumour formation in micelacking Brca2. Another intriguing finding isthat there is nothing unique about mammarytissue, as the double knockouts developedtumours in skin. Further experiments willuncover how these two important tumoursuppressors normally collaborate to protectagainst cancer.
Cath Brooksbank
References and linksORIGINAL RESEARCH PAPER Jonkers, J. et al. Synergistictumour suppressor activity of BRCA2 and p53 in a conditionalmouse model for breast cancer. Nature Genet. 29, 418–425(2001)FURTHER READING Scully, R. & Livingston, D. In search ofthe tumour suppressor functions of BRCA1 and BRCA2.Nature 408, 429–432 (2000)
Team effort
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