Balmain Lab

Allan Balmain Lab UCSF

Allan Balmain, PhD, FRSBarbara Bass Bakar Distinguished Professorship in Cancer Genetics, UCSF.

Contact:    [email protected]
(415) 502-0229 (asst)
(415) 502-4192 (office)
(415) 502-6791 (lab)
(415) 502-3179 (fax)

1450 3rd St., MC 0128; PO Box 589001
San Francisco, CA 94158-9001
deliveries: 1450 3rd Street, HD-320; San Francisco, CA 94158

Allan Balmain, PhD, FRS

A major goal of our research for many years has been to use mouse genetics to understand the relationships between exposure to environmental carcinogens, including chemicals and radiation, and cancer susceptibility. We have generated mouse models that recapitulate the genetic heterogeneity in human populations, with a view to develop approaches to personalized diagnosis and treatment. The focus of our most recent projects is the development of “Systems Genetics” approaches to analysis of normal tissue biology, and the changes in the genome mutational landscapes and gene expression architecture that are induced by carcinogen exposure.

These seek to integrate multidimensional data sets to provide a network view of normal genetic architecture in mouse and human tissues, and the perturbations that take place during development of benign tumors and their progression to metastasis. Studies to date have revealed important roles for genes and pathways linked to stem cell fate decisions, cell cycle control, inflammation, and immune regulation. These integrated systems approaches represent a novel and highly promising route to the identification of the critical interacting components of signaling pathways, and identification of the critical changes in expression networks that are linked to cancer.

They will further provide a platform, using chemically induced tumors that have many point mutations, to design and test novel approaches to combinatorial therapy including radiotherapy and immunotherapy, and the development of biomarkers of responsiveness. Finally, analysis of mouse tumors induced by different carcinogens can provide a deeper understanding of the etiology and mutational origins of human cancers.

This summary will focus on: 1) Genetic modifiers of cancer susceptibility; 2) Development of transgenic and knockout models for multistage tumor development; 3) Genetic alterations and biological changes during tumor invasion and metastasis

1. Identification of Genetic Modifiers of Cancer Susceptibility

Epidemiological evidence has shown that the major causes of cancer include environmental agents such as radiation, or chemicals of natural or synthetic origin The particular genetic background of each individual is also extremely important in determining his or her overall cancer susceptibility. One person in three in an average lifespan develops cancer, but two out of three do not. Certain individuals may therefore be predisposed to develop tumors, by virtue of their particular genetic background, while others appear to be more resistant. These susceptibility genes may control intrinsic cellular growth functions, or may influence the ways in which environmental carcinogens interact with and cause mutations in target cells. The isolation of such genes is one of the major goals of human cancer research worldwide, since they have the potential not only for use in identification of high risk individuals, but for development of novel strategies for prevention or therapy.

One of the major challenges facing the pharmaceutical industry is the identification of appropriate genetic "targets" for drug discovery. The human genome project will in the foreseeable future provide us with a database of many thousands of genes, but the validation of specific genes as candidates for drug screening is an enormous task, particularly in the case of multigenic diseases such as cancer. Our research program is concentrated on the identification of germline susceptibility genes for cancer, which are by definition pre-validated targets since we know, even without having cloned these genes, that they affect the incidence or progression of human cancers.

Such genes will be difficult to find from studies on human families, since they do not segregate as single mendelian traits which can be easily mapped. However, the mouse is ideally suited to studies of polygenic traits such as diabetes, obesity, osteoporosis, and in particular cancer. In addition, since these tumor modifier genes presumably control multiple facets of tumor development such as tumor incidence, growth rate, capacity for angiogenesis or ability to metastasize, their identification could provide us with new opportunities to intervene at specific stages of tumor formation in humans. Our goal is therefore to carry out a comprehensive survey of the numbers and locations of tumor modifier genes using several of the best characterized models of carcinogenesis in the skin, lung, prostate, and lymphoid system.

In previous work on skin carcinogenesis, we have mapped at least ten independent tumor resistance loci in the spretus genome. Some of these affect the early development of tumors, while others influence primarily tumor progression. A specific subset of three of these modifier loci dramatically prolongs the survival of tumor-bearing mice, suggesting that they modify either tumor growth rate or the ability to invade and metastasize. Candidate genes have been identified for several of these loci and further studies on their identification are in progress. Other groups have mapped susceptibility genes for lung, colon or liver cancer to the same genomic loci, thus raising the possibility that some of these modifier genes may have pleiotropic effects on multiple tumor systems. At present, a total of around 80 tumor susceptibility loci have been mapped in the mouse genome, and it is expected that further loci will be uncovered as the mapping proceeds.

At present, it is not feasible to carry out whole genome scans to find polymorphisms in linkage disequilibrium with multiple cancer phenotypes in human populations. Candidate regions of the genome, and candidate genes therein will therefore chosen on the basis of results of mouse modifier studies for testing in human cancer patients and control groups. Candidate genes will be sequenced to find polymorphisms in the human population, and the frequencies of these polymorphic variants in affected sibs vs unaffected control will give an indication their relevance to susceptibility to the different cancer types. Particular attention will be paid to modifiers that appear on the basis of mouse studies to be common among different mouse strain combinations, and/or to be important for several tumor types. Depending on the accuracy with which the modifiers are mapped, it may be possible to carry out these preliminary studies using polymorphisms in the syntenic regions of the human genome, without necessarily identifying the critical gene. We have therefore initiated collaborations with groups that have access to considerable numbers of samples of both constitutional and tumor DNA from human breast, ovarian, and other cancers, together with a strong interest in (and experience of) the analysis of the effects of low penetrance genes on human cancer susceptibility.

In conclusion, it is anticipated that a large number of prevalidated cancer targets will be identified over the next few years from studies of cancer susceptibility in mouse and human populations. Although positional cloning remains a difficult task, the total sequence of the human and mouse genomes that will be available in the near future, together with microarray technology for screening of differentially expressed genes, will obviate the necessity for much of this work. Accurate mapping of tumor modifier genes is possible now, and is a necessary prerequisite for the eventual identification of these important cancer genes.

2. Transgenic and Knock-out Models for Multistage Cancer Development

Figure 1Tumours develop in both mice and humans as a consequence of sequential genetic alterations at critical genomic loci. These changes involve mutations in oncogenes such as ras, and in tumour suppressor loci, including the p53 and p16 genes. Our strategy has been to investigate the genetic alterations which take place during mouse skin tumour development, and subsequently to use both transgenic and knock-out mice to test the functions of candidate genes in vivo. Genes implicated in multistage carcinogenesis include H-ras, p53 and Transforming Growth Factor Beta (TGFbeta).

The steps involved in sequential induction of benign and malignant mouse skin tumors using initiators and promoters of carcinogenesis have been well characterized. Initiation is accomplished by a single treatment with an initiating dose of the carcinogen Dimethylbenzanthracene (DMBA) followed by multiple treatments with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Initiation results in activation of the mouse cellular H-ras gene by mutation at codon 61, and promotion stimulates the outgrowth of benign papillomas. A proportion of these progress to carcinomas over a period of 6-18 months, and in some cases highly invasive spindle tumors are formed that have lost many of the characteristics of their epithelial cell precursors. Many of the genetic and biological changes that occur during this well defined model system have been identified, and are summarized in Figure 1. We have also made a substantial investment in the isolation and characterization, both genetically and biologically, of cell lines representing each of the morphologically distinguishable stages of carcinogenesis. These have proved invaluable for the dissection of the causal events involved in progression to the invasive spindle stage. In some cases (indicated by arrows on Figure 1), cell lines from different stages were derived from the same original target cell and share common genetic alterations.

Our early studies showed that the same proto-oncogene (H-ras) was consistently activated in multiple independent tumors derived from the mouse epidermis (1,2). This study also showed that quantitative changes in ras gene dosage are important during tumor progression, since the mutant gene was present at relatively low copy number in premalignant lesions, but became duplicated or amplified during tumor progression Amplification of the mutant allele and/or loss of the normal ras allele was specifically associated with a particular morphological change seen during the acquisition of metastatic properties (3,4).

Our attempts to demonstrate the causal nature of these genetic changes in tumorigenesis led us to develop transgenic models in which keratin gene promoters were used to target the expression of specific oncogenes or growth factors to subpopulations of cells within the skin. We have used promoters which target expression of transgenes to the differentiating suprabasal cells either constitutively or inducibly, or to subpopulations of basal cells. These studies have given us some important insights into the biological consequences of mutant ras expression in vivo. The use of a keratin 10 gene promoter, which targets predominantly suprabasal cells, led to the surprising result that the main consequence of ras expression in vivo was induction of a terminal differentiation phenotype. The transgenic animals showed evidence of increased keratinisation without hyperplasia, but subsequently developed focal areas of hyperplasia which progressed to form benign papillomas (5). These lesions however were terminally benign, and failed to give rise to malignancies in the lifetime of the animals. Expression of the same mutant ras transgene in a sub-population of hair follicle cells, which contain the putative stem cells, led to the development of tumors which progressed to malignancy, indicating the importance of the target cell as a major determinant of malignant potential (6).

We have also extensively studied the functions of the p53 tumor suppressor gene in responses to DNA damage and in carcinogenesis in vivo (7,8). This gene is mutated in about 30% of malignant skin tumors, and experiments with knock-out mice have demonstrated that animals deficient in p53 function show increased progression of benign tumors to malignancy. However, the same animals had a decreased incidence of benign tumors, contrary to expectations (7). Hence, although these results illustrated the causal nature of p53 loss in tumor progression, they also highlighted another surprising function for p53 in allowing the outgrowth of early benign tumor cells. Progression to the undifferentiated spindle stage of carcinogenesis is associated with further changes involving amplification of mutant ras together with deletions at the Ink4a locus, suggesting that concomitant alterations of p53 and Rb pathways are involved in progression to invasion and metastasis (9).

3. Genetic Alterations and Biological Changes During Tumor Invasion and Metastasis

Our studies on the biology of mouse skin tumor invasion have demonstrated the existence of at least two pathways leading to the acquisition of invasive properties: a) genetic alterations involving the p16/Ink4a locus and b) overexpression and activation of TGFbeta signaling.

a) A Genetic Basis for Tumor Invasion and Metastasis

The isolation of cell lines representing discrete stages of tumor progression from the same primary tumor has enabled us to dissect some of the critical events required for invasion and metastasis (1). Squamous carcinomas of the skin are relatively poorly invasive and metastasize infrequently. They can however progress to spindle cell carcinomas, a high risk tumor type with dramatically increased invasiveness, and greater metastatic potential (1,2,3). This squamous to spindle cell conversion is the result of EMT of the squamous tumor cells . Some spindle cell lines have acquired these properties through genetic alterations, the most frequent of which involve deletions at the p16INK4 locus together with amplification of the mutant H-ras gene (3,4,5). Others have reached the same end point through alternative genetic changes involving overexpression of the p16 gene, suggesting the presence of other mutation in the Rb pathway (but not in Rb itself, which seems to be intact in these cells). Both of these classes of genetic changes are recessive, as shown by fusion with normal cells, and indicate loss of differentiation-controlling genes during tumor progression (4). Our results suggest a novel role for p16/Ink4a in control of tumor invasion and cell adhesion rather than (or in addition to) its known functions in cell cycle control.

b) The Role of Transforming Growth Factor beta in Tumor Suppression and Tumor Progression

Mice expressing TGFbeta develop fewer papillomas than non-transgenic littermates, but have elevated rates of tumour progression. This indicates that TGFbeta can act either positively or negatively at different stages of carcinogenesis. We have proposed that TGFbeta can act as a negative regulator of cell cycle progression during the early phase of tumorigenesis, but positively induces invasion and angiogenesis during progression to malignancy. This possibility is supported by studies involving transfection of dominant negative TGFbeta type II receptor constructs into invasive carcinoma cells. This results in suppression of the invasive phenotype and the restoration of expression of some adhesion proteins such as E-cadherin. We have shown that TGFbeta signalling can also be upregulated by genetic alterations in highly malignant spindle carcinoma cells. Many of the features characteristic of the invasive, metastatic phenotype of these cells can be reversed by the introduction of dominant negative interfering mutants that disrupt Smad signalling. Thresholds of Smad activity are important at distinct stages of tumor progression, cooperating with activated ras to induce invasion or dissemination to distant sites.

  1. Rose Li Y, Halliwill KD, Adams CJ, Iyer V, Riva L, Mamunur R, Jen KY, Del Rosario R, Fredlund E, Hirst G, Alexandrov LB, Adams D, Balmain A. Mutational signatures in tumours induced by high and low energy radiation in Trp53 deficient mice. Nat Commun. 2020 Jan 20;11(1):394. View on PubMed
  2. Reeves MQ, Kandyba E, Harris S, Del Rosario R, Balmain A. Multicolour lineage tracing reveals clonal dynamics of squamous carcinoma evolution from initiation to metastasis. Nat Cell Biol. 2018 Jun;20(6):699-709. View on PubMed
  3. Huang PY, Kandyba E, Jabouille A, Sjolund J, Kumar A, Halliwill K, McCreery M, DelRosario R, Kang HC, Wong CE, Seibler J, Beuger V, Pellegrino M, Sciambi A, Eastburn DJ, Balmain A. Lgr6 is a stem cell marker in mouse skin squamous cell carcinoma. Nat Genet. 2017 Nov;49(11):1624-1632.  View on PubMed
  4. Halliwill KD, Quigley DA, Kang HC, Del Rosario R, Ginzinger D, Balmain A. Panx3 links body mass index and tumorigenesis in a genetically heterogeneous mouse model of carcinogen-induced cancer. Genome Med. 2016; 8(1):83. View on PubMed
  5. Quigley DA, Kandyba E, Huang P, Halliwill KD, Sjölund J, Pelorosso F, Wong CE, Hirst GL, Wu D, Delrosario R, Kumar A, Balmain A. Gene Expression Architecture of Mouse Dorsal and Tail Skin Reveals Functional Differences in Inflammation and Cancer. Cell Rep. 2016 Jul 14. View on PubMed
  6. Wang MT, Holderfield M, Galeas J, Delrosario R, To MD, Balmain A, McCormick F. K-Ras Promotes Tumorigenicity through Suppression of Non-canonical Wnt Signaling. Cell. 2015 Nov 19; 163(5):1237-51. View on PubMed
  7. McCreery MQ, Halliwill KD, Chin D, Delrosario R, Hirst G, Vuong P, Jen KY, Hewinson J, Adams DJ, Balmain A. Evolution of metastasis revealed by mutational landscapes of chemically induced skin cancers. Nat Med. 2015 Dec; 21(12):1514-20. View on PubMed
  8. Adams CJ, Yu JS, Mao JH, Jen KY, Costes SV, Wade M, Shoemake J, Aina OH, Del Rosario R, Menchavez PT, Cardiff RD, Wahl GM, Balmain A. The Trp53 delta proline (Trp53?P) mouse exhibits increased genome instability and susceptibility to radiation-induced, but not spontaneous, tumor development. Mol Carcinog. 2016 Sep; 55(9):1387-96. View on PubMed
  9. Hiraoka K, Inoue T, Taylor RD, Watanabe T, Koshikawa N, Yoda H, Shinohara K, Takatori A, Sugimoto H, Maru Y, Denda T, Fujiwara K, Balmain A, Ozaki T, Bando T, Sugiyama H, Nagase H. Inhibition of KRAS codon 12 mutants using a novel DNA-alkylating pyrrole-imidazole polyamide conjugate. Nat Commun. 2015; 6:6706. View on PubMed
  10. Letteboer TG, Benzinou M, Merrick CB, Quigley DA, Zhau K, Kim IJ, To MD, Jablons DM, van Amstel JK, Westermann CJ, Giraud S, Dupuis-Girod S, Lesca G, Berg JH, Balmain A, Akhurst RJ. Genetic variation in the functional ENG allele inherited from the non-affected parent associates with presence of pulmonary arteriovenous malformation in hereditary hemorrhagic telangiectasia 1 (HHT1) and may influence expression of PTPN14. Front Genet. 2015; 6:67. View on PubMed
  11. Chen J, Hackett CS, Zhang S, Song YK, Bell RJ, Molinaro AM, Quigley DA, Balmain A, Song JS, Costello JF, Gustafson WC, Van Dyke T, Kwok PY, Khan J, Weiss WA. The genetics of splicing in neuroblastoma. Cancer Discov. 2015 Apr; 5(4):380-95. View on PubMed
  12. Westcott PM, Halliwill KD, To MD, Rashid M, Rust AG, Keane TM, Delrosario R, Jen KY, Gurley KE, Kemp CJ, Fredlund E, Quigley DA, Adams DJ, Balmain A. The mutational landscapes of genetic and chemical models of Kras-driven lung cancer. Nature. 2015 Jan 22; 517(7535):489-92. View on PubMed
  13. Quigley D, Silwal-Pandit L, Dannenfelser R, Langerød A, Vollan HK, Vaske C, Siegel JU, Troyanskaya O, Chin SF, Caldas C, Balmain A, Børresen-Dale AL, Kristensen V. Lymphocyte Invasion in IC10/Basal-Like Breast Tumors Is Associated with Wild-Type TP53. Mol Cancer Res. 2015 Mar; 13(3):493-501. View on PubMed
  14. Hackett CS, Quigley DA, Wong RA, Chen J, Cheng C, Song YK, Wei JS, Pawlikowska L, Bao Y, Goldenberg DD, Nguyen K, Gustafson WC, Rallapalli SK, Cho YJ, Cook JM, Kozlov S, Mao JH, Van Dyke T, Kwok PY, Khan J, Balmain A, Fan Q, Weiss WA. Expression quantitative trait loci and receptor pharmacology implicate Arg1 and the GABA-A receptor as therapeutic targets in neuroblastoma. Cell Rep. 2014 Nov 6; 9(3):1034-46. View on PubMed
  15. Balmain A, Yuspa SH. Milestones in skin carcinogenesis: the biology of multistage carcinogenesis. J Invest Dermatol. 2014; 134(e1):E2-7. View on PubMed
  16. Huang PY, Balmain A. Modeling cutaneous squamous carcinoma development in the mouse. Cold Spring Harb Perspect Med. 2014 Sep; 4(9):a013623. View on PubMed
  17. Hiatt RA, Porco TC, Liu F, Balke K, Balmain A, Barlow J, Braithwaite D, Diez-Roux AV, Kushi LH, Moasser MM, Werb Z, Windham GC, Rehkopf DH. A multilevel model of postmenopausal breast cancer incidence. Cancer Epidemiol Biomarkers Prev. 2014 Oct; 23(10):2078-92. View on PubMed
  18. Sjölund J, Pelorosso FG, Quigley DA, DelRosario R, Balmain A. Identification of Hipk2 as an essential regulator of white fat development. Proc Natl Acad Sci U S A. 2014 May 20; 111(20):7373-8. View on PubMed
  19. Song IY, Balmain A. Cellular reprogramming in skin cancer. Semin Cancer Biol. 2015 Jun; 32:32-9. View on PubMed
  20. Quigley DA, Fiorito E, Nord S, Van Loo P, Alnæs GG, Fleischer T, Tost J, Moen Vollan HK, Tramm T, Overgaard J, Bukholm IR, Hurtado A, Balmain A, Børresen-Dale AL, Kristensen V. The 5p12 breast cancer susceptibility locus affects MRPS30 expression in estrogen-receptor positive tumors. Mol Oncol. 2014 Mar; 8(2):273-84. View on PubMed
  21. Lessard JC, Piña-Paz S, Rotty JD, Hickerson RP, Kaspar RL, Balmain A, Coulombe PA. Keratin 16 regulates innate immunity in response to epidermal barrier breach. Proc Natl Acad Sci U S A. 2013 Nov 26; 110(48):19537-42. View on PubMed
  22. Saunders SP, Goh CS, Brown SJ, Palmer CN, Porter RM, Cole C, Campbell LE, Gierlinski M, Barton GJ, Schneider G, Balmain A, Prescott AR, Weidinger S, Baurecht H, Kabesch M, Gieger C, Lee YA, Tavendale R, Mukhopadhyay S, Turner SW, Madhok VB, Sullivan FM, Relton C, Burn J, Meggitt S, Smith CH, Allen MA, Barker JN, Reynolds NJ, Cordell HJ, Irvine AD, McLean WH, Sandilands A, Fallon PG. Tmem79/Matt is the matted mouse gene and is a predisposing gene for atopic dermatitis in human subjects. J Allergy Clin Immunol. 2013 Nov; 132(5):1121-9. View on PubMed
  23. Wong CE, Yu JS, Quigley DA, To MD, Jen KY, Huang PY, Del Rosario R, Balmain A. Inflammation and Hras signaling control epithelial-mesenchymal transition during skin tumor progression. Genes Dev. 2013 Mar 15; 27(6):670-82. View on PubMed

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