[Cell Cycle 3:10, e45-e46, EPUB Ahead of Print: http://www.landesbioscience.com/journals/cc/abstract.php?id=1169; October 2004]; ©2004 Landes Bioscience Metastasizing and Non-Metastasizing Tumors Likely Evolve from DNA Phenotypes via Independent Pathways Perspective This manuscript has been published online, prior to printing for Cell Cycle, Volume 3, Issue 10. Definitive page numbers have not been assigned. The current citation is: Cell Cycle 2004; 3(10): http://www.landesbioscience.com/journals/cc/abstract.php?id=1169 Once the issue is complete and page numbers have been assigned, the citation will change accordingly. KEY WORDS metastasis, tumorigenesis, DNA structure, prostate cancer Fourier transform-infrared 3-methylcholanthrene te ibu Bio ACKNOWLEDGEMENTS sci FT-IR MCA The classic concept that a primary tumor gives rise to metastasis via clonal selection of a few rare cells1 persists to this day and has profoundly influenced the direction and interpretation of cancer research.2 However, this concept has been challenged on the basis of gene expression profiles of human breast tumors.3-6 These studies suggest that the proclivity for metastasis might develop in progenitor cells early in multistep tumorigenesis, as put forth by Bernards and Weinberg.7 We have now discovered a metastatic cancer DNA phenotype in histologically normal tissues from prostates with tumors having confirmed distant metastases.8 These matched normal and tumor tissues from the same individual were found to have virtually identical DNA base and backbone structures. This surprising discovery was made using statistical models of Fourier transform-infrared (FT-IR) spectra of DNA having the proven ability to identify subtle modifications in DNA structure.8-11 The presence of a metastatic cancer DNA phenotype in normal tissues of prostates with metastasizing tumors implies that this phenotype likely arises early in tumor evolution and signals a significant risk for metastasis. Using the FT-IR models, evidence for the early development of an analogous phenotype for non-metastasizing tumors (a primary cancer DNA phenotype) was obtained with BALB/c mice injected in the hind leg with the carcinogen 3-methylcholanthrene (MCA).10 Strikingly, this primary phenotype appeared in histologically normal leg tissues about eight weeks before the mean occurrence of palpable sarcomas and was indistinguishable from the DNA structure of these tumors. We also found that the prodrug cyclophosphamide delayed tumor formation by about 30%. Concomitantly, the MCA-injected mice receiving the cyclophosphamide showed almost complete suppression of the primary phenotype.10 This finding firmly established that a relationship exists between the appearance of this phenotype and the development of a tumor. We reported in 1995 that the DNA structure of histologically normal tissue adjacent to breast tumors (invasive ductal carcinomas) was spectrally indistinguishable from that of the tumor.12 This evidence appears to be the first demonstration that a cancer DNA phenotype occurs in normal tissues surrounding a tumor, and is consistent with data showing that benign tissue in a cancerous breast has an elevated risk for developing a tumor.13 In a recent study,9 we showed that 42% of cancer-free men (ages 55–80) had a primary cancer DNA phenotype in histologically normal prostates, thus implying a high risk for prostate cancer. The prostates of healthy younger men (ages 16–36) did not exhibit this phenotype. The discovery of the primary8-10 and metastatic8 cancer DNA phenotypes clearly calls into question the widely-held concept that primary tumors somehow acquire mutations that yield a few rare cells with the ability to metastasize. If metastasis were indeed to arise in this way from a primary tumor, then the DNA structure of the metastasizing tumor (as evinced by FT-IR spectroscopy) would be essentially identical to that of the primary tumor. However, the DNA base and backbone structures of the non-metastasizing (primary) and metastasizing tumors are, in fact, substantially different.8 Most significantly, the DNA structures of the corresponding histologically normal tissues surrounding these tumors exhibited virtually the same differences.8 These data challenge the classic theory en ABBREVIATIONS No tD ist r Received 08/12/04; Accepted 08/13/04 We have recently discovered, using statistical models of Fourier transform-infrared spectra, two distinct cancer DNA phenotypes in histologically normal tissues surrounding non-metastasizing primary tumors and tumors with evidence of distant metastases. Structural comparisons of the DNA from these tumor types imply that each evolves via a separate pathway from DNA phenotypes originating in progenitor cells. These findings challenge the widely-held concept of metastasis. Do Correspondence to: Donald C. Malins; Biochemical Oncology Program; Pacific Northwest Research Institute; 720 Broadway; Seattle, Washington 98122 USA; Tel.: 206.726.1240; Fax: 206.726.1235; Email: [email protected] ABSTRACT ce. Donald C. Malins © 20 04 La nd es I am thankful to Katie M. Anderson, Edward A. Barker, Naomi K. Gilman, Virginia M. Green, Karl Erik Hellström, Thomas R. Sutter and Edward L. Weber for their helpful comments. e45 Cell Cycle 2004; Vol. 3 Issue 10 INDEPENDENT PATHWAYS FOR DEVELOPMENT OF METASTASIZING AND NON-METASTASIZING TUMORS 20 04 La nd es Bio sci en ce. Do No tD ist r ibu te of metastasis and support the hypothesis that primary and metastasizing tumors evolve along separate pathways established early in tumorigenesis (Fig. 1). How then might the primary and metastatic cancer DNA phenotypes develop? We found that the DNA base and backbone structures from the prostates of cancer-free older men were markedly different from those of younger men.9 In addition, we have shown that free radical-induced base lesion concentrations (e.g., mutagenic 8-hydroxyadenine) were considerably higher in the prostate Figure 1. Two-pathway model for non-metastasizing and metastasizing tumor development. (A) Changes in DNA of older men.9 The introduction DNA as a function of genetic and epigenetic modifications produce distinctly different structures (1) that result of just a single oxygen (a known radical- in the emergence of two independent tumor pathways. (B) Non-metastasizing tumor pathway. Progenitor cells induced lesion) in an adenine or guanine expressing the primary cancer DNA phenotype (2) develop into a non-metastasizing primary tumor. (C) of a 25-base DNA strand has been Metastasizing tumor pathway. Progenitor cells expressing the metastatic cancer DNA phenotype (3) develop reported to significantly disrupt vertical into a metastasizing tumor. (See text for details) base stacking interactions, as well as to induce perceptible conformational changes in the backbone.14 10. Malins DC, Anderson KM, Gilman NK, Green VM, Barker EA, Hellström KE. Development of a DNA cancer phenotype prior to tumor formation. Proc Natl Acad Sci Other factors (e.g., hypermethylation)15 can also contribute to modUSA 2004; 10721-5. 16,17 ifications of DNA structure. Collectively, these and possibly 11. Malins DC, Polissar NL, Su Y, Gardner HS, Gunselman SJ. A new structural analysis of other structural perturbations arising largely as a function of increasDNA using statistical models of infrared spectra. Nat Med 1997; 3:927-30. 12. Malins DC, Polissar NL, Nishikida K, Holmes EH, Gardner HS, Gunselman SJ. The etiing age would be expected to create substantial genetic instability. ology and prediction of breast cancer. Fourier transform-infrared spectroscopy reveals proThis instability may well provide a fertile environment for generatgressive alterations in breast DNA leading to a cancer-like phenotype in a high proportion ing the primary and metastatic cancer DNA phenotypes. The genetof normal women. Cancer 1995; 75:503-17. 13. Henderson IC. Risk factors for breast cancer development. Cancer 1993; 71:2127-40. ic and other structural features unique to each of these DNA phe14. Malins DC, Polissar NL, Ostrander GK, Vinson MA. Single 8-oxo-guanine and 8-oxo-adenotypes would then determine the biological properties of the tumor nine lesions induce marked changes in the backbone structure of a 25-base DNA strand. formed. These properties may be regulated via gene de-repression Proc Natl Acad Sci USA 2000; 97:12442-5. and include, for example, aggressiveness in primary tumors, or dis15. Yegnasubramanian S, Kowalski J, Gonzalgo ML, Zahurak M, Piantadosi S, Walsh PC, et al. Hypermethylation of CpG islands in primary and metastatic human prostate cancer. tant site preference18 and cellular heterogeneity19 in metastasizing Cancer Res 2004; 64:1975-86. tumors. 16. Forde G, Gorb L, Shiskin O, Flood A, Hubbard C, Hill G, et al. Molecular structure and In conclusion, we see no inconsistency between our proposed properties of protonated and methylated derivatives of cytosine. J Biomol Struct Dyn 2003; 20:819-28. two-pathway model (Fig. 1) and recent findings based on gene 17. Banyay M, Gräslund A. Structural effects of cytosine methylation on DNA sugar pucker expression profiles,3-5,20 or Slaughter’s 1953 concept of field cancerstudied by FTIR. J Mol Biol 2002; 324:667-76. ization21 for which supportive genetic evidence has been recently 18. Kang Y, Siegel PM, Shu W, Drobnjak M, Kakonen SM, Cordón-Cardo C, et al. A multigenic program mediating breast cancer metastasis to bone. Cancer Cell 2003; 3:537-49. provided.22,23 It appears that sufficient data now exist to justify that 19. Fidler IJ. The pathogenesis of cancer metastasis: The ‘seed and soil’ hypothesis revisited. more attention be paid to mechanisms leading to the formation and Nat Rev Cancer 2003; 3:453-8. development of the DNA phenotypes described. Overall, we believe 20. Ramaswamy S, Ross KN, Lander ES, Golub TR. A molecular signature of metastasis in prithat the novel perspective presented here for the evolution of metasmary solid tumors. Nat Genet 2003; 33:49-54. 21. Slaughter DP, Southwick HW, Smejkal W. Field cancerization in oral stratified squamous tasizing and non-metastasizing tumors from DNA phenotypes in epithelium; clinical implications of multicentric origin. Cancer 1953; 6:963-8. histologically normal cells has potentially profound implications for 22. Braakhuis BJ, Tabor MP, Kummer JA, Leemans CR, Brakenhoff RH. 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