"The more simplistic the more complex." A quote John Coltrane was noted as saying when asked about the variety of his "riffs" on the jazz set. Such can be said about the influence of hormones on mammary tumor development.
The mouse mammary tumor biologists were instrumental from the very beginning of experimental cancer research in pursuing the hormonal influences on the mammary gland and its tumors. However, the significance of hormones in mouse mammary tumorigenesis has been overshadowed by the detection of the hormone response elements (HREs) in the MMTV-LTR (Yamamoto et al., 1983). Since the HRE involve the hormonal regulation both of virus production and of activated oncogenes, most experiments with "spontaneous" mammary tumors are difficult to interpret because it is impossible to separate primary and secondary effects of hormones. Since the hormones influence both virus and abnormal growth, many of the historical studies of endocrine regulation of mouse mammary gland development and tumorigenesis are open to question. How does one distinguish between effects that are primarily on the host cells and effects that are mediated through the presence of the virus?
The initial focus was dictated by clinical observations. Remarkably, the first observation was made in 1895 and published in 1896 by Beatson who detected the regression and then recurrence of a breast lump in a 33-year-old Glasgow woman following removal of her ovaries. The temporary effect led Beatson to opine that "We must look in the female to the ovaries as the seat of the exciting cause of carcinoma, certainly of the mamma, " (Beatson, 1896). Some 40 years after the Beatson report, clinicians were administering either androgens or estrogens and/or removing the adrenals or pituitary in women with breast cancer (Haddow et al., 1944; Huggins and Bergenstal, 1952; Loeser, 1938; Ulrich, 1939).
Loeb and Lathrop performed the same experiment as Beatson 20 years later in mice (Lathrop and Loeb, 1916). Surgical ablation became the modus operandi for studying the hormonal effects of various endocrine organs. Loeb continued performing such "physiological experiments" without the advantage of isolated chemical hormones and without inbred mouse strains (Loeb and Kirtz, 1939; Shimkin, 1945). Like the human, the mouse required ovaries for the development of breast cancer but in contrast to human, mouse mammary tumorigenesis was driven by pregnancy (Lacassagne, 1936).
The early insights on the spontaneous mammary cancer in mice also contributed significantly to understanding estrogen and its secretion from the ovary. Once removed, the incidence of tumors was reduced, while the grafting of syngeneic ovarian tissue or, when synthetic hormones became available, the injection of estrogen into male mice which normally do not form mammary tumors induced tumorigenesis (Lacassagne, 1932; Lathrop and Loeb, 1916; Murray, 1928).
With the development of chemically pure estrogens, numerous investigators led by Lacassagne, starting in 1932, verified that the ovarian function could be replaced by estrogens (Lacassagne, 1932). However, administration of estrogens seemed to accelerate tumorigenesis in susceptible strains but, in itself, was not carcinogenic. When the milk factor was elucidated, tumors induced by estrogen were found to be dependent on the presence of the milk agent. In modern terms, the MMTV-LTR promoter element has a number of HREs that drive MMTV expression and promote or enhance/activate upstream or downstream oncogenes.
The MMTV-HRE also created the endocrine paradox between human and mice. Early pregnancy and lactation protects the human from breast cancer but increases the risk of breast cancer in susceptible strains of mice (Nandi et al., 1995). Further, a higher percentage of breast cancers in humans are estrogen receptor alpha (ER-alpha) positive and estrogen responsive while the majority of "spontaneous" MMTV-induced mouse tumors are ER-alpha negative and hormone independent (Cardiff, 2001). The use of the MMTV-LTR as an organ-specific promoter to drive oncogenic transgenes has continued to produce hormone-independent mammary tumors (Cardiff etal., 2000a). The utilization of whey acidic protein and various "knockins" behind native promoters may encourage the development of ER-alpha positive, hormone-dependent tumors in GEM (Lin et al., 2004).
During the turbulent years leading up to and following World War II, the major question was whether only ovarian hormones such as progesterone and estrogen were the inducers and controllers of normal breast growth or were there other elements? Lyons, in 1958, clearly defined the minimal hormonal growth requirements for normal mammary development in mice as being estrogen, adrenocortical steroids, growth hormone, and cortisol (Lyons, 1958; Lyons et al., 1958). Muhlbock in the Netherlands was particularly interested in the role of the pituitary in mammary gland physiology (Dux and Muhlbock, 1969a,b; Muhlbock, 1956).
Foulds found the pregnancy-dependent plaque in his hybrid BR mice in 1947, which he used to describe the basic rules of neoplastic progression (Foulds, 1949, 1954, 1958). His observations of the hormone-dependent tumors were confirmed in the GR strain by Muhlbock in the Netherlands and in RIII mice by Squartini in Italy (Foulds, 1975). Again, the initiating oncogene appears to be activated with a germ line insertion of the MMTV (Morris et al., 1990).
The preneoplastic HAN is also regulated by hormones (Nandi et al., 1960a). Early experiments related the HAN to pregnancy and when used for infectivity assays, either pituitary implants or hormone injections induced the HAN. However, the HAN does not regress after withdrawal of hormones but, in contrast to the normal mammary gland, persists in the presence of the constitutive levels of native hormones. Bern and Nandi provided a detailed analysis of the endocrinology of the HAN (Bern and Nandi, 1961; Nandi et al., 1960a,b). The more subtle effects of MMTV on normal mammary gland required morphometric analysis to demonstrate that the presence of the virus is associated with alveologenesis (Squartini, 1962; Squartini and Bistocchi, 1977; Squartini et al, 1963, 1981, 1983).
On researching this chapter, we have discovered a researcher, the aforementioned Leo Loeb, who devoted his life to mammary biology and thus is probably the first true mammary biologist. Dr. Loeb spent some 60 years studying the mammary glands of mice. Loeb began his career in 1895 with Ribbert in Zurich and participated in the early transplantation experiments but immigrated to Chicago in 1896. He had far reaching interests that included some of the first attempts at tissue culture of tumor cells on blood clots (Loeb, 1958; Witkowski, 1983). In 1903, Loeb manipulated adult and embryonic mice to understand the attributes of tumor growth and sustainability. Along with Lathrop, he discovered ovarian influence on the development of mouse mammary tumors (Lathrop and Loeb, 1916). Twenty-eight years later, he furthered his observations by demonstrating that pituitary transplantation drives mammary tumor incidence and that variability in MMTV-induced tumor growth potentials varies in different mouse strains. Loeb also made major contributions to tissue culture and genetics of individuality (Loeb, 1953). Although rarely mentioned or referenced in today's literature, this field is deeply indebted to Leon Loeb's triumphant years of scientific observations.
Significant credit also must be given to Jull who, in early 1954, observed the effects of ovarian secretion on carcinogen-induced mouse mammary tumors (Jull, 1954), and later along with Bonser and Dossett discovered that forced breeding and pregnancies may also enhance the growth of spontaneous mammary tumors (Bonser et al., 1961). These observations have been very insightful for those studying mammary tumors in today's multiparous transgenic mice.
In the 1960s, a period of transition occurred from the mouse to the rat as an experimental model for endocrinology and chemical carcinogens (Shimkin, 1979b). Many of the observations on chemical-induced, hormone-dependent or hormone-independent mammary tumors occurred in the rat systems during this period. For the sake of this chapter, we will only mention that in the rat model, a single intragastric or intravenous injection of DMBA induces estrogen-dependent adenocarcinomas 60-100 days postinjection. This tumor model developed by Huggins in 1959 was aptly named after him (Huggins et al., 1959).
Russo used the rat model to develop the "window of susceptibility" concept which postulates that the mammary gland is highly sensitive to carcinogens between puberty and the first pregnancy (Russo and Russo, 1995; Russo et al., 1982). This model has been confirmed in mouse strains free of MMTV expression (Dandekar et al., 1986; Gardner et al., 1985). Almost parenthetically, specific mutations of the Ras gene have been observed in both the rat and the mouse models (Dandekar et al., 1986). In modern terms, the DMBA-induced tumors have activation of the Wnt-1 pathway (Currier et al., 2005).
IX. THE NATIONAL CANCER INSTITUTE AND THE BIRTH OF MOLECULAR BIOLOGY: 1970-1980
The modern mammary tumor biologist has been well served by the NCI and other funding agencies organized to support cancer research. We must remember the extraordinary dedication of the pioneers in this field. Dr. and Mrs. Leonel Strong spent their honeymoon caretaking in the church rectory with Lionel's priceless mice stored under the church pews (Morse, 1978; Strong, 1978). Descriptions of the early days at the Jackson Laboratory recount the community vegetable gardens used to feed staff and mice (Crow, 2002). The Dutch recall that their mouse colonies were taken home and kept during most of the German occupation because the Netherlands Cancer Institute (NKI) did not have heat, electricity, or food.7 Otto Muhlbock, a German national, had to serve as a medical officer during the occupation caring for the German nurses while sheltering and adopting a Jewish boy.7 CRGL of University of California, Berkeley was initially housed in one of the temporary buildings in Strawberry Canyon just east of the football stadium. At times, people who came looking for him were "knocked for a loop" when they found "Dr. De Ome" identifying himself as the person mopping the animal room floor. But all of this was soon to change.8
In 1937, the US NCI was founded (Shimkin, 1977a). C. C. Little had become the managing director of the American Society for the Control of Cancer (founded in 1913) in 1929, which became the American Cancer Society in 1945, a calling he embraced with his usual enthusiasm and energy. In 1955, Little, a dedicated pipe-smoker, also became the first director of the Tobacco Research Council because he wanted to study the influence of genetics on susceptibility to smoking in humans (Crow, 2002).
In 1938, the Jackson Laboratory received the first extramural grant from the newly formed US NCI. These events reflected the increasing cancer awareness of the American public and marked the beginning of large-scale federal and private support for cancer research. The newly formed NCI took the lead and published the proceedings of a symposium on mouse mammary tumors in 1945 (Moulton, 1945). After the war, the funding, tools, and concepts of scientific research underwent major changes that led to the current era.
7 Personal communications from R. van Nie and P. Hageman.
The post-World War II infusion of cancer research funds encouraged the development and rise of mammary gland biology-focused centers located on the campuses of University of California, Berkeley, NCI, NIH, M.D. Anderson, Texas, NKI, and the University of Minnesota. Each of these centers assessed and contributed to transplantation assays, virus detection techniques, and hormonal influences on mammary gland development.
In the United States, the west coast contingent was led by Kenneth B. De Ome who had moved from Purdue to the University of California, Berkeley and persuaded Chancellor Robert Sproul to support a CRGL, incorporating a west coast production facility for inbred mice (Cardiff et al., 2002). The CRGL faculty included endocrinology (Howard Bern), EM (Dorothy Pitelka), and De Ome who is best known for his work with the HAN. Later, other scientists such as Satyabrata "Ranu" Nandi (endocrinology) and immunologists David Weiss and Phyllis Blair joined the CRGL faculty. On the east coast, Dan Moore at the Rockefeller Institute in New York was working on the biophysical isolation and characterization of the virus. In Texas, Leon Dmochowski, the flamboyant Polish emigre, held sway with his emphasis on EM and viral structure. In the Netherlands, Otto Muhlbock was the director of the NKI. Muhlbock, a gynecologist, was particularly interested in the pituitary. The NKI developed many investigators whose major contributions are recorded in the monograph edited by Hilgers and Sluyser. Of course, the NCI had Heston, Andervont, and Bryant leading the effort in Bethesda. Francisco Squartini led the Italian mouse mammary biologists. Oddly, with Bittner's return to the Midwest in 1942, the Jackson Laboratory chose to concentrate on mouse genetics and MHC, thus abdicating its leadership in the field of mammary tumorigenesis.
In retrospect, the "schools" that developed in the 1950s and became prominent in the 1960s were led by the last of the natural historians of disease. De Ome and Muhlbock began holding informal "MTV Meetings" starting in 1955. By 1964 the group became international and met in California (Fig. 1). The group has maintained its international orientation and has become the International Association for Breast Cancer Research which is holding its twenty-fifth congress this year (2006) in Montreal, Canada. The group in Fig. 1 is counted as the "Third Congress." These leaders viewed mouse mammary tumor biology as a subject worthy of study in and of itself. They were disease oriented and understandably unprepared for the impact of molecular biology on scientific thinking and progress.
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