Hormone Dependence and Independence of Mammary Tumors in Mice

INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 103

Hormone Dependence and Independence of Mammary Tumors in Mice AKIOMATSUZAWA Laboratory Animal Research Center, The Institute of Medical Science, The University of Tohyo, Tokyo 108, Japan I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Development of Hormone-Dependent Mammary Tumors. . . . . . A. Mouse Mammary Tumor Virus (MMTV) B. Carcinogens ..... .. . . ..... . . . . . . . . ...... . . . _ . . . . . . . . . C. Hormones .. . .. .. . ... . .. .. .. . . .. . ... . .. . .. . . . .. . . . . . . 111. Growth of Hormone-Dependent Mammary Tumors . . . . . . . . . . . A. Hormonal Requirements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Behavior in Virgin Mice.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Response to Endocrine Therapies .. IV. Hormone Receptors in Dependent Ma .. A. Estrogen Receptors (ER) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Progesterone Receptors (PgR). . . C. Prolactin Receptors (PrlR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Other Hormone Receptors.. . . . . V. Progression from Dependence to Independence . . . . . . . . . .. . . . A. Mechanism of Progression.. . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Alteration of Hormone Receptors with Progression. . . . . . . C. Alteration of Responsiveness to Hormones and Therapeutics with Progression .............. D. Markers for Progression.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Conclusions. .... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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303 307 307 309 310 310 310 314 316 318 318 320 322 323 323 323 325 328 331 334 336

I. Introduction The study of mammary tumors in mice has a long history. The first case came from a wild mouse described by Cripp in 1858 (as cited by Dunn, 1945). One of the reasons for this may be easy palpation and visualization of the tumor because of its localization in a circumscribed area under the skin. The development, differentiation, and function of the mammary gland are under the control of complicated interactions among many hormones from the pituitary, ovary, adrenal, and other organs. As such, the hormones are involved in mammary tumorigenesis in mice (for reviews 303 Copyright D 19x6 by Academic Press, h i . All rights of reproduction in any form reserved.

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see Nandi and McGrath, 1973; Matsuzawa, 1982). The existence of genetic influences on the tumorigenesis has been established by the studies using inbred strains of mice developed by Little, Strong, and others (Statts, 1966). In 1936, Bittner made the epoch-making observation that the extrachromosomal factor transmitted through mother’s milk is concerned with development of mammary tumors in mice. The factor is now called mouse mammary tumor virus (MMTV), which is an RNA-containing tumor virus and has a life cycle similar to that of other characterized retroviruses (Hynes et al., 1984). It has also been shown that chemical carcinogens can induce mammary tumors in mice (Bonser et ul., 1961). It is, however, natural that many studies have emphasized MMTV, because most mouse inbred strains have the virus in their milk and all have the proviral information for it in their DNA (Nandi and McGrath, 1973; Zotter et al., 1981). These studies have confirmed that hormones can accelerate induction of mammary tumors by MMTV, although the resulting tumors are freed of their control. This may be partly explained by the use of inbred strains of mice originating in the United States or mainly of C3H mice. In fact, most of the hormone-responsive mammary tumors reported have come from European mice, as seen in Table I. In addition, the table clearly reveals that pregnancy responsiveness is highly usual in the generality of mouse mammary tumors. Sporadic examples of pregnancy-responsive mammary tumors, which are characterized by arrested or reversed growth following parturition, were noted by Haddow (1938) in dba mice (now called DBA) and by Gardner (1941) in (C57 x DBA)FI mice. Foulds (1947, 1949) first reported the existence of conditional, hormoneresponsive mouse mammary tumors by transplantation experiments. In his observation, some mammary tumors arising in (C57BL X RIII)F, and their reciprocal hybrid mice could grow when implanted into intact female or hormone-treated gonadectomized mice, but not when implanted into intact male or untreated gonadectomized mice, and a few additional transplanted tumors grew during pregnancy and regressed after parturition. The latter finding motivated him to conduct a more systematic study on the growth of mammary tumors in inbred descendants of (C57BL x RIII)F, mice, which has led to the confirmation that many spontaneous tumors in sitic grow during pregnancy and regress after delivery. Such types of tumors are called pregnancy-dependent tumors. Subsequently, many reports on development of pregnancy-dependent mammary tumors have appeared from other mouse strains, RIII (Squartini, 1962), DD (Heston ei a / . , 19641, BR6 (Lee, 1970). which is a inbred descendant of the hybrid mice used by Foulds, GR (Van Nie and Dux, 1971), and DDD (Matsuzawa et al., 1974). These strains have been noted in their origina-

HORMONE DEPENDENCE AND INDEPENDENCE O F MMTs

305

TABLE I REPORTEDHORMONE-DEPENDENT OR HORMONE-RESPONSIVE MAMMARY TUMORSI N MICE Date

Authors

I938 1941 I947

Haddow Gardner Foulds

1962 1963 I964 I965

Squartini Squartini ef al. Heston ei al. Van Nie and Thung Bentvelzen and Daams Lee Van Nie and Dux Matsuzawa el al. Sluyser and Van Nie

I969 1970 1971 I974 1974 1977 I977

Watson ei al. Matsuzawa et al.

1980

Matsuzawa ef al. Medina et al.

1984

Matsuzawa

1978

Strains"

Conditions for tumorigenesis

Dependent on or responsive to

DBA (C57 X CBA)F, RIll hybrids

Breeding Breeding Breeding

RlII BALB/cfRIII DD GR hybrids

Breeding Breeding Breeding Breeding

Pregnancy Pregnancy Pregnancy, estrogen Pregnancy Pregnancy Pregnancy Pregnancy

BALBIcfGR

Breeding

Pregnancy

BR6 GR

Breeding Breeding

Pregnancy Pregnancy

DDD

Breeding

Pregnancy

Estrone and progesterone treatment Urethan, pituitary i sografts U rethan, breeding

Estrone and progesterone

Ovary

Breeding

Pregnancy

DMBA, pituitary isografts Breeding

Ovary

GR, GR hybrids (C57BL

X

DBA/Zf)F,

BALBIc DDD hybrids (C57BL x DBA/Zf)F, BALBIcfDDD BALBIcfGR BALBlcfFM

Ovary

Pregnancy

BALBlcfRIII is a strain established by foster nursing BALBIc babies to an RIIl mother.

tion in Europe. For example, the DDD strain, on which more light is thrown by a stable pregnancy-dependent mammary tumor line, TPDMT4, in the present review, was established in the Institute of Medical Science, the University of Tokyo, from a pair of albino mice imported into Japan from Germany more than half a century ago (Matsuzawa et al., 1970). More interestingly, mammary tumors of the same sort have developed

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in hybrid mice with these strains, i.e. (C57BL x RIII)FI (Foulds, 1947), tGR x RIII)FI (Van Nie and Thung, 1963, and (BALBkfDDD x DDD)FI (Moriyama and Matsuzawa, unpublished observation), and in BALBic mice infected with MMTVs from these strains of mice by the natural route or foster nursing, i.e., BALBkfRIII established by foster nursing BALB/c babies to an RIII mother (Squartini et al., 1963), BALBkfGR (Bentvelzen and Daams, 1969; Matsuzawa, 1984), and BALB/cfDDD (Matsuzawa. 1984). Sluyser and Van Nie (1974) have demonstrated that continuous treatment with estrone ( E l )and progesterone (Pg) following ovariectomy induces hormone-dependent mammary tumors at a high frequency in GR mice and their hybrids. Their growth is still dependent on these hormones in the course of several passages (Sluyser et c d . , 1976). In rats, it is well known that a single feeding of a carcinogen, 7,12dimethylbenzanthracene (DMBA), around SO days of age induces hormone-dependent mammary tumors (Huggins et a l . , 1961). In mice, Matsuzawa et ( I / . ( 1977) and Watson et ( I / . (1977) succeeded coincidentally but independently in induction of ovarian-dependent mammary tumors using the same carcinogen, urethan, in BALB/c and (CS7 x DBADF, mice, respectively. Subsequently, Medina et a / . f 1980) have also induced mammary tumors of the same sort with another carcinogen, DMBA, in the same F, mice. As mentioned above, many investigators have used the terms deperident and rc7sponsiue to express the degree of hormone responsiveness of mouse mammary tumors. However, the definition of the terms is not clear. and differs from one investigator to another or from one case to another even by the same investigators. The effects of hormones or endocrine organs on mammary tumors are not an all-or-none phenomenon, but quantitatively are gradual and qualitatively vary. It is, therefore, difficult to define them in an absolute manner. The extent of dependence of mouse mammary tumors on hormones or endocrine organs producing them has been generally graded as follows. A tumor is called horniona or ‘orgcin dependent when it can grow in the presence but not in the absence of a specified hormone or organ, hortnonr or organ responsive when it can grow in the presence and absence of a specified hormone or organ but significantly more rapidly in its presence, and hormonc or orgciri independent or circtonornoirs when it can grow at a similar rate in the presence and absence of a specified hormone or organ. In a special case, a tumor is designated horrnonc or orgoti setisifive when it can grow significantly faster in the absence than in the presence of a specified hormone or organ (Medina et d., 1980).

HORMONE DEPENDENCE AND 1NDEPENDENCE OF MMTs

307

11. Development of Hormone-Dependent Mammary Tumors

A. MOUSEMAMMARY TUMOR VIRUS(MMTV) Frequent occurrence of hormone-responsive mammary tumors have been reported in RIII, GR, BR6, DD, and DDD mice and their F, hybrids (Table I). Nearly all primary mammary tumors appear during pregnancy and regress totally or partially after parturition in GR (Bentvelzen and Daams, 1969) and BR6 (Lee, 1970) breeding mice, and 80% of tumors produce a similar growth habit in RIII breeders (Squartini, 1962). All these tumors derived from GR breeders are still dependent on hormones after transplantation (Van Nie and Dux, 1971). Matsuzawa et al. (1974) used a transplantation test to examine the extent of pregnancy dependence of 22 tumors from DDD breeders, a strain characterized by the low and late incidence of mammary tumors. Six of them grew during pregnancy and regressed or were in arrested growth after parturition. Four of them were specially classified as completely pregnancy-dependent tumors because of no growth in virgin mice. As a result, a transplantable pregnancy-dependent tumor line was established from one of them and designated as TPDMT-4, which has been characterized by exceptionally stable hormone dependence and served as a model for analysis of hormone requirements of hormone-dependent mouse mammary tumors. These pregnancy-dependent tumors have been morphologically related to a plaque in their origin (Foulds, 1956). The plaque is a disk-like lesion which appears during pregnancy, measures 0.5-1 .O cm in diameter and 0.2-0.3 cm in thickness, and histologically comprises ductal and alveolar-like elements. The unique lesions have been also found in RIII (Squartini et af.,1963), GR (Van Nie, 1981), BR6 (Foulds, 1979, DD (Heston et a / . , 1964), and DDD mice (Matsuzawa et al., 1970). Interestingly, all these strains are of European origin, suggesting the possible existence of some common factors which have a role in development of hormone-responsive tumors. Thus, MMTVs transmitted through milk have been suspected and tested. For this purpose, MMTVs have been introduced into BALB/c mice from these strains by the natural route of infection or foster nursing, since BALB/c mice carry no MMTV in the milk and develop mammary tumors at a high frequency when infected with the virus. Many new strains thus established, BALB/cfRIII (BALBlc foster nursed by RIII mothers), BALBkfGR, BALBIcfDD, and BALB/cfDDD, have been investigated for development of pregnancy-dependent mammary tumors in various laboratories. As summarized in Table 11, the substantial proportion vary-

308

AKlO MATSUZAWA TABLE I 1 MAMMARY TUMORS RESPONSIVE TO PREGNANCY I N BALBk MICEBY MOUSEMAMMARY TUMORVIRUS(MMTV) INFECTED FROM VARIOUS STRAINS OF MICEBY FOSTER NURSING

INDUCTION OF

Source of MMTV RIIl GR C3H DD C3H GK DDD FM C3H

Number of tumors observed

Number of pregnancy dependent tumors (97)

378 20

Number of pregnancyresponsive tumors 1%) 246 ( 6 5 )

60

17 f8.5) 6 (10)

223 226 46 39 40 30

82 (37) 10 (4) 6 (14) 9 (23) 7 (18) 2 (7)

10 10 5 0

(22) (26)

113) (0)

Reference Squartini et ol. ( 1981 P3 Bentvelzen and Daams (1969V’ Bentvelzen and Daams (l969Y Heston and Vlahakis (l97I)’l Heston and Vlahakis (1971)* Matsuzdwa (1984)’ Matsuzawa (1984)’ Matsuzawa (1984)’ Matwzawa (l984p

Response of tumors to pregnancies was examined in their own hosts. Tumors arising from plaques were considered as pregnancy-responsive tumors by histologic examination. Pregnancy dependence of tumors was graded by comparing their growth between breeding and virgin mice after transplantation. ir

I

ing from 35 to 85% of the tumors induced with MMTVs from these strains are pregnancy dependent or responsive in support of the significant role of the virus in development of hormone-responsive tumors. In contrast, the proportion of such tumors is at most 10% when MMTV comes from C3H mice which rarely develop pregnancy-responsive tumors. In consideration of the strain difference, Bentvelzen and Daams (1969) have proposed calling MMTV transmitted by the European mice MMTV-P (MMTV-inducing plaques or pregnancy-dependent tumors). However, hormone dependence of mouse mammary tumors cannot be explained completely only by a variant of MMTV, because autonomous or hormone-unresponsive tumors develop in the European mouse strains and BALB/c mice infected with MMTV-P. In GR mice, a single dominant gene located on chromosome 18, M f u - 2 , has been proved to be directly involved in the expression of MTV-P particles and induction of hormonedependent mammary tumors by E l plus Pg treatment (Michalides and Nusse, 1981). In support of this, neither MTVs nor mammary tumors emerge under the same conditions in a congeneic line of the GR mouse strain without the gene (Van Nie and De Moes, 1977). However, it appears impossible to give the Mru-2 gene top priority for hormone dependence of mouse mammary tumors, since its introduction into another

HORMONE DEPENDENCE AND INDEPENDENCE OF MMTs

309

European strain, DDD, has not increased the incidence of pregnancydependent tumors (Matsuzawa and Sayama, unpublished observation). MMTV belongs to the group of retroviruses which do not carry an oncogene. Thus, the most plausible mechanism by which MMTV transforms mammary epithelial cells is insertional mutagenesis. It has been suggested that their transformation is related to the integration site of the MMTV viral DNA in the host genome. In GR mice, certain extra MMTVDNA fragments may be related with hormone dependence of tumors (Michalides et af.,1982). However, no specific extra MMTV-DNA copies have been found in relation to pregnancy dependence in TPDMT-4 tumors in DDD mice (Matsuzawa et al., 1986). Peters et al. (1984) showed that MMTV provirus integration within int-2, a specific viral DNA integration site in the host DNA, has already occurred at the earliest appearance of pregnancy-dependent tumors in BR6 mice. Further studies remain to be made to elucidate the role of MMTV proviruses and the viral DNA integration sites such as int-2 in induction and maintenance of hormonedependent tumors. B. CARCINOGENS Only three reports have been published on successful induction of hormone-responsive mammary tumors with chemical carcinogens in mice. Matsuzawa et al. (1977) gave urethan in drinking water at 0.05% to BALB/c breeding mice and isolated a transplantable, ovary-dependent mammary tumor line, UHDMT-26, from one of the tumors induced. The line has been characterized by progressive growth in virgin and breeding mice but insignificant growth in ovariectomized mice. Five of the seventeen tumors examined produced a similar growth habit (Matsuzawa, unpublished observation). However, no ovarian-responsive tumors have been induced with the same experimental procedures in milk-transmitted MMTV-free DDDf mice, which develop pregnancy-dependent mammary tumors in the presence of MMTV in the milk (Matsuzawa, unpublished observation). Watson et al. (1977) injected the same carcinogen into (C57BL x DBA/2f)FI mice carrying a pituitary isograft (PI) under the kidney capsule and demonstrated that 10 of 11 tumors were responsive to ovarian hormones. They have also established a transplantable, ovarydependent tumor line, MXT, from one of them. Medina er af. (1980) have also succeeded in the induction of mammary tumors responsive to ovariectomy by feeding DMBA to the same F, mice in the same endocrine condition, although the incidence of ovarian-responsive tumors was as low as 17%. Interestingly, 28% of tumors were sensitive to the ovary; they grew significantly better in the absence than in the presence of the ovary. However, they have failed to induce hormone-responsive mam-

310

AKlO MATSUZAWA

mary tumors with DMBA in BALB/c mice. It is noted that the mice used in these observations were free from milk-borne MMTV. In BALB/ cfDDD mice, urethan simulates mammary tumorigenesis by MMTV in terms of both latency period and incidence, but the proportion of pregnancy responsive tumors is decreased (Matsuzawa, unpublished observation). These results suggest that certain combinations of carcinogens and mouse strains will be favorable to the development of hormonedependent mammary tumors, although it is difficult to predict what combination is best.

C. HORMONES Bern (1960) has suggested that hormones may play a permissive role rather than a causative one in mouse mammary tumorigenesis. In support of this, there is no clear evidence to date that hormones act as mutagens or carcinogens. Many observations have confirmed that MMTV and chemical carcinogens can induce hormone-responsive mammary tumors in certain circumstances in mice. It is notable in these observations that the host mice developing these tumors were exposed to higher levels of hormones. All pregnancy-dependent and pregnancy-responsive tumors have appeared in breeders and hormone-dependent tumors have developed only in mice continuously treated with hormone (Table I). In addition. ovarian-dependent tumors can be induced with chemical carcinogens in mice repeating pregnancies (Matsuzawa ef o/., 1977) and in these carrying an ectopic PI (Watson ct 01.. 1977; Medina et al., 1980).The PI, free from the hypothalamic control, secretes prolactin (Prl) which is both luteotropic and mammotropic and produces a hormonal millieu mimicking that of pregnancy (Miihlbock and Boot. 1959; Heston, 1961).On the other hand. nearly all mammary tumors arising in virgin GR mice are independent of hormonal control or autonomous (Van Nie and Dux, 1971). Thus, the presence of hormones at higher levels in the inductive phase is a prerequisite for development of hormone-responsive mammary tumors in mice. However, it should be pointed out that not all of the mammary tumors arising in these conditions are dependent on or responsive to hormones, as seen in mammary tumors of C3H breeding mice. 111. Growth of Hormone-Dependent Mammary Tumors

A. HORMONALREQUIREMENTS Spontaneous hormone-responsive mouse mammary tumors all produce growth responsive to pregnancies. They grow during pregnancy and re-

HORMONE DEPENDENCE AND INDEPENDENCE OF MMTs

311

gress partially or totally, or discontinue to grow after parturition with a tendency to reach higher growth peaks in subsequent pregnancies (Fould, 1969; Van Nie, 1981). The rapid growth in the latter half of the pregnancy suggests the importance of placental lactogen for their growth. Yanai and Nagasawa (1977) have shown that the combination of estradiol (Ez)and Pg supported the growth and DNA synthesis of primary mammary tumors after ovariectomy in late-pregnant GR mice. Thus, it is likely that placental hormones may act on the tumors not only directly but also indirectly through their effects on secretion of estrogen and progestin by the ovary. For more detailed investigation of their response to hormones transplanted tumors have been used. Matsuzawa and Yamamoto (1974) established a stable, pregnancy-dependent mammary tumor line, TPDMT-4, in DDD mice of European origin. Twenty-two spontaneous mammary tumors from retired DDD breeding mice under I year of age were examined for their pregnancy dependence by comparing their growth after transplantation in breeding and virgin mice (Matsuzawa and Yamamoto, 1974). Four tumors grew in pregnant mice but not at all in virgin mice. The TPDMT-4 tumor came from one of them. TPDMT-4 tumors do not grow to palpable volumes and survive long in a quiescent state in virgins. In contrast, they grow during pregnancy and regress sharply after parturition, reaching ascending growth peaks in subsequent pregnancies in breeders. However, they continue to regress to insignificant or fixed volumes when the hosts do not become pregnant again after parturition. The growth characteristics have been maintained up to the fiftieth transplant generation. Moreover, TPDMT-4 tumors can grow without regression in mice carrying either ectopic PIS or a hormone pellet containing E? and either Pg or deox ycorticosterone acetate. These systems have served for analysis of the hormonal requirement of TPDMT-4 tumors (Matsuzawa and Yamamoto, 1975). As summarized in Table 111, PI-bearing, pseudopregnant mice allow TPDMT-4 tumors to grow progressively. However, significant tumor growth never occurs when they are ovariectomized. Tumor growth is rescued from the inhibitory effect of ovariectomy by injections of both Ez and Pg but not by injections of either of them. Continuous treatment with E2combined with Pg or deoxycorticosterone acetate in a pellet form gives rise to tumor growth in intact but not in hypophysectornized female mice. These results indicate that E2 and Pg from the ovary and pituitary hormones, primarily Prl, are essential for the growth of TPDMT-4 tumors and that placental lactogen may have a significant role in their rapid growth during the latter half of pregnancy. In the earlier transplantation study, Foulds (1947, 1949) demonstrated that a few transplanted mammary tumors derived from (C57BL x RIII)FI and reciprocal F, mice grew well in virgins but tardily or not at all in intact or castrated males. The

312

AKlO MATSUZAWA TABLE 111 EFFECTOF HORMONES ON GROWTHOF PREGNANCY-DEPENDENT TPDMT-4 MOUSEMAMMARY TUMORS

Hormonal condition,'

Number of mice with tumor growt hlnum her of mice used ~

Virgins Breeders PI PI + ovariectomy PI + ovariectomy + E2 PI + ovariectomy + Pg PI + ovariectomy + E? + Pg E:Pg pellet Hypophysectomy + E,Pg pellet EzDCA pellet Hypophysectomy + E2DCA pellet

~~~

0132 717 24125 01s 01.5

01s 415 718

Oil0 515 016

PI, lmplantation of three pituitary isografts with a tumor graft into the inguinal fat pad; E2, I7p-estradiol injection. 2 pg daily; pg. progesterone injection. 500 pg daily; E2Pg or E2DCA pellet, subcutaneous implantation of a pellet containing 39.90 mg Pg or deoxycorticosterone acetate (DCA), 0.16 mg El. and 9.94 mg cholesterol.

growth of these responsive tumors was stimulated by implantation of a diethylstilbestrol pellet. Van Nie (1981) conducted an observation in the same line with mammary tumors from RIII breeding mice and found that the growth of two transplantable, pregnancy-responsive tumor lines which grew better in breeders than in virgins was accelerated by either treatment with E l alone or implantation of PIS in gonadectomized mice. The effect of progesterone alone on these responsive tumors has not been investigated. Mammary tumors of GR mice have been examined far more extensively for their response to hormones. Transplanted hormone-dependent tumors from GR breeding mice which are characterized by no growth in ovariectomized mice can grow only when pituitary grafting is added to treatment with El and Pg at the first generation, but can grow when both steroids are present without PIS at the second and later generations (Van Nie and Dux, 19711. The addition of Prl or growth hormone to a combination of E2 and Pg displays no further stimulatory effect on transplanted pregnancydependent tumors as compared with t h e effect of the E: and Pg combina-

HORMONE DEPENDENCE AND INDEPENDENCE OF MMTs

313

tion alone (Aidells and Daniel, 1976b).Organ culture studies have demonstrated that Pg is essential for the growth of the pregnancy-dependent tumors (Harbell and Daniel, 1978). Mammary tumors induced with El and Pg in ovariectomized GR mice are mostly hormone dependent for the first few serial transplantations (Sluyser and Van Nie, 1974). These hormone-dependent tumors grow in El plus Pg-treated but not at all in untreated ovariectomized mice. It is notable that they can grow in the absence of pituitary hormones or in hypophysectomized mice if both E l and Pg are present (Van Nie, 1981). In support of the importance of estrogen and Pg for the growth of the hormone-dependent tumors, 17tr-ethynyl-lPnortestosterone,which has a pronounced progestational activity combined with a relatively low estrogenicity, causes these tumors to grow in ovariectomized mice (Van Nie and Hilgers, 1976; Van Nie, 1981). On the other hand, Briand et al. (1977) have reported that similarly induced and transplanted hormone-dependent mammary tumors of GR mice respond to ovine Prl by significant growth in ovariectomized mice and that bromocryptine, a Prl-suppressing drug, inhibits the tumor growth induced by El and Pg in ovariectomized mice. The transplantable, ovarian-dependent mammary tumor line. UHDMT26, which was established from an urethan-induced tumor of a BALB/c mouse, has been characterized by growth in virgins and more rapid growth without postpartum regression in breeders as well as by insignificant growth in ovariectomized mice. In ovariectomized or hypophysectomized hosts E2 and Pg cause the tumors to grow when given together but not when given separately (Matsuzawa, 1982). Thus, UHDMT-26 tumors are similar to hormone-dependent GR mouse mammary tumors. Other mammary tumor lines of the same sort, MXT and MXT-3590, which were established from mammary tumors induced with the same carcinogen in (C57BL x DBA/2f)FI mice, are different from the UHDMT-26 line in responsiveness to ovarian steroids. These tumors produce significant growth in ovariectomized mice when at least either Ez or Pg is present (Watson et al., 1977, 1979). Collectively, these observations indicate that the combined action of Prl, estrogen, and progestin is the most important effect on the growth of hormone-dependent or hormone-responsive mouse mammary tumors. Whether a certain tumor responds to none, one, two, or all of the hormones may be determined by a number of factors including mouse strains, carcinogenic agents, chemical and viral, target cells, and endocrine environment. In this sense, the TPDMT-4 tumor can be considered as a prototype of hormone-dependent mouse mammary tumors, since its growth is under the strict control by all of these hormones.

3 14

AKlO MATSUZAWA

B. BEHAVIOR I N VIRGINMICE Completely pregnancy-dependent mouse mammary tumors are characterized by tumor formation in the endocrine environment of pregnancy and no growth in that of virgin. In fact, TPDMT-4 tumors do not grow to palpable sizes in virgin hosts. However. tumor growth occurs immediately when they become pregnant or treated with the appropriate hormone combination even as long as 6 months after the implantation. This suggests that hormone-dependent tumor cells can survive in a quiescent state at lower hormone levels and are similar to hyperplastic alveolar nodules, considered as preneoplastic lesions in many strains of mice including C3H (Medina, 1973; Cardiff, 1984). To clarify the preneoplastic nature of TPDMT-4 tumors, their behavior in fat pads of virgin mice has been fully examined (Matsuzawa et d.,1982). As shown in Fig. IA and C, TPDMT-4 tissue pieces from late-pregnant hosts grow out in all directions and form mammary gland-like structures consisting of ducts, lobules, and acini in mammary gland-free fat pads. The appearance of the structure varies from one outgrowth to another in predominance of each component depending on the hormone levels of the hosts. In contrast, the tissue pieces in intact fat pads do not grow out and remain as traces in the host mammary gland (Fig. IB). There may be some interaction between TPDMT-4 cells and normal mammary epithelial cells resulting in inhibited outgrowth of the former by the latter. In support of the interaction, TPDMT-4 cells also block the expansion of normal mammary ducts when placed in the not-yet-occupied site of the fat pad at 3 weeks of age (Matsuzawa. 1984). Aidells and Daniel (1974, 1976a.b. 1978) conducted similar experiments with early hormone-dependent mammary tumors from pregnant or E? plus Pg-treated GR mice. The grafts of these tumors develop into a network of ducts resembling normal mammary glands of nonpregnant mice. Dependent tumor and normal mammary tissues interact with each other when they are implanted into a single gland-free fat pad of a virgin mouse. Dependent mammary tumor tissue pieces transplanted into mammary fat pads already containing normal mammary ducts usually cannot be localized or, at best. display minimal growth. Normal mammary gland and hormone-dependent tumor transplants in a single glandfree fat pad show normal regulatory behavior: they produce ductal outgrowths displaying mutual avoidance behavior in which ducts do not touch and are normally spaced. These results clearly demonstrate that pregnancy- or hormone-dependent mouse mammary tumor cells express preneoplastic properties in an environment where hormone levels are too low to produce tumorous growth and suggest that they may originate in ductal cells. In this respect,

HORMONE DEPENDENCE AND INDEPENDENCE OF MMTs

315

FIG. 1. Outgrowths from TPDMT-4 tissue grafts implanted into inguinal fat pads 12 months previously. (A) Wholemount preparation of the outgrowth in gland-free fat pad. Note the formation of apparently normal mammary gland comparable to early pregnant state. (B) Wholemount preparation of the graft in intact fat pad. Note complete suppression of outgrowth from the graft (arrow) by normal mammary parenchyma. (C) Histologic section of the outgrowth in gland-free fat pad. Note ductal-alveolar structures, secretion in lumina, and absence of tumorous foci. (A,B) X 1.8: (C) x 140.

it is important that MMTV- and carcinogen-related hormone-dependent tumors in a growth phase produce hyperplastic ductal structures in many areas (Foulds, 1969; Van Nie and Dux, 1971; Aidells and Daniel, 1978; Matsuzawa et al., 1977; Watson er a/., 1977). In addition, TPDMT-4 tumors form a tubular and papillary architecture in a maximally regressed and resting state after parturition, and produce ducts and end buds of normal appearance in hypophysectomized mice (Matsuzawa and Yamamoto, 1974, 1977).

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C. RESPONSE TO ENDOCRINE THERAPIES It is of clinical importance to elucidate the action mechanism by which endocrinotherapeutic agents suppress the growth of hormone-dependent breast cancer. Models which are stable in hormone dependence and produce progressive growth are indispensable to this purpose. It is therefore expected that TPDMT-4 mammary tumors in mice carrying PIS or E, plus Pg pellets are available as a model for study on endocrine therapy of breast cancer. In an experiment the steroidal antiestrogen with androcaused genic activity, epitiostanol (2a,3a-epithio-5a-androstan-17/3-ol). immediate regression of TPDMT-4 tumors with ovarian atrophy accompanied at daily dose of 100-1000 pg in PI-bearing mice (Matsuzawa and Yamamoto, 1976). Testosterone, although less effective, also gave rise to tumor regression under the same condition. Either steroid did not reverse but suppressed the growth of tumors induced by an E2plus Pg pellet. Van Nie (1981) reported that testosterone inhibited the El-induced but not the PI-induced growth of hormone-responsive mammary tumors of RIIl mice. In contrast, the androgen had no influence on the growth of hormone-dependent tumors of GR mice induced with E l and Pg (Van Nie, 1981). These results indicate that the androgenic agents can inhibit the growth of hormone-dependent mammary tumors by direct action on tumor cells themselves and by suppressive effects on the endocrine organs. I]- I ,'-diphenylbut- 1Tamoxifen ( I-[4-(2-dimethylaminoethoxy)phenyl ene citrate), a therapeutic agent more widely used in the clinical field, has been examined for the antitumor effect on TPDMT-4 tumors under similar conditions (Matsuzawa and Yamamoto. 1979: Matsuzawa ef d., 1981). Treatment with the agent at a daily dose of 200-800 p g caused complete arrest of tumor growth instantly followed by gradual regression and accompanied the atrophy of the ovary in Pi-bearing mice, and it suppressed the tumor growth in E2 plus Pg-treated mice. In PI-bearing mice the autitumor effect attained was comparable to that of ovariectomy in spite of the fact that tamoxifen has an estrogenic activity in the mouse (Terenius. 1971). Tamoxifen treatment inhibited the growth of hormone-responsive mammary tumors induced with a combination of El and Pg in GR mice (Sluyser. 1979) and monohydroxytamoxifen. a metabolite of tarnoxifen, manifested a stronger inhibitory effect than tamoxifen in this model system (Sluyser et a / . , 1981b). Thus, the action mechanism of the estrogenic antiestrogen for hormone-dependent mouse mammary tumors is considered to be the same as that of the androgenic agents. It is, however, notable that the former is less effective than the latter in inhibiting the growth of the ovarian-responsive tumors. T4-OR26, isolated from the TPDMT-4 tumor (Matsuzawa and Ikecla. 1983). In contrast, another

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therapeutic agent, medroxyprogesterone acetate (6a-methyl-17a-acetoxyprogesterone), enhanced the growth of TPDMT-4 tumors at a daily dose even as high as 3 mg in PI-bearing mice (Matsuzawa, unpublished data). The result is conceivable, since medroxyprogesterone has a progestogenic activity (Babcock et al., 1958) and Pg is important for the tumor growth (Matsuzawa, 1982). Interestingly, the gonadotropin-releasing hormone (GnRH) agonist analog (D-leucy16,des-glycyl-NH:", prolylethylamide9)GnRH, displays both enhancing and reversing effects on the growth of TPDMT-4 tumors in PI-bearing hosts depending on the time after administration is started (Matsuzawa and Yamamoto, 1982). The GnRH analog enhanced the tumor growth for the first 2 weeks and subsequently caused rapid tumor regression as seen after parturition. However, only the enhancing effect was manifested in mice implanted with an E2 plus Pg pellet. The dual action of the polypeptide on TPDMT-4 tumors can be explained by its stimulatory effect on the pituitary gland during the first growth-enhancing phase and by its direct suppressive effect on the ovary during the second growth-reversing phase. Only the latter inhibitory effect seems to appear in carcinogen-induced hormone-dependent mammary tumor models in rats (Johnson et al., 1976; DeSombre et al., 1976). Bromocryptine (2-Br-a-ergokryptine-methansulfonate) inhibited the growth of hormone-dependent mammary tumors in GR mice treated with E2 plus Pg (Briand et al., 1977).This indicates that inhibition of Prl release from the pituitary gland can lead to suppressed growth of hormone-dependent tumors. Regression of hormone-dependent mammary tumors occurs following parturition, elimination of hormonal stimulation, or treatment with endocrinotherapeutic agents, supporting the possibility of reversing their growth by endocrine manipulation. The rate of regression varies with different endocrine environments. The TPDMT-4 tumors grown in the presence of both E2 and Pg regressed faster when both hormones were deprived than when either of them was deprived. It is likely that Pg delays the tumor regression as compared to E2 (Matsuzawa, 1982). To clarify the mechanism of tumor regression, Janik et al. (1975) have calculated the total cell loss on the basis of growth rate, pulse-labeled mitosis curves, and labeling index, noting no difference in cell loss rate between growing and regressing tumors and have ascribed the regression of hormonedependent tumors to the relatively more reduced cell production by deprivation of the proper hormones. Schiilein et al. (1976) investigated the biochemical changes during regression and regrowth of hormonedependent GR mouse mammary tumors. The RNA content decreased during regression and increased during regrowth, whereas the DNA and protein contents showed no variation. The DNA, RNA, and protein syn-

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theses decreased in parallel during regression with the most pronounced reduction in DNA synthesis. Regrowth of regressed tumors induced by readministration of hormones was accompanied by an immediate increase in RNA synthesis followed by the increase in DNA synthesis about 24 hours later. The result implies that hormonal regulation of DNA synthesis (tumor growth) is preceded by increased RNA synthesis in hormonedependent mouse mammary tumors.

IV. Hormone Receptors in Dependent Mammary Tumors A. ESTROGEN RECEPTORS(ER)

In the mouse mammary tumors responsive to pregnancy or hormones, ER. the specific binding protein for estrogen, has been demonstrated by various assay methods in support of the involvement of the hormone in their growth. Terenius ( 1972) incubated the slices of pregnancy-dependent mammary tumors of GR mice with [)HIEz in the presence and absence of excess nonradioactive Ez and demonstrated that all of these tumors bound E2 to a significant extent in a specific fashion. Sluyser and Van Nie (1974) and Sluyser et cil. (1976) applied the dextrancoated charcoal (DCC) method to assay ER in the cytosols of mammary tumors induced with E2 plus Pg treatment in GR mice and obtained the average ER contents of 48 and 32 fmol/mg cytosol protein in hormone-dependent and hormone-responsive tumors. respectively. Daehnfeldt and Briand ( 1977) utilized the same assay method in similarly induced hormone-responsive mammary tumors and detected higher levels of ER in the cytosol while noting no significant difference between unoccupied and total (unoccupied plus occupied) receptors. Richards el 01. (1974) detected significant levels of cytoplasmic ER sedimenting at 8 S by the sucrose density gradient (SDG) analysis in spontaneous mammary tumors of GR and RIIl mice, although they did not mention the hormone requirement of these tumors. Watson ef ul. (1977) determined the cytoplasmic ER by the DCC method in the urethan-induced, ovarian-dependent mammary tumor, MXT, obtaining the average level of 8.03 fmolhg tissue. In addition, they have confirmed that the receptor was translocated to the nucleus after E? injection in the same tumor. With regard to the nuclear translocation, Sluyser and Tulp (1979a.b) have shown that the nuclei of hormone-responsive tumors of GR mice took up E2during all phases of the cell cycle. Watson and Clark (1980) have confirmed the localization of two types of ER, called types I and 11, in both cytoplasmic and nuclear compartments in another urethan-induced, ovarian-dependent mammary tumor, MXT-

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3590. Type I ER, so-called “classical ER,” has been characterized by higher affinity and lower binding capacity and type I1 by lower affinity and lower binding capacity (Clark et al., 1979). More extensive studies on ER have been conducted in pregnancy-dependent TPDMT-4 mammary tumors in DDD mice (Matsuzawa et ul., 1980; Matsuzawa, 1982; Hayakawa and Matsuzawa, unpublished observation). The cytosols from growing tumors in pregnant hosts were incubated with increasing amount of [3H]E2and analyzed by the SDG containing no KCl. The radioactivity was preferentially incorporated into the 8 S region at lower concentrations. At higher concentrations the 8 S region was saturated and excess radioactivity was recovered in the fractions near the top. The 8 S peak was completely abolished by excess nonradioactive E2or epitiostanol. The cytoplasmic ER sedimented at 4 S in a SDG containing KCI. The binding specificity of ER has been confirmed by a high affinity to the steroid (Kd0.7 nM) in the DCC method with Scatchard analysis. Activation of the E2-ER complex, which is considered to be an essential event (Jensen et al., 1968) and to have a biological regulatory function (Weichman and Notides, 1980; Rochfort and Borgna, 1981) in estrogen action, has been proved to occur at a high temperature and in ammonium sulfate precipitation. As a result, the ER complex underwent a 4-5 S change in sedimentation coefficient in a high salt medium, displayed a decrease in dissociation rate of E2 from ER (see Fig. 3A), and acquired an augmented ability to bind to DNA. The activation was inhibited by sodium mulybdate as observed in ER of the normal mouse mammary tissue (Haslam et al., 1984). Cytoplasmic ER contents have been determined by the DCC method in various endocrine environments. The ER level remains at 20-70 fmol/mg cytosol protein regardless of the status of tumor growth, growing, regressing, or static: it does not decline along with tumor regression after parturition in breeders and after ovariectomy in PI-bearing mice. ER is also detectable at significant levels in tumors in a quiescent state. This finding is noteworthy, since DMBA-induced, hormone-dependent rat mammary tumors show a significant reduction in cytoplasmic ER in parallel with regression induced by ovariectomy (Vignon and Rochfort, 1976). However, the normal mouse mammary gland produces no major changes in specific estrogen binding activity throughout pregnancy and lactation when it is expressed on the basis of cytosol protein (Hunt and Muldoon, 1977; Muldoon, 1978). Translocation of the activated ER into the nucleus is also a prerequisite for full expression of the biological functions of estrogen (Jensen et al., 1968). Evidence for occurrence of this event in the TPDMT-4 tumor is available. When TPDMT-4 tumors grew to significant sizes, the hosts received an injection of E? with previous ovariectomy. Analysis of the

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KCI extract of the nuclear fraction from the tumors thus obtained with [3H]D2by the SDG method has demonstrated the specific binding protein with a sedimentation coefficient of about 5 S. The nuclear translocation has been also confirmed by a nuclear exchange assay. Taken together, these results suggest that the machinery of estrogen action may be intact in hormone-dependent mouse mammary tumor cells, although the recent observations of exclusive localization within the nucleus of what is known as cytosolic ER (King and Greene, 1984; McClellan r f a / . , 1983) may require some modification of the currently accepted two-step mechanism for the interaction of steroid hormones with their target cells (Jensen, 1984).

B. PROGESTERONE RECEPTORS(PgR)

It is intriguing to investigate PgR in pregnancy-dependent and hormone-dependent mouse mammary tumors, since Pg plays a significant role in their growth (see Section 111,A). Initially, the receptor assay was hampered by the binding of radioactive Pg to the corticosteroid binding globulin. Recently, a new potent synthetic progestin, promegestone ( 17,21-dimethyl- 19-nor-4,9-prognadiene-3,20-dione), which does not bind specifically to the globulin (Philbert and Raynaud, 1973; Raynaud, 1977). has enabled an easier PgR assay and prompted studies on PgR in both experimental and clinical fields. Sluyser et d. (1976) first applied the compound to hormone-induced mammary tumors of GR mice and demonstrated significant levels of cytoplasmic PgR in transplanted hormonedependent and hormone-responsive tumors. Daehnfeldt and Briand (1977) have also detected the receptor at an average level of about 60 fmol/mg protein in these tumors. Matsuzawa et d.(1978) assayed PrR with the ligand in the cytosois from a number of transplantable. pregnancy-dependent and ovarian-dependent mammary tumor lines. The receptor levels varied from 250 to 550 fmol/mg cytosol protein in MMTVinduced pregnancy-dependent lines, TPDMT-4, TPDMT- 185, and TPDMT-G8, which were growing in late-pregnant DDD, (BALB/cfDDD x DDDIF,. and BALBicfGR mice, respectively. it averaged 170 and 79 fmol/mg protein in two urethan-induced, ovarian-dependent lines, UHDMT-26 and UHDMT-38. respectively, in BALB/c mice. In these tumors, PgR was reduced to very low or undetectable levels after ovariectomy. Watson cf a / . (1979) have confirmed the presence of cytoplasmic PgR at significant levels by both DCC and SDG methods using radiolabeled Pg and its reduction after ovariectomy in the urethan-induced, ovarian-dependent mammary tumor line. MXT-3590, in (C57BL x DBAflF, mice.

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32 1

PgR of the TPDMT-4 tumor has been extensively investigated in relation to changed hormonal environments (Matsuzawa et a / . , 1978; Matsuzawa, 1982; Koseki et al., 1982). Cytoplasmic PgR sedimenting around 4 S in a SDG is demonstrated at significant levels in growing tumors from P1-bearing, intact mice but at an insignificant level in static tumors from PI-bearing, ovariectomized mice. Unlike ER, PgR shows a dramatic decrease after parturition when tumors are regressing, and becomes almost undetectable 4 weeks later. The similar postpartum reduction in PgR levels has been reported in the normal mammary gland in mice (Shyamala and Haslam, 1980) and in rats (Levy and Glick, 1977). In PI-bearing mice, ovariectomy causes reduction in PgR content along with gradual regression of TPDMT-4 tumors. The ovariectomy-dependent reduction in the receptor level has been also observed in other pregnancy-dependent (Matsuzawa et af., 1978) and ovarian-dependent (Matsuzawa et al., 1978; Watson et al., 1979) mouse mammary tumors. These findings suggest that PgR synthesis is under the control of estrogen in hormone-dependent mouse mammary tumors as in the normal target tissues (Toft and O’Malley, 1972; Rao et a / . , 1973; Horwitz and McGuire, 1977) and in DMBAinduced, hormone-dependent rat mammary tumors (Kelley et al., 1977; Koenders et al., 1977; McGuire et al., 1977). In agreement with the suggestion, El injection increases the PgR levels of these pregnancy-dependent and ovarian-dependent tumors in ovariectomized mice (Matsuzawa et al., 1978; Watson et al., 1979). A time course study of PgR induction by Ez has been conducted using TPDMT-4 tumors with the results illustrated in Fig. 2 (Matsuzawa, 1982). PI-bearing mice with tumors were given a

0

20

40

60

Hours after estradiol injection

FIG.2. Induction of progesterone receptors (PgR) by estradiol (E2)in TPDMT-4 mammary tumors at transplant generation 32. Pituitary isograft-bearing mice with tumors, ovariectomized a week previously, received a single sc injection of 3 pg ELat time 0. Tumors were excised at indicated times for PgR assay. Each value is mean & SE for two to four tumors.

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single sc injection of El I week after ovariectomy and sacrificed for PgR assay at various times after the injection. PgR levels rose rapidly after E? injection and reached a peak around 25 hours later, which is 7-8 times as high as the basal level. Subsequently, it decreased precipitously to a plateau, a slightly higher level over the basal level. Actinomycin D, an inhibitor of RNA synthesis, suppressed the Ez-induced PgR synthesis by more than 75% when given simultaneously with E? and by 60-80% when given 4 hours after Ez. This suggests that Ez-dependent PgR synthesis is also a process involving gene regulation in hormone-dependent mouse mammary tumor cells. In contrast, Pg has no significant influence on cytoplasmic PgR of pregnancy-dependent mammary tumors in the absence of E2 (Matsuzawa ef al., 1978). In the light of the action mechanism of steroid hormones, attempts have been made to clarify whether or not cytoplasmic PgR produced in response to Ez can be translocated to the nucleus in hormone-dependent mammary tumors. Koseki et al. (1982) have detected a significant amount of PgR in the nucleus in growing TPDMT-4 tumors from pregnant mice. In addition, they have confirmed that cytoplasmic PgR is translocated to the nucleus after Pg injection in uiuo and after treatment of tumor slices with Pg in uirro. Matsuzawa (1982) has demonstrated the nuclear translocation of E:-induced PgR in TPDMT-4 tumors. These results clearly demonstrate that the pathway of nuclear translocation of PgR is effective in hormone-dependent mammary tumor cells in mice.

RECEPTORS(PrlR) C. PROLACTIN Prl is important for growth of hormone-dependent mouse mammary tumors, as suggested by the fact that TPDMT-4 tumors can grow progressively in PI-bearing mice but not at all in virgin mice or even in the presence of both El and Pg in hypophysectomized mice (Matsuzawa and Yamamoto, 1977). Thus, Costlow et (11. (1977) have identified PrlR in the crude membrane fraction of El plus Pg-induced mammary tumors of GR mice. The PrlR level is highest (16 fmol/mg protein) in primary, hormonedependent tumors and declines gradually in transplanted hormone-dependent tumors and transplanted hormone-responsive tumors. This is the only report on PrlR in hormone-responsive mouse mammary tumors, although many reports have been published on the receptor in rat hormone-dependent mammary tumors (Costlow and McGuire, 1978). It is. however, very interesting that TPDMT-4 tumors, especially at early generations, have significantly higher basal levels of PgR in the absence ofthe ovarian hormones and produce significantly more PgR in response to Ez in PI-bearing mice than in multiparous, nonpregnant mice (Matsuzawa, 1982). since the Prl level is considered to be far higher in the former.

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D. OTHERHORMONE RECEPTORS Androgen receptors have been detected at far lower levels as compared with those of ER and PgR in hormone-dependent and hormone-responsive mammary tumors of GR mice (Sluyser et al., 1976). There has been no evidence to indicate that androgens are involved in growth of hormone-dependent mouse mammary tumors except for the unique androgen-dependent Shionogi carcinoma, SC-115 (Mineshita and Yamaguchi, 1965; Smith and King, 1972). On the other hand, testosterone gives rise to regression of TPDMT-4 tumors (Matsuzawa and Yamamoto, 1976). It is plausible that the androgen may display the antitumor effect through the receptors in view of the presence of Sa-reductase in mouse mammary tumors (Abul-Haj and Kiang, 1982). Glucocorticoids are essential for the development and function of the mammary gland (Topper and Freeman, 1980). Specific binding proteins for the hormone have been detected in lactating mammary glands of mice in support of it (Shyamala, 1973). Shyamala (1974) has clearly demonstrated that glucocorticoid receptors are present in the cytosols of spontaneous mammary tumors from GR breeding mice, most of which are considered to be hormone dependent at least at their onset (Van Nie, 1981). Glucocorticoids stimulate production of MMTV by mouse mammary tumor cells in culture (Dickson et al., 1974; Parks et al., 1974; Ringold et al., 1975a,b). It is generally accepted that stimulation of MMTV production by glucocorticoid hormones is a result of the direct action of the steroid-receptor complex to MMTV proviral DNA. Thus, the experimental system has provided a good model for studying the mechanism by which steroid hormones regulate gene expression on the molecular basis (see the review by Ringold, 1983). However, it remains to be elucidated whether or not glucocorticoids may manifest any influence on malignant growth of hormone-dependent mouse mammary tumors through their stimulating effect on MMTV production.

V. Progression from Dependence to Independence A. MECHANISM OF PROGRESSION Many studies have reached the general consensus that hormone-dependent tumor progress toward full autonomy via responsive stages. Foulds (1969) pointed out: “Hormone responsiveness is not an all-or-none phenomenon; the responses are qualitatively graded, as well as qualitatively varied.” Foulds (1969) reported that many spontaneous mammary tumors of BR mice were completely pregnancy dependent at the first detection by

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palpation but progressed to be less dependent and finally independent or autonomous in the course of repeated pregnancies. Spontaneous mammary tumors with similar growth characteristics have been observed in RlII (Squartini, 1962), BALBIcfRIII (Squartini et ul., 1981), and BR6 (Peters et al., 1984) breeding mice. In GR mice, spontaneous pregnancydependent mammary tumors are also converted to independent tumors in a similar course during repeated pregnancies in the same hosts (Aidells and Daniel, 1978; Van Nie, 1981). This phenomenon has been ascribed to the appearance of unresponsive cells in foci. Progression to full autonomy occurs during transplantation of pregnancy-dependent and hormone-dependent mammary tumors. Foulds (1947, 1949) isolated mammary tumors which grew well in females but tardily or not at all in males in (C57BL x RIII)F, mice. These tumors lost the sex dependence following several times of transplantation in females. Urethan-induced, ovarian-dependent mammary tumor lines, UHDMT-26 and UHDMT-38, have progressed to be independent via responsive stages after 15 and 4 passages in virgin mice, respectively (Matsuzawa et (11.. 1977, 1978: Matsuzawa, 1982). Watson et cil. (1980) have established an ovarian-independent subline from a similarily induced, ovarian-dependent mammary tumor, MXT-3590, by 5 repeated passages through ovariectomized mice starting at transplant generation 10. Transplanted pregnancy-dependent mammary tumors of GR mice acquire the ability to grow autonomously after 6-8 repeated pregnancies in the same hosts (Aidells and Daniel. 1978). Hormone-dependent GR mouse mammary tumors induced by continuous E, plus Pg treatment have been widely used as a model for studying the mechanism of tumor progression. In general, these tumors lose their hormone responsiveness when passaged through more than four generations in hormone-treated mice (Briand and Daehnfield. 1973; Sluyser and Van Nie, 1974; Sluyser ef ul., 1976: Van Nie, 1981; Kiang et d . , 1982). Briand ef al. (1982) have demonstrated that about half of these dependent tumors regress to be unpalpable within 1-3 months after discontinuation of hormone treatment and regrow as independent tumors after a dormancy period varying from 1 to 8 months. Progression toward greater autonomy of these transplanted dependent tumors with advanced generations may be explained by heterogeneity of cell populations. In GR mice, hormone-dependent mammary tumors already contain a small population of autonomous cells when detected, and they progress to a responsive state and subsequently to an autonomous state as a result of a gradual increase in the proportion of these cells during serial transplantation (Sluyser er al., 1976). However, it remains unclear how the heterogeneity of tumor cells is produced. In contradistinction to these tumors, the transplantable pregnancy-dependent mouse mammary tumor, TPDMT-4, has been noted in its excep-

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tionally stable hormone dependence, since the tumors have maintained pregnancy dependence up to transplant generation 50; they grow during pregnancy and regress after parturition in breeders, but produce no significant tumors for at least 3 months in virgins. However, the tumor grafts in the thirtieth and later generations form palpable tumors sporadically after latency periods longer than 4 months in virgin mice. Importantly, almost all of the resulting tumors are still dependent on the ovary (Matsuzawa et al., 1983). The morphological study indicates that TPDMT-4 tumors have become less secretory and more malignant with serial passages (Matsuzawa et al., 1977). Thus, progression of the tumor toward autonomy may be very slow under physiological conditions. In addition, analysis of proviral MMTV genomes and chromosomes has demonstrated that TPDMT4 tumors are monoclonal in origin differing from GR mouse mammary tumors (Matsuzawa et al., 1986). As such, the tumor has served as a model for studying the factors affecting tumor progression. Brief exposure of the tumor grafts to chemical carcinogens in uirro followed by a few passages in uivo in virgins had led to development of autonomous tumors (Matsuzawa et al., 1977). Continuous exposure of the tumors to hormones has accelerated the appearance of autonomous cells: TPDMT-4 tumor cells have acquired autonomy significantly earlier when serially transplanted in mice carrying an Ez plus Pg pellet than in breeding mice (Matsuzawa et al., 1983). More interestingly, enzyme dissociation of the tumors has led to development of autonomous tumors without delay: free cells prepared by dissociating TPDMT-4 tumors with collagenase, hyaluronidase, and pronase form autonomous and responsive tumors more frequently with shorter latency periods in virgins (Matsuzawa ct d., 1986). It has been noted that the progression-enhancing effect of the enzyme dissociation is far stronger on intermediate generation tumors than on later generation ones and is not expressed in ovariectomized hosts. Collectively, these observations indicate that both endogenous and exogenous factors may cause neoplastic cells comprised in a tumor more heterogeneous and display significant influences on the tumor progression. The process peculiar to oncogenesis may be a result of the appearance of new types of cells with different potency and selection of a special population of cells under environmental pressures. Hormone-dependent mammary tumors which are composed of more heterogeneous cells at the time of detection can progress toward full autonomy for a shorter period of time (Sluyser et al., 1976; Sluyser, 1979).

B. ALTERATION OF HORMONE RECEPTORSWITH

PROGRESSION

The hormone receptors are essential for the initial interaction between the hormone and the target cell and function to trigger the biochemical

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chain of events characteristic for the particular hormone. Therefore, the presence of the specific receptors for one hormone may be evidence that the tumor growth is under the control of the hormone. Clinically, this has been well supported by the fact that ER-negative breast cancers do not respond to endocrine therapies at all but not by the fact that only about half of ER-positive breast cancers respond to them (McGuire ef (11.. 1974: Horwitz and McGuire, 1978). Thus, it is important for a deeper understanding of hormone dependence of tumors to know how the levels, properties, and functions of the receptors change along with tumor progression. Studies of this type have been conducted with transplanted mammary tumors in GR mice. Sluyser and collaborators (Sluyser and Van Nie, 1974: Sluyser rt d . , 1976) have demonstrated that cytoplasmic ER contents were lower in hormone-dependent, hormone-responsive, and hormone-independent tumors in this order and that autonomous tumors contained lower levels of the receptor than the hormone-dependent tumors from which they were derived. Cytoplasmic PgR levels are also higher in hormone-dependent than in hormone-independent tumors. It is noteworthy that low but significant levels of ER were maintained during many generations whereas PgR became undetectable soon after progression to autonomy. However, Kiang ct (11. (1982)reported that ER and PgR levels showed cyclic changes during serial transplantation even after the tumors progressed to full autonomy, although the degree was less con5picuous in ER and the peak PgR levels seemed to be lower with advanced generation. Costlow rt al. (1977) have observed changes of PrlR with tumor progression under the same experimental conditions. The PrlR levels were highest in primary, hormone-dependent tumors and declined gradually with transition to responsive and then to autonomous states during serial transplantation. The binding in autonomous tumors is approximately 5% of that in dependent tumors. In (C57BL x DBAOF, hybrid mice, Watson ef nl. (1980) have isolated a hormone-independent variant from the urethan-induced, ovarian-dependent MXT-3590 mammary tumor and demonstrated that the autonomous variant still had ER, both types I and 11. and PgR. In the pregnancy-dependent TPDMT-4 mammary tumors of DDD mice, changes in both receptor levels have been investigated in the course of progression to ovarian-dependent, ovarian-responsive, and autonomous states under various experimental conditions (Matsuzawa rt ul., 1978, 1980. 1982, 1983, 1986; Matuszawa, 1982). AH ovarian-dependent and ovarian-responsive tumors contain levels of ER and PgR similar to those of TPDMT-4 tumors regardless of the conditions under which they have arisen. It has been noted that almost all autonomous tumors with PgR originated in TPDMT-4 tumors transplanted in the presence of

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continuous E2 plus Pg stimulation. In general, these tumors lost PgR without delay during transplantation in virgin mice. In contrast, ER tends to be maintained for a longer period and some autonomous tumors have possessed significant levels of the receptor for 30 or more generations. The earlier loss of PgR than ER during progression is reasonable, since the synthesis of PgR is controlled by estrogen (Fig. 2). These observations clearly demonstrate that a significant proportion of autonomous mouse mammary tumors can synthesize significant levels of ER consistently, as seen in about half of hormone-unresponsive human breast cancer. This raises the question of what stage of the two-step mechanism of estrogen action might be defective in these tumors. Shyamala (1972) have shown that the nuclear translocation of ER might be defective; ER in the cytoplasm could not be translocated to the nucleus after incubation of tumor tissues with E2 in spontaneous hormone-independent mammary tumors of GR mice. In contrast, Vignon and Rochfort (1978) found that the nuclear translocation was intact in spontaneous autonomous C3H mouse mammary tumors. Baskevitch et al. (1983) have extended the study to demonstrate significantly higher affinity for DNA of ER in these tumors than in the uterus but no differences in the dissociation rate of E2from the nonactivated or activated E2-ER complex and the density in a metrizamide isopycnic gradient of ER between the tumors and the uterus. They have suggested that the lack of E2-induced PgR synthesis in spite of nuclear ER translocation might be ascribed to the increased affinity of ER for nonspecific DNA sites. In different types of hormone-independent mouse mammary tumors derived from urethan-induced, ovarian-dependent tumors, however, estrogen can stimulate PgR synthesis (Matsuzawa et al., 1978; Watson et a / . , 1980). Importantly, Watson et a / . (1980) have confirmed that Ez translocates cytoplasmic ER of both types I and I1 to the nucleus and increases cytoplasmic PgR, which is in turn translocated to the nucleus by Pg in such a tumor. Kiang et al. (1984) have compared the nuclei from three types of GR mouse mammary tumors, ER-positive, hormone-dependent, and hormone-independent tumors, and ER-negative hormone-independent tumors, for their ability to bind to an activated E2-ER complex and found a defect in ER-positive, hormone-independent tumors but not in the others. Interestingly, a nonhistone chromosomal protein with a molecular weight of 31,000 was markedly diminished or abolished in parallel with loss of the ability. A systematic comparative study on ER from the pregnancy-dependent TPDMT-4 tumor and its autonomous subline, T401320, is in progress. The subline derives from enzyme-dissociated TPDMT-4 cells and produces ER consistently but does not synthesize PgR in response to E2. Preliminarily, the parent and subline tumors do not

378

A K l O MATSUZAWA 100

100

50

50

E! n 10

10

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z

3 0

m -1

Q

n

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s -1

A

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90

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TIME ( m i n )

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FIG. 3 . Comparison of ['Hlestradiol (E.)-receptor diswciation kinetics between the pregnancy-dependent TPDMT-4 mammary tumor ( A ) and its autonomous subline. T4-01320 ( B ) . Cytosols prepared from growing tumors were equilibrated with 5 nM ['HIE? with or without unlabeled E2 at 0°C in the absence (circle) and presence (triangle) of 10 mM Na2Moo4. Dissociation of ('HIE2 from the nonactivated (open symbol) and heat-activated (filled symbol) receptors was measured at 3 ° C after addition of 5 g M unlabeled E2 for the indicated length of time. Receptor inactivation was measured by parallel incubation of aliquots (dotted line) at 25°C wirhout the addition of unlabeled E?. Each dissociation measurement was corrected for nonspecific binding and receptor inactivation.

differ from each other in the dissociation rate of E? from nonactivated and activated E2-ER complexes (Fig. 3), and sucrose gradient pattern and nuclear translocation of ER. but they seem to be slightly different in the interaction of the E2-ER complex with nuclei (Hayakawa and Matsuzawa, unpublished observation). Thus, the defect appears to be at the level of the nucleus in hormone-independent T4-01320 tumors. In summary, it is suggested that the machinery of estrogen action may be disturbed at various steps in hormone-independent mouse mammary tumors with ER and that ER loss may be the result of progression to autonomy but not the cause of it.

C. ALTERATION OF RESPONSIVENESS TO HORMONES AND THERAPEUTICS W I T H PROGRESSION Since its isolation the TPDMT-4 mammary tumor has been maintained on the criterion of pregnancy-dependent growth in breeders and no appreciable growth in virgins. Morphologically, however, the tumors have progressed from differentiated to undifferentiated states along with loss of

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secretory activity (Matsuzawa and Yamamoto, 1977). In addition, the tumor implants have produced sporadic growth after a latency period Of 4 months in virgins since around transplant generation 30. Actually, an ovarian-responsive subline, T4-OR26, was successfully isolated and has served as an experimental model (Matsuzawa et al., 1983). These observations suggest that TPDMT-4 tumors have gradually progressed to acquire the ability to grow at lower hormone levels and to be less dependent on Prl. Along with the progression, the tumors have become more resistant to an androgenic antiestrogen, epitiostanol. The antiestrogen gave rise to immediate tumor regression at generations 7 and 8, but it only partially suppressed tumor growth at generations 23 and 24 (Matsuzawa and Yamamoto, 1976, 1977). In this context, it is of great significance that TPDMT-4 tumors have acquired resistance to an estrogenic agent, tamoxifen, earlier than to androgenic agents, epitiostanol and testosterone, during serial transplantation (Matsuzawa and Yamamoto, 1976; Matsuzawa et al., 1981; Matsuzawa and Ikeda, 1983). In addition, the tumor regression following ovariectomy was smaller and the regrowth of the regressed tumors induced by E2 or Pg alone was larger in extent at late than at early generations in PI-bearing mice (Matsuzawa et af., 1980). However, the growth-enhancing effect of Pg alone on the late generation tumors appears inconsistent, since it was comparable to that of the combination of E2 and Pg in one experiment (Matsuzawa et al., 1980) but weaker than it and comparable to that of E2 alone in the other (Table IV). In accord with these data, the basal level in an ovariectomized state and the stimulated level by either Ez and Pg of DNA synthesis were significantly higher in late generation than in early generation tumors (Table IV). Interestingly, Pg alone was equally or more stimulative in terms of tumor growth but less so in terms of DNA synthesis as compared with E2 alone (Table IV), suggesting the possibility that Pg may inhibit the degradation of DNA synthesized. In this respect, it is noted that Pg retarded the tumor regression following discontinuation of E2 plus Pg treatment. However, the mechanism by which Pg causes tumor growth and stimulation of DNA synthesis is ovariectomized hosts with very low or insignificant levels of PgR (Matsuzawa et al., 1978; Watson et al., 1979) remains to be elucidated. Estrogen control of PgR synthesis is an important marker for intactness of estrogen action. The ability to produce PgR in response to E2 was compared among early and late generation TPDMT-4 tumors and the ovarian-dependent subline in the presence or absence of elevated levels of Prl secreted by ectopic PIS. The ability was greater at later generations, although it declined after progression to ovarian dependence. More significantly, the early generation tumors contrasted with the late generation and ovarian-dependent tumors in that the former produced significantly

370

AKIOBMATSUZAWA

EFFECT OF OF

T A B L E IV ESTRADIOL 4 N D PROGF\TERONE ON GROWTH A N D DNA SYNTHESI5 PREGNANCY-DEPENDENT TPDMT-4 MAMMARY TUMORS AT EARLY(F,,)A N D LATE(F15)TRANSPLANT GENERATIONS Change in tumor volume during I-weeh treatment (5%)

Treatment.' Control E2 pe E: plus Pg

Early -3.5.7

2

5.8

+4.7 5 10.7

-6.7 i 2.1 ~ 7 0 . 0f 10.9

Incorporation of ['Hlthymidine (dpm/pg D N A )

Late

Early

-32.2 c 3.9 +63.9 2 11.1 +66.0 2 18.9 +134.1 2 22.8

3.2 5 0.5 14.1 5 1.4

8.4 c 1.5 41.3 t 4.3

Late

11.0 t36.3 -C 21.1 2 38.7 2

3.3 8.1

3.x 4.8

Mice received implant of a tumor graft and three pituitary isografts into the right inguinal fat pad. When tumors grew to significant volumes, ovariectomy allowed them to regress. A week later mice received sc implant of a pellet containing cholesterol (control). estradiol (E'). progesterone (Pg). o r E2 and Pg ( E 2 plus Pg). An additional week later mice received an ip injection of 50 yCi ['Hlthymidine 2 hours before their sacrifice. Tumor diameters were determined at implantation of a pellet and sacrifice of mice to estimate a change in tumor volume. Mean f SE for six or seven tumor$.

more PgR in response to E? in the presence than in the absence of PIS (Matsuzawa, 1982). This indicates that TPDMT-4 tumors have progressed to be less dependent on prolactin in terms of PgR synthesis. In addition, it has been noted throughout these observations that the late generation tumors gave more widely distributed values in the determination of parameters such as growth rate, ER, PgR. and DNA synthesis, suggesting that neoplastic cells comprised in TPDMT-4 tumors have become more heterogeneous with progression during serial transplantation. With regard to the response to chemotherapy, Sluyser and Benckhuysen ( 1977) reported that hormone-dependent and independent mammary tumor cells of GR mice showed similar sensitivity to cyclophorphamide. Moreover, Sluyser el al. (1981a) have investigated the effect of the cytostatic agent on hormone-dependent tumors through several transplant generations and found that tumors might be especially susceptible to chemotherapy at the time of transition from hormone dependence to independence. The autonomous subline of the TPDMT-4 tumor, T4-01320. is also sensitive to cyclophosphamide. However, it changed into a resistant tumor with a higher growth rate after several times of transplantation through mice treated with the chemotherapeutic agent (Takeda and Matsuzawa, unpublished observation). Tumor progression may be an endless phenomenon.

HORMONE DEPENDENCE AND INDEPENDENCE OF MMTs

33 1

D. MARKERS FOR PROGRESSION Hormone-dependent mammary tumors have the specific receptors for the hormones on which they are dependent. However, the presence of the hormone receptors does not necessarily mean the dependence of tumors on specific hormones. For clarification of the mechanisms of hormonal control of tumor growth and release of hormone-dependent tumors from hormonal control, biochemical and biological markers which can discriminate horrnone-dependent from hormone-independent tumors have been searched for with little success in the experimental and clinical fields (Briand, 1983).

1 . Iodide Uptake Thorpe (1976) reported that the uptake of lZsI injected into the hosts was about 20 times higher in hormone-dependent than in hormone-independent mammary tumors of GR mice. When the hormone-dependent tumors were serially transplanted, their ability to concentrate iodide declined gradually and was lost simultaneously with their progression to horpone independence (Thorpe and Briand, 1984). The tumors concentrate iodide in a free form but not in a protein bound form (Lyttle et al., 1979). The iodide taken up by the tumors may be present in the outer membrane and in the intercellular spaces (Sluyser, 1981). Of particular interest in this regard is the decrease in amounts of extracellular matrices during progression of pregnancy-dependent TPDMT-4 mammary tumors to less dependent or autonomous states with a higher growth rate (Matsuzawa and Amano, unpublished observation). 2 . Enzymes Lyttle el al. (1979) reported that peroxidase activity was about 10 times greater in hormone-dependent mammary tumors than in hormone-independent GR mouse mammary tumors. However, Sluyser (1981) has not detected the enzyme activity at appreciable levels in these tumors. In support of this observation, Strum and Becci (1979) have demonstrated no peroxidase-positive tumor cells by a cytochemical technique in either hormone-dependent or hormone-independent tumors. In our laboratory, the enzyme activity has been assayed in the TPDMT-4 tumor and its sublines differing in extent of hormone responsiveness. The TPDMT-4 tumors growing in a proper endocrine milieu have slightly higher levels of peroxidase varying from 0.2 to 1.5 units/g tissue as compared with those reported by Lyttle et al. (1979) in hormone-responsive tumors of GR mice. The levels tend to decline with tumor regression following delivery, ovariectomy, or discontinuation of hormonal treatment and the declined

331

AKIO MATSUZAWA

levels appear to recover after injection of E? and Pg alone or in combination. In contrast, the enzyme activity is markedly different from one autonomous subline to another: it is lower in some and far higher in some as compared with that of the parent tumor. However, in evaluation of these data, attention should be paid to infiltration of granulocytes in tumor tissues, since eosinophils are rich in peroxidase (Rytomaa and Teir, 1961) and have a significant role in E?-dependent changes in the enzyme activity in the uterus (Lyttle rt al., 1984). Thus, endogenous mammary peroxidase may not be a reliable marker for hormone dependence of mammary tumors as expected earlier. Smith and King (1970b) have observed in BR6 mice that lactate dehydrogenase activity was lower in pregnancy-dependent than in pregnancy-independent mammary tumors whereas isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase, and phosphohexose isomerase activities were not different between them. I n GR mice, the activities of the enzymes involved in glycolysis seem to be higher in hormone-independent than in hormonedependent mammary tumors, although only the difference in hexokinase activity reached a significant level (Briand and Daehnfeldt, 1973). This observation and the significantly higher lactate accumulation in hormoneindependent tumors suggest that the glycolytic activity may augment with transition from dependence to autonomy. In contrast, G6PDH activity is significantly higher in hormone-dependent than in hormone-independent tumors of GR mice. Lactose synthetase A-protein was detected at similar levels but Bprotein was undetectable in hormone-dependent and hormone-independent tumors (Schiilein et al., 1974). Kiang et 01. (1982) have observed cyclic changes in thymidine kinase activity during serial transplantation of tumors after their transition from hormone-dependent to hormone-independent states in GR mice. Abul-Haij and Kiang (1982) investigated the metabolism of testosterone by GR mouse mammary tumors and found that the proportion of Sa-reduction decreased with the transition from hormone-dependent to hormone-independent states. It is noteworthy that E? is synthesized from testosterone by hormone-independent tumors but not by hormone-dependent tumors (Abul-Haij and Kiang, 1982). 3. Proteins, Nucleic Acids, and Others Smith and King (1970a,b) reported higher histone content, lower protein content. lower levels of nicotinamide adenine nucleotide, and lower rates of total protein synthesis and amino acid incorporation into the nuclear protein in pregnancy-dependent than in pregnancy-independent mammary tumors of BR6 mice. However, no apparent differences have

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333

been found in chromatographic elution patterns of H 1 histones (Sluyser, 1977) and in transfer RNA levels (Quist et al., 1976a) between hormonedependent and hormone-independent tumors of GR mice. There are differences in the number of isoacceptor peaks of some transfer RNA between these tumors (Quist et al., 1976b). In addition, Smets et a/. (1977) have noted certain differences in membrane glycoproteins between them and Sluyser et al. (1979) have revealed a lower concanavalin A-mediated agglutinability in hormone-dependent tumor cells. 4. MMTV Expression and Proviral Information Sluyser et al. (1977) assayed the production of MMTV particles (types A and B) and MMTV antigens by serially transplanted mammary tumors of GR mice without noting any changes specific for their transition from hormone-dependent to hormone-independent states. Michalides et al. (1982) found that the hormone-dependent tumors contained extra MMTV proviral DNA which was lost at the time of transition to a hormoneindependent state. Pregnancy-dependent TPDMT-4 mammary tumors can progress toward ovarian dependence and full autonomy without major changes in MMTV integration into the host DNA (Matsuzawa el ul., 1986). It still remains unclear whether MMTV plays a prominent role in progression to autonomy of hormone-dependent mouse mammary tumors.

5. Chromosomes Chromosomal abnormalities have been reported in many mouse tumors (Miller, 1983).Trisomy of chromosome 13 has been described for MMTVinduced mammary tumors from GR and C3H mice (Dofuku et al., 1979) and for urethan-induced mammary tumors from BALB/c mice (Dofuku and Matsuzawa, 1983). Although the tumors analyzed were all hormone independent, tumor cells with trisomy of chromosome 13 have also been found in pregnancy-dependent TPDMT-4 tumors (Matsuzawa and Kaneko, unpublished observation). This abnormality is likely derived from endomitosis of chromosome 13 and loss of one chromosome I3 (Dofuku and Matsuzawa, 1983). Hormone-dependent mammary tumors of GR mice have been used to assess the changes of chromosomal pattern during their progression to hormone independence in the course of serial transplantation (Kiang et al., 1982). The appearance of polyploid cells was not associated with the tumor progression and their proportion changed cyclically at intervals of four to six generations. In contrast, a longer marker chromosome appeared after transition to a hormone-independent state and the proportion of cells with the chromosome continued to increase to 100% during serial transplantation of the hormone-indepen-

334

AKlO MATSUZAWA

dent tumors. Pregnancy-dependent TPDMT-4 tumors can progress to autonomy without accompanying either polyploidy or the marker chromosome. However, it should be pointed out that a significant number of polyploid cells are found in autonomous tumors derived from TPDMT-4 tumors which had been passaged in the presence of continuous hormonal stimulation (Matsuzawa et al., 19861, since G R mouse mammary tumors have been transplanted under a similar condition (Kiang ef d.,1982).

6 . Angiogeriic Activity Formation of new blood vessels is important for growth of neoplastic tissues and most malignant solid tumors have the ability to induce angiogenesis (Folkman, 1974, 1985). Thus, it is intriguing to know whether the angiogenic activity of mouse mammary tumors will change during their transition from hormone-dependent to horrnone-independent states. To answer this question, the angiogenic activity has been assayed in pregnanc y-dependent TPDMT-4 mammary tumors and hormone-independent subline tumors using the rabbit cornea as described by Gullino (1981). These tumors have similar levels of angiogenesis activity regardless of their hormone responsiveness, which are far lower than that of hormoneindependent tumors from C3H mice (Oikawa et al., 1985). This finding suggests that hormone-dependent mammary tumors can progress toward autonomy without accompanying enhanced angiogenesis. However, further studies will be needed utilizing different assay methods to reach a final conclusion with regard to the role of angiogenic activity in progression to more malignant states, since a chorioallantoic membrane assay has been used to demonstrate augmentation in the activity along with transition from hormone dependence to independence in the GR mouse mammary tumor system (Strum, 1983). VI. Conclusions Hormone dependence of mammary tumors had been considered a rare phenomenon in mice. However, the concept has been exploded by frequent development of pregnanc y-dependent or hormone-dependent mammary tumors in European mouse strains including G R , RIII, BR6, DD, and DDD. Endocrine milieus with higher hormone levels such as pregnancy, ectopic pituitary isografts, and continuous treatment with estrogen and progesterone are prerequisites for the development of these tumors. A variant of mouse mammary tumor virus transmitted by these strains has an appreciable role in induction of the tumors. Chemical carcinogens can also induce hormone-dependent mammary tumors. Therefore it has been

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335

suggested that certain combinations of carcinogenic agents and mouse strains may be favorable to the development of hormone-dependent mammary tumors, although it is impossible at present to predict what combination is best. Prototypic hormone-dependent mouse mammary tumors have the following characteristics.

1 . Their growth is controlled primarily by prolactin, progesterone, and estrogen. Hence, it is reversed or suppressed by depletion of one or more of the hormones or by administration of endocrinotherapeutic agents. 2 . They have prolactin, progesterone, and estrogen receptors in support of the importance of these hormones to their growth. 3. Hormone-dependent tumor cells survive long in a quiescent state in the endocrine milieu where the hormonal stimulation is insufficient to cause tumorous growth: they produce an interaction with the normal mammary epithelium and form the structures mimicking the mammary gland in the gland-free fat pad. Hormone-dependent mouse mammary tumors progress toward autonomy via less dependent stages with time. The tumor progression, which may be explained by the appearance of new types of cells with different potency and selection of a special population of cells under environmental pressures, is associated with the following events.

I . Hormone-dependent tumors are released from the controls by prolactin, progesterone, and estrogen in this sequence. In accordance with it, prolactin, progesterone, and estrogen receptors are lost sequentially, although estrogen receptors can be maintained long after transition to autonomy. 2 . Tumors in intermediate stages of the progression may respond to one or more of the hormones to a significant degree. 3. They acquire resistance to endocrinotherapeutic agents along with advanced progression. As described above, there is no doubt that estrogen and progesterone enhance growth of hormone-dependent mouse mammary tumors in viuo. However, there is no evidence to date that they display mitogenic activity for hormone-dependent tumor cells in uirro. Recently, a large body of evidence has been presented to indicate that growth factors are directly involved in proliferation of hormone-dependent normal and neoplastic cells (Sirbasku et al., 1985). Thus, further studies remain to be conducted to elucidate the mechanism by which the steroids control hormone-dependent growth of mammary tumors on a molecular basis.

336

AKIO MATSUZAWA ACKNOWLEDGMENTS

1 take this opportunity to express my gratitude to the late Prof. T. Yamamoto for introducing me to mammary tumor research and his continued interest in my professional activities f o hi3 last moment. Thanks are also due to Ms. Y. M . lkeda and Mr. T. Kaneko for their excellent technical assistance. and Miss M. Matsuzawa for her secretarial work in preparation of the manuscript. The author’s investigations cited here have been supported by a grant-in-aid for cancer research from the Ministry of Education. Science. and Culture of Japan.

REFERENCES Ahul-Hajj, Y. J.. and Kiang. D. T . (1982). Ctrticer Rcs. 42, 3510-3513. Aidells. B. D.. and Daniel, C. W. (1974). J. N u f l . Cctnc,er In.tf.52, 1855-1863. Aidells. B. D.. and Daniel, C. W. (1976a). J. N u t / . Cnncer Insr. 57, 519-526. Aidells. B. D.. and Daniel. C . W. (l976b). J. Nor/. f t i r i c e r Iricf. 57, 527-537. Aidells. B. D.. and Daniel. C . W. (1978). J . N a r f . Cttnc-rr Inst. 60, 1345-1350. Babcock. J. C.. Gutsell. E. S.. Herr, N. H., Hogg. J. A.. Stucki. J. C.. Barnes. L. E.. and Dulin. W. E. (1958). J. A m . Chew. Soc.. SO, 2904-2905. Baskevitch. P. P.. Vignon, F.. Bousquet. C . . and Rochfort. H. (1983). Ctriiwr Rex. 43, 230-2297,

Bentvelren. P.. and Daams. J. H. (1969). J . N o r / . Ctrncc~I r i v r . 43, 1025-1035. Bern. H. A. (1960). Science 131, 1039-1040. Bittner. J. J . (1936). Srience 84, 162-164. Bonser. G. M.. Dossett, J. A , , and Jull. J. W. (1961). “Human and Experimental Breast 1 Cancer.” Pitman. London. Briand. P. (1983). Anricttncw Rrs. 3, 273-282. Hriand. P.. and Daehnfeldt. J . L. (1973). Etrr. J . Coriwr 9, 763-770. Briand. P.. Thrope. S. M . . and Daehnfeldt. J. L. (1977). Br. 1. C r m w 35, 816-821. Briand. P., Rose. C.. and Thrope. S. M. (1982). Ertr. J. C n n w Clin. O n c d . 18, 1391-1393. Cardiff. R . D. (1984). Adu. Cunccr Res. 42, 167-190. Clark, J. H.. Hardin. J. W.. Erikson. H.. Upchurch, S.. and Peck. E. J. (1979). 111 “Ontogeny of Receptors and Reproductive Hormone Action“ (T. H. Hamilton, J. H. Clark. and W. A. Sadler, eds.). pp. 65-77. Raven. New York. Costlow. M. E.. and McGuire, W. L. (1978). I n “Endocrine Control in Neoplasia” (R. K. Sharma and W. E. Riss, eds.), pp. 121-150. Raven, New York. Costlow.. M . E.. Sluyser. M..and Gallagher, P. E. (1977). E n d o ~ r Rvs. . C~nirntur.4, 285294.

Daehnfeldt. J . L.. and Briand. P. (1977). In “Progesterone Receptors in Normal and Neoplastic Tissues” ( W . L. McGuire. J. P. Raynaud. and E. E. Baulieu. eds.). pp. 59-69. Raven. Ne* York. DeSombre, E. R.. Johnson. E. S., and White. W. F. (1976). Cuiicer Rrs. 36, 3830-3833. Dickson. C.. Haslam. S . . and Nandi, S. (1974). Virctlogy 62, 242-252. Dofuku. R.. and Matsuzawa, A. (1983). Anricctncer Rex. 3, 17-34. Dofuku. R..Utakoji. T.. and Matsuzawa. A. (1979). J . N u r f . Ctrriccr I n s r . 63, 651-656. Dunn, T. B. (1945). “Mammary Tumors in Mice.” pp. 13-38. Publ. No. 22, Amer. Assoc. Advance. Sci., Washington, D.C. Folkman. J . (1974). Adu. Cancer R ~ s 19, . 331-358. Folkman. J. (1985). Ado. Cuncw RPS.43, 175-203.

HORMONE DEPENDENCE AND INDEPENDENCE OF MMTs

337

Foulds, L. (1947). Br. 1.Cancer 1, 362-370. Foulds, L. (1949). Br. J. Cancer 3, 240-246. Foulds, L. (1956). J. Null. Cancer Insf. 17, 701-801. Foulds, L. (1969). In “Neoplastic Development” (L. Foulds. ed.), Vol. I . Academic Press, New York. Foulds. L. (1975). In “Neoplastic Development” (L. Foulds, ed.), Vol. 2, pp. 457-458. Academic Press, New York. Gardner, W. U. (1941). Cunrer Res. 1, 345-358. Gullino, P. M. (1981). In “Handbook of Experimental Pharmacology” (R. Baserga. ed.). Vol. 57, pp. 427-449. Springer-Verlag, Berlin and New York. Haddow, A . J. (1938). farhol. Bacferiol. 47, 553-565. Harbell, J. W., and Daniel, C. W. (1978). In V i m 14, 361. Haslam, S. Z., Gale, K. J., and Dachtler, S. L. (1984). Endocrinology 114, 1163-1172. Heston, W. E. (1961). J. Natl. Cancer Inst. 36, 1273-1284. Heston, W. E., and Vlahakis, G. (1971). f n t . J . Cancer 7, 141-148. Heston, W. E., Vlahakis, G., and Tsubura, Y. (1964). J. Null. Cancer Inst. 32, 237-251. Horwitz, K. B., and McGuire. W. L. (1977). In “Progesterone Receptors in Normal and Neoplastic Tissues” (W. L. McGuire, J. P. Raynaud. and E. E. Baulieu, eds.), pp. 103124. Raven, New York. Horwitz, K. B., and McGuire, W. L . (1978). In “Breast Cancer” (W. L. McGuire, ed.), Vol. 2, pp. 155-204. Plenum, New York. Huggins, C.. Grand, L. C., and Brillantes, P. F. (1961). Nuture (London) 189, 204-207. Hunt, M. E., and Muldoon, T. G. (1977). J . Steroid Biocliem. 8, 181-186. Hynes, N. E., Groner, B., and Michalides, R. (1984). Adu. Cancer Res. 41, 155-184. Janik, P., Briand, P., and Hartmann, N. R. (1975). Cancer Res. 35, 3698-3704. Jensen, E. V. (1984). Lab. inurst. 51, 487-489. Jensen, E. V., Suzuki, T., Kawashima, T., Stumpf, W. E., Jungblut. P. W., and DeSombre, E. R. (1968). f r o c . Narl. Acad. Sci. U . S . A . 59, 632-638. Johnson, E. S., White, W. F., and DeSombre. E. R. (1976). Science 194, 329-330. Kelly, P. A., Asselin, J., Labrie, F., and Raynaud. J. P. (1977).In “Progesterone Receptors in Normal and Neoplastic Tissues” (W. L. McGuire, J. P. Raynaud. and E. E. Baulieu, eds.), pp. 85-101. Raven, New York. Kiang, D. T., King, M., Zhang, H. J., Kennedy, B. J.. and Wang. N. (1982). Science 216, 68-70. Kiang, D. T., Handschin, B., and Zhang, H. 3 . (1984). Cancer Res. 44, 4118-4123. King, W. J., and Greene, G. L. (1984). Nuture (London) 307, 745-747. Koenders, A. J., Moespot, G. A,. Zolingen. S. J., and Benraad, T. J. (1977). In “Progesterone Receptors in Normal and Neoplastic Tissues” (W. L. McGuire, J . P. Raynaud, and E. E. Baulieu, eds.). pp. 71-84. Raven, New York. Koseki, Y., Costlow, M. E., Cole. D.. and Matsuzawa, A. (1982). J . Endornnol. 94, I 11123. Lee. A. E. (1970). Br. J . Cancer 24, 561-567. Levy, J., and Glick, S. M. (1977). In *‘Progesterone Receptors in Normal and Neoplastic Tissues” (W. L. McGuire. J. P. Raynaud, and E. E. Baulieu, eds.), pp. 21 1-225. Raven, New York. Lyttle, C . R., Thrope, S. M., DeSombre, E. R., and Daehnfeldt, J . L. (1979). J. Nut/. Cancer Inst. 62, 1031-1034. Lyttle, C. R., Medlock, K. L., and Sheehan. D. M. (1984). J. Biol. Ciiem. 259, 2697-2700. McClellan, M. C., West, N. B., Tacha, D. E., Greene, G. L., and Brenner, R. M. (1984). Endocrinology 114, 2002-2014.

338

AKlO MATSUZAWA

McGuire, W. L., Carbone, P. P., Sears, M. E., and Escher, G. C. (1974). In ”Estrogen Receptors in Human Breast Cancer” (W. L. McGuire. P. P. Carbone. and E. P. Vollmer. eds.), pp. 1-7. Raven, New York. McGuire, W. L., Horwitz, K. B., Peason, 0. H., and Segaloff, A. (1977). Cancer 39, 29342947. Matsuzawa, A. (1982). In “Hormonal Regulation of Mammary Tumors” (B. Leung. ed.), Vol. I , pp. 183-215. Eden Press, Montreal. Matsuzawa, A. (1984). Onrologia 10, 54-71 (in Japanese). Matsuzawa, A,, and Ikeda, Y. (1983). Cancer Res. 43, 3680-3686. Matsuzawa, A., and Yamamoto, T. (1974). Gann 65, 307-315. Matsuzawa, A., and Yamamoto, T. (1975). J. Natl. Cancer Insi. 55, 447-453. Matsuzawa, A., and Yamamoto, T. (1976). Cancer Res. 36, 1598-1606. Matsuzawa, A., and Yamamoto, T. (1977). Cancer, Res. 37, 4408-4415. Matsuzawa, A., and Yamamoto, T. (1979). Gann 70, 387-388. Matsuzawa, A., and Yamamoto, T. (1982). Eur. J . Concer Clin. Oncol. 18, 495-505. Matsuzawa, A., Yamamoto, T., and Suzusi, K. (1970). Jpn. J . Exp. Med. 40, 159-181. Matsuzawa, A.. Yamamoto, T., and Suzusi, K. (1974). J. Natl. Cancer I n s f . 52, 449-456. Matsuzawa, A., Yamamoto, T., and Mizuno, Y. (1977). Garin 68, 523-524. Matsuzawa, A., Yamamoto, T., and Mizuno, Y. (1978). I n ”Hormones, Receptors, and Breast Cancer” (W. L. McGuire, ed.), pp. 263-279. Raven, New York. Matsuzawa, A., Mizuno, Y., Yamamoto, T.. Hori. T., and Suzuki, T. (1980). Cancer Re.\. 40, 3361-3368. Matsuzawa, A,, Mizuno, Y., and Ysmamoto, T. (1981). Cancer Res. 41, 316-324. Matsuzawa, A., Kaneko, T., Ikeda, Y., and Yamamoto, T. (1982). Gann 73, 372-376. Matsuzawa, A., Kaneko, T., and Ikeda, Y. (1983). Cancer Res. 43, 2283-2289. Matsuzawa, A., Ikeda, Y., Kaneko, T., Murakami, A., and Tanaka, H. (1986). Cancer R ~ J . Submitted. Medina, D. (1973). Merhods Cancer Res. 7, 3-53. Medina, D., Butel, J. S.. Socher, S. H., and Miller, F. L. (1980). Cancer Res. 40, 368-373. Michalides, R., and Nusse, R. (1981).In “Mammary Tumors in the Mouse” (J. Hilgers and M. Sluyser, eds.), pp. 465-503. Elsevier, Amsterdam. Michalides, R.. Wagenaar, E., and Sluyser, M. (1982). Cancer Res. 42, 1154-1158. Miller, D. A. (1983). Adu. Cancer Res. 39, 153-182. Mineshita, T., and Yamaguchi, K. (1965). Cancer Res. 25, 1168-1 177. Muhlbock, O., and Boot, L. (1959). Cancer Res. 19, 402-412. Muldoon, T. G. (1978). J . Steroid Biochem. 9, 485-494. Nandi, S., and McGrath, M. (1973). Adu. Cancer Res. 17, 353-414. Oikawa, T., Matsuzawa, A., and Iwaguchi, T. (1986). Br. J . Cancer. Submitted. Parks, W. P., Scolnick, E. M., and Kozikowski, E. H. (1974). Science 184, 158-160. Peters, G., Lee, A. E., and Dickson. C. (1984). Nature (Lundon)309, 273-275. Philbert, D., and Raynaud, J. P. (1973). S/eroids 22, 89-98. Quist, R.. Palin, C., and Heiberg, I. (1976a). Cancer Bioctiem. Biophys. 1, 215-222. Quist, R . , Palin, C., and Heiberg. I. (1976b). Canter Biochem. Biophys. 1, 317-329. Rao, B. R., Wiest, W. G., and Allen, W. M. (1973). Endocrinology 92, 1229-1240. Raynaud, J. P. (1977). In “Progesterone Receptors in Normal and Neoplastic Tissues” (W. L. McCuire and J. P. Raynaud, eds.), pp. 9-21. Raven, New York. Richards, .I.E., Shyamala, G.. and Nandi, S. (1974). Cmcer Re.\. 34, 2764-2772. Ringold, G. M. (1983). Curr. Top. Microbiol. I!?zml4nO/. 106, 79-103. Ringold, G . M., Lasfargues, E. Y., Bishop, J. M., and Varmus, H. E. (1975a). Virology 65, 135-147.

HORMONE DEPENDENCE AND INDEPENDENCE O F MMTs

339

Ringold, G. M., Yamamoto, K. R., Tomkins, G. M., Bishop, J. M.. and Varmus, H. E. (1975b). Cell 6, 299-305. Rochfort, H., and Borgna, J. L. (1981). Nature (London) 292, 257-259. Rytomaa, T., and Teir, H. (1961). Nature (London) 192, 271-272. Schiilein, M., Daehnfeldt, J. L., and Briand, P. (1974). Int. J . Cancer 14, 372-378. Schiilein, M., Daehnfeldt, J. L., and Briand, P. (1976). Int. J . Cancer 17, 120-128. Shyamala, G . (1972). Biochem. Biophys. Res. Commun. 46, 1623-1630. Shyamala, G. (1973). Biochemisiry l2, 3085-3090. Shyamala, G. (1974). J . Biol. Chem. 249, 2160-2163. Shyamala, G., and Haslam, S . Z. (1980). In “Perspectives in Steroid Receptor Research” (F. Bresciani, ed.), pp. 193-216. Raven, New York. Sirbasku, D. A., Ikeda, T., and Danielpour, D. (1985). In “Mediators in Cell Growth and Differentiation” (R. J. Ford and A. L. Maizel, eds.). pp. 213-232. Raven, New York. Sluyser. M. (1977). Cancer Lett. 2, 147-151. Sluyser, M. (1979). Biochim. Biophys. Aria 560, 509-529. Sluyser, M. (1981). In “Mammary Tumors in the Mouse” (J. Hilgers and M. Sluyser. eds.), pp. 265-299. Elsevier, Amsterdam. Sluyser, M., and Benckhuysen, C. (1977). Cuncer Treur. Rep. 61, 861-867. Sluyser, M., and Tulp, A. (1979a). J . Steroid Biochern. 10, 305-309. Sluyser, M., and Tulp, A. (1979b). J. Steroid Biocheni. 11, 343-350. Sluyser, M., and Van Nie, R. (1974). Cancer Res. 34, 3253-3257. Sluyser, M., Evers, S. G., and De Goeij, C. C. J. (1976). Nature (London) 263, 386-389. Sluyser, M., Nouwen, T., Hilgers. J., and Calafat, J. (1977). Cancer Res. 37, 1986-1990. Sluyser. M., Van der Valk, M., and Van Bllitterswijk. W. L. (1979). Br. J . Cuncer 41,348355. Sluyser, M., De Goeij, C. C. J., and Evers, S. J. (1981a). J . Nut/. Cancer Inrt. 66,327-330. Sluyser, M., Evers, S. G.. and De Goeij, C. C. J. (1981b). Eur. J . Cancer 17, 1063-1065. Smets, L. A., Van Beek. W. P., and Van Nie, R. (1977). Cancer Lett. 3, 133-138. Smith, J. A., and King, R. J. B. (1970a). Br. J. Cancer 24, 861-868. Smith, J. A., and King, R. J. B. (1970b). Cuncer Res. 30, 2055-2060. Smith. J . A., and King. R. J. B. (1972). Excerpta Med. I n t . Congr. Ser. 256, 20. Squartini. F. (1962). J . Nail. Cancer Inst. 28, 91 1-926. Squartini, F., Rossi, G., and Paoletti, I. (1963). Nature (London) 197, 505-506. Squartini, F.. Bistocchi, M.. and Buongiorno, L. (1981). J . N d . Cancer Inst. 66, 31 1-319. Staats, J . (1966). In “Biology of the Laboratory Mouse” (E. L. Green, ed.), pp. 1-9. McGraw-Hill, New York. Strum, J. M. (1983). Am. J . Patho/. 111, 282-287. Strum, J . M., and Becci, P. J. (1979). Virchonis Arch. Abt. B 31, 135-142. Terenius, L. (1971). Acta Endocrind. 66, 431-447. Terenius. L. (1972). Eur. J . Cancer 8, 55-58. Thorpe, S. M. (1976). Int. J . Cancer 18, 345-350. Thorpe, S. M., and Briand, P. (1984). Int. J . Cuncer 34, 127-131. Toft, D. D., and O’Malley. B. W. (1972). Endocrinology 90, 1041-1045. Topper, Y. J . , and Freeman, C. S. (1980). Physiol. Rev. 60, 1049-1106. Van Nie, R. (1981). I n “Mammary Tumors in the Mouse“ (J. Hilgers and M. Sluyser, eds.), pp. 201-266. Elsevier, Amsterdam. Van Nie, R., and De Moes, J. (1977). Inr. J . Cuncer 20, 588-594. Van Nie. R., and Dux,A. (1971). J . Narl. Cancer fnsr. 46, 885-897. Van Nie, R., and Hilgers, J. (1976). J . Nail. Cancer Inst. 56, 27-32. Van Nie, R., and Thung, P. J. (1965). Eur. J . Cancer 1, 41-50.

340

AKlO MATSUZAWA

Vignon. F.. and Rochfort. H . (1976). Endwrinolog.v 98, 722-729. Vignon. F.. and Rochfort. H . (1978). Cancer Re.,. 38, 1808-1814. Watson. C. S . . and Clark. J . H . (19801. J . Recepror Res. 1, 1-9. Watson. f.S . , Medina. D.. and Clark. J . H . (19771. Concer R ~ J37, . 3344-3348. Watson. C. S . . Medina. D.,and Clark. J . H . (1979). Cunrer Res. 39, 4098-4104. Wahon. C. S . , Medina. D . . and Clark. J . H . (1980). Endoi~rinolog.~ 107, 1432-1437. Wrichman. B . M . . and Notides. A . C. (1980). Etidocrinology 106, 434-439. Yanai. R . . and Nagasawa. H . (1977). Ertr. J . Cuncw 13, 813-816. Zotter. S . . Kemmer. C . . and Miillcr. M. (1981). E x p . Pnlhol. Sirpp. 6 , 1-164.