The clinical biology of hormone-responsive breast cancer

Cawr

7’reatmnt

Reticws

The clinical cancer

( 1988)

15,33-5

biology

1

of hormone-responsive

breast

R. J. Epstein University Department and MRC Unit of Clinical Council Centre, Cambridge CBZ P&H, U.K.

Oncology & Radiotherapeutics,

Medical

Research

Introduction The notion of breast cancer as a systemic disease is a relatively new one. Originally born out of disenchantment with the long-term results of aggressive local therapy on disease outcome (60), this view has gained further support from recent clinical and laboratory observations: (a) breast cancer seems likely to be incurable in the biological (if not the actuarial) sense, commonly recurring after disease-free intervals exceeding two decades (22); (b) primary tumours are often multicentric in origin (177) and can be detected as such in up to 40% of patients following excision of macroscopically localized disease (76); (c) skin fibroblasts from patients with breast cancer display abnormal growth properties even prior to diagnosis (9); and (d) peripheral blood lymphocytes of affected patients exhibit defective DNA repair (110). Findings such as these have led to renewed interest in both the biology (189) and the systemic therapy (190) of breast cancer. One expression of this interest has been an abundance of clinical trials. Such trials have contributed significantly to our understanding of breast cancer biology (13) but, unfortunately, major advances in clinical progress have been less forthcoming. In the case of cytotoxic chemotherapy, initial enthusiasm generated by high tumour response rates has been tempered by negative reports of survival benefit (164) and serious doubts regarding palliative efficacy ( 102). This therapeutic cul-de-sac hints at the limitations of a purely empirical approach to this malignancy and suggests that the development of more effective systemic chemotherapy may depend upon combining insights gained from both basic and clinical research. This cooling of enthusiasm for aggressive cytotoxic management of breast cancer contrasts with the evergreen popularity of hormone therapy. Hormonally induced regression of breast cancer remains one of the most gratifying experiences in clinical oncology, while the minimal morbidity and relative cheapness of hormone therapy have ensured its continuing favour as a subject for clinical research. Moreover, well-characterised laboratory models of hormone-responsive human breast cancer now exist, permitting analysis of hormone action at the level of cellular and molecular biology. Since many of the developments which have revolutionized our ideas about breast cancer have arisen from the study of this fascinating entity, it seems timely to review the way in which clinical and 030557372/88/010033

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+ 19 $03.00/O 33

1988 Academic

Press Limited

34

laboratory research breast cancer.

R. J. EPSTEIN

have helped

The

elucidate

oestrogen

the role played

receptor:

state

by hormonal

factors in human

of the art

Hormonal responsiveness of breast tumours appears to be mediated primarily by the oestrogen receptor, a 66-kilodalton protein consisting of a central DNA-binding domain, a carboxy-terminal oestrogen-binding domain, and an amino-terminal ‘modulating’ domain of obscure function. The structure of the receptor has been evolutionarily conserved to a high degree, exhibiting significant homology both with other hormone receptors (including thyroid hormone receptor; 174) and across species (113). This is particularly true for the DNA-binding domain; it is the steroid-binding domain, however, which represses receptor function in the absence of hormone (83). Although such an inactive receptor has for many years been thought to reside in the cytoplasm and to undergo nuclear translocation on binding hormone, it now seems that active and inactive receptor alike are situated predominantly within the nuclei of target cells (107, 211). Binding of oestrogen increases affinity of the hormone-receptor complex for DNA ( 186)) perhaps by precipitating removal of an attached protein and thereby inducing the appearance of two DNA-binding zinc-cysteine ‘fingers’ on the receptor surface (135); as yet, however, there is no direct (e.g. X-ray crystallographic) evidence for this model. Similarly, the popular speculation that the receptor’s modulating domain interacts with the transcriptional machinery (e.g. RNA polymerase) is attractive but unproven. There is some evidence that active transformation of the receptor facilitates its interaction with a ribonucleic acid moiety ( 116)) probably transfer RNA (3)) and that the half-lives ofcertain messenger RNA molecules may be affected-whether the latter phenomenon reflects a direct interaction between receptor and message, however, remains uncertain. Specific DNA sequences at the 5’ end of hormone-responsive genes form the ultimate target for activated receptor. Several of these ‘hormone response elements’ (HREs), or enhancers, may be present within the same gene, and these sequences may be situated as far as two kilobases apart from one another. Electron microscopy has suggested that DNA-bound receptor dimers undergo cooperative binding, thereby linking these distant HREs to form DNA loops which may then participate in interactions between other truns-acting transcriptional factors and gene promoter regions (200). Cellular oestrogen-responsiveness appears to be influenced by the configuration of DNA sequences adjacent to such promoters (18 1). Though traditionally regarded as markers of residual differentiation in breast cancer (2 18)) oestrogen receptors are now recognized to be markedly overexpressed in malignant compared with normal tissues (94) and to share structural homology with the product of the v-erb-A oncogene (70). Moreover, it is recognized that modification of c-erb-A to the oncogenic v-erb-A results in alteration of the receptor’s hormone-binding domain, indicating that this oncoprotein may play a central role in breast carcinogenesis by constitutively interfering with transcriptional regulation of important target genes (69)even in the absence of oestrogen! Since levels of oestrogen receptors in breast tumours vary inversely with those of epidermal growth factor receptors (172)-the latter being homologous to the v-erb-B oncogene product which cooperates with v-erb-A in inducing neoplastic transformation in a variety of experimental systems (16, 62, 98)-disease progression in viva may also be mediated by an interplay between cellular oncogenes. Yet,

HORMONE-RESPONSIVE

BREAST

CANCER

35

intriguing as such speculations are, we are still a long way from formulating a coherent gene-based algorithm of tumour evolution (52). Levels of progesterone receptor are rarely measurable in breast tumours lacking the oestrogen receptor (31). Since oestrogen appears to be required for synthesis of the progesterone receptor ( 130)) this finding is consistent with the view that expression of this receptor denotes functional integrity of the associated oestrogen receptor (84). Paradoxically, then, the recognition that progesterone receptor positivity correlates more specifically with clinical hormone-responsiveness than does presence of oestrogen receptor alone (58) now seems more relevant to the physiology of oestrogen action than to that of progesterone. Despite these advances, several anomalies indicate that our knowledge of hormoneDNA interaction remains incomplete. One controversial clinical study described oestrogen-induced growth stimulation of receptor-negative breast tumours (45); another study reported oestrogen-induced potentiation of cytotoxic cell kill in both receptor-positive and -negative breast cancer cell lines (90); oestrogen stimulation of receptor-negative nonmalignant cells has been documented in uivo (15) and in vitro (68); while the two largest clinical trials of adjuvant hormonal therapy demonstrated survival benefit in both receptor-positive and -negative disease (145,179). C onversely, failure ofreceptor-positive cells to respond uniformly to oestrogen stimulation has been associated with functionally abnormal receptors (143), tumour cell heterogeneity (180), ‘byp assing’ of hormone sensitivity by oncogene activation (loo), presence of growth inhibitors (192), or alterations of target cell chromatin structure (185). Such app arent inconsistencies may also arise due to errors in measuring either growth stimulation (128, 188) or receptor levels (159). Nonetheless, the gap between predicted outcome and actual response remains disconcertingly wide, implying the existence of corresponding gaps in traditional models of hormone action (19 1). Oestrogen

receptors

in clinical

practice

Much of the literature regarding clinical use of receptor determinations has been contradictory. The number of potential confounding variables between studies is large: these include differences in receptor assays, lack of inter-laboratory standardization, variable methods of handling and processing specimens, variations in tumour cellularity, and heterogeneity in receptor distribution. Equally numerous are the methods currently availtechable for assaying receptor levels (197), which include newer immunocytochemical niques that correlate impressively with traditional assays (106) as well as with clinical response to endocrine manipulation ( 13 1). State-of-the-art reproducibility using commercial ‘*“I-oestrogen kits is characterised by inter-assay variation of around 10% (197) but major discordances in receptor status are reported in only 3% of samples assayed (91). In contrast, coefficients of variation as high as 86% have been reported for quantitative receptor analyses obtained by multiple microsampling of a single tumour (206). This in turn contrasts with the high concordance rate between simultaneous assays of multiple metastatic sites when receptor status has been quantified simply as positive (greater than 10 fmol/mg protein) or negative (4). Nevertheless, given that absolute receptor level correlates with hormonal responsiveness more strongly than does receptor positivity alone (28), it is difficult to ignore the observed variations. At the same time, continuing methodologic uncertainties preclude a firm interpretation of intratumoural receptor heterogeneity on the basis of radioligand incorporation assays alone ( 149, 162) since variability may also arise due to differences in the cell/stroma ratio within biopsies (202).

36

R. J. EPSTEIN

Primary breast turnouts are more commonly oestrogen receptor-positive in older (60% of post-menopausal as compared with 30% of pre-menopausal) patients with well-differentiated disease. Receptor-positive tumours have also been reported to exhibit lower DNA ploidy than receptor-negative tumours (147), though many studies report only weak correlations (40) while others have found none at all (129). Using flow cytometric determination of S-phase fraction to assess tumour proliferation, receptor-positive tumours have been characterized as having lower proliferative rates (148); although this correlation has been reported to be stronger for progesterone than for oestrogen receptor status (139), the opposite has been reported for tumour proliferation rates measured by thymidine labelling (129). Involvement of regional lymph nodes in primary disease-an acknowledged major prognostic factordoes not appear to be strongly influenced by receptor status (32, 215). Hence, notwithstanding the claims made for the potential clinical utility of ploidy (77), S-phase fraction (134) and thymidine labelling index (133) as prognostic indicators in breast cancer, the oestrogen receptor remains the most commonly measured parameter-almost certainly because it is the variable most germane to clinical management decisions. Improved overall survival is associated with receptor-positive disease, and this seems most likely to reflect hormone-responsiveness following relapse (19). In one recent study, overall survival benefit in receptor-positive primary disease was restricted to a subset of patients with lymph node involvement (215). Despite early reports of improved diseasefree survival following mastectomy in receptor-positive patients (18, log), recent studies have failed to confirm any association of receptor status with this outcome (2, 27). Moreover, patients relapsing with receptor-positive disease unresponsive to hormone manipulation have identical overall survival to receptor-negative patients (86). Metastatic deposits generally exhibit similar receptor status to primary tumours when assayed simultaneously (81), though considerable discordance (3040%) has been reported between assays of primary disease and subsequent metastases (95, 171). The natural history of metastatic disease is influenced by receptor status, with receptor-positive disease showing predilection for bone while being inversely correlated with liver and brain recurrence (194). Furthermore, liver metastases tend to be unresponsive to hormone manipulation even in receptor-positive disease, whereas relatively ‘responsive’ sites of metastasis include bone, soft tissue and pleura (178). Responses are seen in 5CL60°/0 of oestrogen receptor-positive patients; approximately half of these will also be progesterone receptor-positive, and 75% of this subgroup will respond to hormonal manipulation (20). In patients who have responded to endocrine therapy, about 50% will respond to a subsequent hormonal manipulation on relapse (103). Median duration of response in previously untreated disease averages around 12 months (78). Numerous studies have shown that endocrine therapy markedly reduces hormone receptor levels in tissue assayed within three months of treatment discontinuation (146, 199, 203), though whether this reflects selective suppression of receptor-bearing cells or some less specific effect is not known with certainty. Cytotoxic therapy, on the other hand, does not reduce receptor levels (146). R esp onsiveness to cytotoxic therapy has been claimed to be more common in receptor-negative disease, but this is not associated with improved disease-free or overall survival ( 142) and is at variance with conclusions of other published reports (39) which indicate clearly that response to chemotherapy is independent of receptor status. Irradiation of breast cancer cells leads to dose-dependent reduction of receptor concentrations in vitro (96)) but the significance ofsuch a local effect in the context of systemic disease seems limited.

HORMONE-RESPONSIVE

Endocrine

BREAST

management

CANCER

of established

37

disease

Response to hormonal therapy is the major determinant of survival in breast cancer patients undergoing systemic treatment (157). A variety of such therapies have been successfully deployed, but antioestrogens, aromatase inhibitors and progestins have emerged as the dominant modalities. Ablative surgical management, though effective, has declined in popularity in recent years: hypophysectomy and adrenalectomy are now outmoded (210) while the long-assumed superiority of oophorectomy to antioestrogens for pre- and peri-menopausal patients now seems rather less certain (25, 93). Radiationinduced menopause is inconvenient, slow in onset of action, and relatively cost-ineffective, and cannot be recommended as routine first-line management for patients requiring palliation of symptomatic metastatic disease. Therapeutic use of androgens or oestrogens has seldom been necessary since the advent of alternative drugs with less morbidity. Corticosteroids are commonly used in palliation of metastatic breast cancer. As in other malignancies, steroids may be useful in cerebral metastases, nerve and spinal cord compression, pulmonary lymphangitic spread, hypercalcaemia, during and following irradiation, and as part of antiemetic management of chemotherapy-induced nausea. In breast cancer, however, glucocorticoids also appear to have an oncolytic effect which is best recognized in bone and soft tissue disease. Since these sites of recurrence correlate with receptor-positivity (29) and commonly regress with conventional hormone manipulation, this action of corticosteroid therapy may relate to its ACTH-inhibitory effect which leads to adrenal suppression with reduction of androgen secretion (17). Prednisolone has accordingly been advocated as a second-line hormonal modality for palliation of breast cancer in elderly patients (136) or as concomitant therapy in patients undergoing antioestrogen treatment (195); overall, however, corticosteroids continue to be most frequently prescribed in conjunction with cytotoxic chemotherapy (49). The survival improvement reported in pre-menopausal women with stage II primary breast cancer following adjuvant cytotoxic therapy (85) has been noted to occur predominantly in patients developing amenorrhoea in the context of receptor-positive disease ( 152). This is consistent with the mechanism of response being related to ovarian suppression (50, 170) rather than to cytotoxicity per se. Antioestrogen therapy, on the other hand, does not appear to function via an effect on ovarian function in pre-menopausal patients, since benefit may be seen in either hypophysectomised (105) or oophorectomised patients (175), while gonadotrophin levels are not consistently affected (65, 126). Nonetheless, the reported survival benefit of adjuvant antioestrogen therapy in pre-(as well as post-) menopausal breast cancer ( 145, 179)) can only enhance the credibility of an endocrine-based mechanism for the observed survival benefit of adjuvant chemotherapy.

Antioestrogens

as antineoplastic

agents

The only antioestrogen routinely used in breast cancer is tamoxifen, a non-steroidal aminoether derivative of polycyclic phenols. It is generally agreed that tamoxifen exerts its cytostatic effect by binding to the oestrogen receptor (11, 34); this effect of tamoxifen remains demonstrable even in the complete absence ofoestrogen, however, suggesting that its mechanism of action may not be strictly ‘antioestrogenic’ but rather mediated by receptor-based anti-growth factor activity (208). In contrast, the in vitro cytotoxic potential of the drug has been ascribed both to receptor-mediated ( 12) and non-receptor pathways,

38

R. J. EPSTEIN

the latter including those involved in polyamine (82) and protein (72) synthesis. Receptor conformation differs when bound to oestrogen or tamoxifen (57, 97), raising the possibility that antioestrogen-induced receptor modification directly influences receptor-DNA interaction (7). Cells treated with tamoxifen appear to become sequestered in the quiescent phase of the cell cycle (198), consistent with the observation that oestrogen-dependent tumours in experimental animals fail either to progress or regress following tamoxifen administration ( 15 1). However, this purely cytostatic mechanism of action remains consistent with the common observation ofclinical tumour regression, since increased ‘reactive’ stromal fibrosis has also been documented in animal tumours exhibiting no significant cytotoxic changes (150). More recently, antioestrogen therapy has been associated with reduction in tumour ploidy ( 10). T amoxifen has also been reported to induce hormonesensitive breast cancer cells to secrete transforming growth factor-p (108), a growth factor which inhibits proliferation of both receptor-positive and receptor-negative breast cancer in vitro. The clinical significance of this development is considerable, hinting as it does at the future potential for molecular biological manipulation of neoplastic growth. Administration of tamoxifen to pre-menopausal women leads to an increase in plasma oestradiol (184) while post-menopausal patients exhibit only an increase in cortisol (114) and reductions in LH and FSH concentrations (216). Male patients incur an increase in gonadotrophin, androgen and oestrogen levels (207). That the drug does have some agonist properties is suggested both by evidence of in vivo oestrogenicity (depression of post-menopausal gonadotrophins, induction of hormone-binding globulins, (61)) and by its biphasic effect on cell proliferation in vitro (166). Indeed, this latter observation has been mooted as an explanation for hormone-induced tumour ‘flare’-a syndrome originally reported with oestrogen use (79) but now more commonly precipitated by tamoxifen (33)-which typically manifests as bone pain, hypercalcaemia, or progression ofsoft-tissue disease in patients with hormone-responsive metastases. This intriguing phenomenon has recently been clarified by the finding that both oestrogens and antioestrogens stimulate bone resorbing activity in human breast cancer cells via the release of E series prostaglandins (205). A more difficult mechanistic issue is raised by the observation that breast cancer may respond to tamoxifen withdrawal following initial response (193). Breast cancer in males is particularly sensitive to antioestrogen therapy, being associated with a 45% overall response rate (121), similar to orchiectomy, and inducing remissions in occasional patients not responding to castration (80) or adrenalectomy (156). The undisputed pre-eminence of tamoxifen in hormonal management of breast cancer stems more from its relative lack of toxicity than from superior efficacy. Similar response rates have been reported using oestrogens (92), progestins (140, 154) and aminoglutethimide (120), but each ofthese agents is associated with significantly greater potential morbidity. Like antioestrogens, LHRH agonists induce remissions in pre-menopausal patients as efficiently as oophorectomy (74) but no decisive advantage has emerged to make this class of drugs competitive with more established therapies. No convincing additive benefit has been seen with loading-dose or high-dose tamoxifen (2 1, 169), or combination endocrine therapy (104), while a number of potentially counterproductive interactions between hormonal modalities have been characterized (5 1, 161). For the foreseeable future, then, tamoxifen is likely to remain the first-line hormonal manipulation for palliation of symptomatic metastatic disease. What of asymptomatic disease? Adjuvant antioestrogen therapy has been reported to prolong disease-free survival in post-menopausal women with both receptorand nodepositive disease (43, 59, 168). However, the two largest trials of adjuvant tamoxifen have

HORMONE-RESPONSIVE

BREAST

CANCER

39

suggested overall survival benefit in unselected patients-including pre-menopausal and receptor-negative disease (145, 179)-though optimal survival remains associated with strongly receptor-positive disease (179). M oreover, although no single trial has demonstrated survival benefit for node-negative patients treated in this fashion, an overview of published results has suggested that the survival benefits of adjuvant antioestrogen may also extend to this therapeutic subset. Since cytotoxic and hormonal therapy act via different mechanisms, the possibility of achieving more frequent and durable remissions with combined therapy seems attractive. In vitro evidence, however, suggests otherwise; tamoxifen has been reported to attenuate the cytotoxicity of commonly used chemotherapeutic agents in both receptor-positive and receptor-negative cell lines (89). These findings have been extended by numerous clinical trials of chemoendocrine therapy which have suggested either unchanged (123, 2 17) or reduced (59) overall survival in pre-menopausal patients, and unchanged (8) or reduced ( 112) survival in post-menopausal patients when compared with cohorts receiving sequential cytotoxic and hormonal therapy. Hence, the reported improvements in response rates and disease-free survival (1, 59, 123) seem unlikely to be realized as significant qualityof-life gains for patients treated with simultaneous chemoendocrine therapy.

Oestrogen

and the origins

of breast

cancer

Exogenous oestrogens are convincingly implicated in the pathogenesis of endometrial carcinoma in probands (22 1) and vaginal carcinoma in offspring (132) while being less firmly associated with mammary tumourigenesis (23). The role of endogenous oestrogen in mediating breast cancer risk is, however, more difficult to assess. Epidemiologic studies have identified high- and low-risk groups using hormonally relevant criteria (parity, menstrual history, oophorectomy), while studies of high-risk families also implicate hormonal factors as a possible basis for genetic predisposition (41)-notwithstanding that many of the reported results are inconsistent and the overall differences between groups small (201). Total plasma oestradiol levels do not vary with breast cancer risk in either pre- or post-menopausal women (26) though significant increases in the bioavailable oestradiol fraction have been reported to correlate with risk in both case-control (137) and cohort ( 138) studies. This is consistent with a model of breast carcinogenesis in which cancer risk is a function of ‘biological breast age’, a concept definable in terms of both chronological age and cumulative oestrogen exposure ( 160). Yet laboratory studies suggest that this epidemiologic model may be an oversimplification. Oestrogens may interact with mitotic spindles to induce aneuploidy in a non-random fashion in vitro (2 13)) though the pharmacological concentrations of oestrogen required make this observation of questionable relevance to human breast cancer. In vivo studies suggest that oestrogenic mitogenicity does not correlate with carcinogenicity (119) and that potentially mutagenic DNA adducts may be induced by both natural and synthetic oestrogens (117); cellular transformation induced in vitro by oestrogen does not, however, reliably predict tumourigenesis in vivo (118). Interestingly, the oestrogen metabolite 16-~hydroxyoestrone has been reported to be elevated in breast cancer patients (176) and has also been found to bind covalently to chromosomal histone proteins (219). These observations suggest that a genotoxic mechanism for oestrogen-mediated breast carcinogenesis should not be discounted, even though hard evidence for involvement of such a mechanism in the evolution of sporadic human carcinomas is weak (54).

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R. J. EPSTEIN

In contrast, promotion of human breast tumour cell growth is well-recognized to be oestrogen-dependent in vivo, even when such growth occurs independently of oestrogen in vitro (182), suggesting that loss of growth inhibition may be at least as important in tumourigenesis as is putative growth stimulation (127). Interpretation of such in vitro data is complicated by the recent finding that the tissue culture medium pH indicator phenol red has weak oestrogenic activity (14); h owever, cells grown in the presence of calf or human serum may also exhibit growth inhibition which is overcome by addition of oestrogen ( 153, 192). The role of receptor-positive stromal cells in mediating the hormone-responsiveness of epithelial cells has only recently begun to be elucidated. A cooperative effect of receptorpositive mesenchyme has been implicated in the stimulation of DNA synthesis in epithelial cells lacking oestrogen receptors in vivo (15), while re-expression of in vivo hormoneresponsiveness has been shown to be dependent upon stromal co-culture in vitro (38). In non-malignant breast epithelium, the presence of fibroblasts appears to be required for oestrogen-dependent cell proliferation or progesterone receptor synthesis (75)) while malignant breast cells may produce a fibroblast chemoattractant (64). The possibility that this latter protein could be platelet-derived growth factor (PDGF; the product of the c-k oncogene which is recognized to induce stromal fibrosis in several clinical syndromes) is strengthened by the finding that this growth factor is frequently expressed in breast cancer cell lines (158). Since breast cancer cells are not known to express PDGF receptors, the growth advantage represented by this phenotype must be mediated by a paracrine mechanism; the involvement of PDGF-regulated breast fibroblasts in such a positivefeedback loop would also help explain the scirrhous pathology of many human breast turnout-s. The recognition of widespread fibroblast abnormalities in preclinical breast cancer patients (9) further emphasises the critical role which stroma may play in the evolution of this malignancy. Oestrogen may induce synthesis of tumour-derived growth factors (44) in an autocrine manner. These growth factors have transforming growth factor-a-like activity (173) and appear to act via the epidermal growth factor receptor (167). Oestrogen-induced growth stimulation and tumourigenicity is bypassed by transfection of the v-Ha-ras oncogene without significant change in levels of receptor for oestrogen or transforming growth factor-a (48), suggesting that more subtle changes in oncogene expression may also come to be implicated in the inconsistent relationship between receptor measurement and hormone responsiveness.

The molecular

basis

of hormone-responsiveness

Enhanced expression of the c-Ha-rus oncogene has been reported to accompany oestrogeninduced growth of breast tumours in vivo, while tumour regression induced by hormone withdrawal may be preceded by suppression of the 21-kilodalton transforming protein encoded by this oncogene (88). Although chemical induction of breast tumours in animal models has been associated with point mutations in the c-Ha-rus locus (196), hormonedependency of both human and animal breast cancer correlates with increased production offunctionally ‘normal’ Y(ESprotein (46), casting doubts on the relevance of this chemically induced model to human breast cancer. Moreover, constant expression of ras protein does not appear necessary for maintenance of the transformed phenotype in human breast tumours (73). Heterogeneity of N-rus gene amplification has been noted in sublines of a

HORMONE-RESPONSIVE

BREAST

CANCER

41

human breast cancer cell parent line (67); as with c-myc (11 l), however, amplification of the oncogene is not a general feature of established breast cancer cell lines. In contrast, biopsy samples of human breast carcinomas commonly reveal amplification of the c-myc gene (55) and enhanced expression of the p-2 1 ras protein product-the latter correlating with disease stage but not with receptor status (124). Amplification of the WI oncogene, a member of the e&-B-like oncogene family, has also been proposed as a better indicator of disease outcome than oestrogen receptor status in lymph-node positive breast cancer patients (187). However, the heterogeneity of oncogene expression in clinical samples and the considerable overlap between levels of expression in benign and malignant specimens (214) suggest that oncogene probes for human breast cancer will remain a research tool for the immediate future. The timing of detectable events following oestrogen stimulation of responsive cells ranges widely. Increased density of microvilli has been documented on scanning electron microscopy within sixty seconds of oestrogen exposure (163), while induction of ~5’2 gene transcription is measurable within 15 minutes of stimulation (24). Physiological stimulation of c-myc expression peaks four hours after oestrogen exposure, and that of cHa-rus after eight hours (204); similarly, breast tumours exhibit reductions in at least four translation products within six hours of oophorectomy (87). In contrast, maximal stimulation of thymidine kinase (99) and dihydrofolate reductase (115) gene expression in vitro is only seen after 24 h oestrogen exposure, a similar delay to that reported for various oestrogen-regulated proteins (30, 2 12); one of these, an autostimulatory 52-kilodalton lysosomal protease ( 141), has been claimed to be a marker protein for high-risk premalignant mastopathies (63). Oestrogen has also been found to inhibit secretion of a specific glycoprotein, similar in size to one stimulated by antioestrogens (183). The diversity and probable interdependence of these events reinforces the impression that oestrogen acts by triggering a cascade of events, some ofwhich may involve activation of pre-existing transcription factors rather than simple dependence on new protein synthesis ( 125). Changes in the higher-order structure of DNA also accompany oestrogen stimulation. Physiological oestrogen-induced increases in chromatin accessibility occur in the vicinity of active gene sequences ( 101) w h i 1e regression of breast tumours due to hormone withdrawal is also associated with alterations in chromatin structure (66). Interestingly, some changes in local chromatin structure represent transient responses to steroid hormone stimulation while other more widespread changes persist following hormone withdrawal and cessation of hormone-specific transcription (220). The functional significance of these indirect structural observations remains to be clarified.

Future

treatment

strategies

in hormone-responsive

breast

cancer

An example of a novel approach to breast cancer management has been the use of tamoxifen synchronization followed by oestrogen ‘priming’ of receptor-positive tumour cells prior to cytotoxic therapy. This approach has been rationally based on in uitro evidence for increased cell kill in oestrogen-stimulated cells exposed to S-phase-active cytotoxic agents (36, 209); the in vivo practicability of such a synchronization strategy has been questioned (61), however, in view of the prolonged biological half-life of tamoxifen in breast cancer patients (56). Such doubts have proven to be well-founded, given that the promising initial reports of this approach (5, 155) h ave been followed by altogether more equivocal studies (37, 53). Yet the finding that the antineoplastic agent m-AMSA induces

42

R. J. EPSTEIN

increased DNA damage in oestrogen-stimulated breast cancer cells (222) suggests an alternative strategy which may be independent of cell synchronisation. m-AMSA is known to interact with the DNA-modifying enzyme topoisomerase II (144); mitoxantrone, a commonly used drug in advanced breast cancer, is now known to inhibit activity of this same enzyme in human breast cancer cells (42), while doxorubicin (a topoisomerase-IIinteractive drug frequently used for palliation of advanced disease) has also been recognized to be potentiated by oestrogen exposure (90). Since both topoisomerase II (165) and the oestrogen receptor (94) app ear to be more abundant in proliferating neoplastic cells than in normal tissue, these observations suggest new avenues for improving both the selectivity and efficacy of systemic therapy in this malignancy. The therapeutic plateau which has been reached in clinical oncology (6, 35) may be a valuable reminder as to the importance of a strong basic research foundation. We are currently well-placed to develop insights gained over the last few years: the structure of hormone receptor proteins, for example, may soon be known in sufficient detail to enable synthesis of analogues or interactive drugs; recognition that hormone and growth factor action are regulated by oncogene expression suggests strategies for manipulating cell behaviour at the molecular level; the characterization of transforming growth factor-b as an inhibitor of neoplastic cell proliferation raises the prospect of cloning the gene and producing the protein recombinantly for clinical trials; while the use of oestrogenic cell recruitment to potentiate the activity of topoisomerase-II-interactive drugs remains a largely unexplored therapeutic possibility. Although it is difficult to assess the feasibility of these possibilities for progress in breast cancer research, it is perhaps equally difficult to appreciate that such possibilities could not have been entertained as recently as five years ago. Whether the next decade will bring any significant improvement in the outlook for breast cancer patients may well depend on the extent to which clinicians and scientists mutually exploit the opportunities for collaboration in this exciting field.

Acknowledgements The author would like to thank Dr Mike Williams and Professor Norman Bleehen for constructive criticism of the manuscript. This work was supported by the Sir Robert Menzies Memorial Trust and in part by the Royal Australasian College of Physicians.

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