Owen W. J. Prall

c-Myc or Cyclin D1 Mimics Estrogen Effects on Cyclin E-Cdk2 Activation and Cell Cycle Reentry

ABSTRACT Estrogen-induced progression through G1 phase of the cell cycle is preceded by increased expression of the G1-phase regulatory proteins c-Myc and cyclin D1. To investigate the potential contribution of these proteins to estrogen action, we derived clonal MCF-7 breast cancer cell lines in which c-Myc or cyclin D1 was expressed under the control of the metal-inducible metallothionein promoter. Inducible expression of either c-Myc or cyclin D1 was sufficient for S-phase entry in cells previously arrested in G1 phase by pretreatment with ICI 182780, a potent estrogen antagonist. c-Myc expression was not accompanied by increased cyclin D1 expression or Cdk4 activation, nor was cyclin D1 induction accompanied by increases in c-Myc. Expression of c-Myc or cyclin D1 was sufficient to activate cyclin E-Cdk2 by promoting the formation of high-molecular-weight complexes lacking the cyclin-dependent kinase inhibitor p21, as has been described, following estrogen treatment. Interestingly, this was accompanied by an association between active cyclin E-Cdk2 complexes and hyperphosphorylated p130, identifying a previously undefined role for p130 in estrogen action. These data provide evidence for distinct c-Myc and cyclin D1 pathways in estrogen-induced mitogenesis which converge on or prior to the formation of active cyclin E-Cdk2-p130 complexes and loss of inactive cyclin E-Cdk2-p21 complexes, indicating a physiologically relevant role for the cyclin E binding motifs shared by p130 and p21.

Lycopene inhibition of cell cycle progression in breast and endometrial cancer cells is associated with reduction in cyclin D levels and retention of p27(Kip1) in the cyclin E-cdk2 complexes.

Numerous studies have demonstrated the anticancer activity of the tomato carotenoid, lycopene. However, the molecular mechanism of this action remains unknown. Lycopene inhibition of human breast and endometrial cancer cell growth is associated with inhibition of cell cycle progression at the G1 phase. In this study we determined the lycopene-mediated changes in the cell cycle machinery. Cells synchronized in the G1 phase by serum deprivation were treated with lycopene or vehicle and restimulated with 5% serum. Lycopene treatment decreased serum-induced phosphorylation of the retinoblastoma protein and related pocket proteins. This effect was associated with reduced cyclin-dependent kinase (cdk4 and cdk2) activities with no alterations in CDK protein levels. Lycopene caused a decrease in cyclin D1 and D3 levels whereas cyclin E levels did not change. The CDK inhibitor p21Cip1/Waf1 abundance was reduced while p27Kip1 levels were unaltered in comparison to control cells. Serum stimulation of control cells resulted in reduction in the p27 content in the cyclin E–cdk2 complex and its accumulation in the cyclin D1–cdk4 complex. This change in distribution was largely prevented by lycopene treatment. These results suggest that lycopene inhibits cell cycle progression via reduction of the cyclin D level and retention of p27 in cyclin E–cdk2, thus leading to inhibition of G1 CDK activities.

Estrogen regulation of cell cycle progression in breast cancer cells

Estrogens are potent mitogens in a number of target tissues including the mammary gland where they play a pivotal role in the development and progression of mammary carcinoma. The demonstration that estrogen-induced mitogenesis is associated with the recruitment of non-cycling, G0, cells into the cell cycle and an increased rate of progression through G1 phase, has focused attention on the estrogenic regulation of molecules with a known role in the control of G1-S phase progression. These experiments provide compelling evidence that estrogens regulate the expression and function of c-Myc and cyclin D1 and activate cyclin E-Cdk2 complexes, all of which are rate limiting for progression from G1 to S phase. Furthermore, these studies reveal a novel mechanism of activation of cyclin E-Cdk2 complexes whereby estrogens promote the formation of high molecular weight complexes lacking the CDK inhibitor p21. Inducible expression of either c-Myc or cyclin D1 can mimic the effects of estrogen in activating the cyclin E-Cdk2 complexes and promoting S phase entry, providing evidence for distinct c-Myc and cyclin D1 pathways in estrogen-induced mitogenesis which converge on the activation of cyclin E-Cdk2. These data provide new mechanistic insights into the known mitogenic effects of estrogens and identify potential downstream targets that contribute to their role in oncogenesis.

Estrogen and progestin regulation of cell cycle progression.

Estrogens and progesterone, acting via theirspecific nuclear receptors, are essential for normalmammary gland development and differentiated function.The molecular mechanisms through which these effects are mediated are not well defined, althoughsignificant recent progress has been made in linkingsteroid hormone action to cell cycle progression. Thisreview summarizes data identifying c-myc and cyclin D1 as major downstream targets of bothestrogenand progestin-stimulated cell cycle progressionin human breast cancer cells. Additionally, estrogeninduces the formation of high specific activity forms of the cyclin E-Cdk2 enzyme complex lacking thecyclin-dependent kinase (CDK)3 inhibitor, p21. Thedelayed growth inhibitory effects of progestins, whichare likely to be prerequisites for manifestation of their function in differentiation, alsoinvolve decreases in cyclin D1 and E gene expression andrecruitment of CDK inhibitors into cyclin D1-Cdk4 andcyclin E-Cdk2 complexes. Thus estrogens and progestins affect CDK function not only by effects oncyclin abundance but also by regulating the recruitmentof CDK inhibitors and, as yet undefined, additionalcomponents which determine the activity of the CDK complexes. These effects of estrogens andprogestins are likely to be major contributors to theirregulation of mammary epithelial cell proliferation anddifferentiation.

EMS1 gene expression in primary breast cancer: relationship to cyclin D1 and oestrogen receptor expression and patient survival.

The EMS1 and CCND1 genes at chromosome 11q13 are amplified in about 15% of primary breast cancers but appear to confer different phenotypes in ER positive and ER negative tumours. Since there are no published data on EMS1 expression in large series of breast cancers we examined the relationship of EMS1 expression with EMS1 gene copy number and expression of mRNAs for cyclin D1 and ER. In a subset of 129 patients, where matched tumour RNA and DNA was available, EMS1 mRNA overexpression was associated predominantly with gene amplification (P=0.0061), whereas cyclin D1 mRNA overexpression was not (P=0.3142). In a more extensive series of 351 breast cancers, there was no correlation between cyclin D1 and EMS1 expression in the EMS1 and cyclin D1 overexpressors (P=0.3503). Although an association between EMS1 mRNA expression and ER positivity was evident (P=0.0232), when the samples were divided into quartiles of EMS1 or cyclin D1 mRNA expression, the increase in the proportion of ER positive tumours in the ascending EMS1 mRNA quartiles was not statistically significant (P=0.0951). In marked contrast there was a significant stepwise increase in ER positivity in ascending quartiles of cyclin D1 mRNA (P=0.030). A potential explanation for this difference was provided by the observation that in ER positive breast cancer cells oestradiol treatment resulted in increased cyclin D1 gene expression but was without effect on EMS1. The relationship between EMS1 expression and clinical outcome was examined in a subset of 234 patients with median follow-up of 74 months. High EMS1 expression was associated with age >50 years (P=0.0001), postmenopausal status (P=0.0008), lymph node negativity (P=0.019) and an apparent trend for worse prognosis in the ER negative subgroup. These data demonstrate that overexpression of EMS1 mRNA is largely due to EMS1 gene amplification, is independent of cyclin D1 and ER expression and, in contrast to cyclin D1, is not regulated by oestrogen. Independent overexpression of these genes may confer different phenotypes and disease outcomes in breast cancer as has been inferred from recent studies of EMS1 and CCND1 gene amplification.

Antiestrogens and the Cell Cycle

Recognition of the involvement of estrogen in the growth of breast cancer stemmed from observations made a century ago, when it was shown that ovariectomy in cases of pre-menopausal breast cancer could lead to tumor regression (1). Subsequent research in experimental models of carcinogeninduced mammary cancer revealed that estrogen was essential for both the initiation and progression of the disease. These observations, together with the demonstration that some breast tumors had a specific binding protein for estrogen, the estrogen receptor (ER), and that ER status was correlated with response to endocrine therapy, provided the rationale for the introduction of the antiestrogen tamoxifen in the treatment of breast cancer (2).

Estrogen/Estrogen Antagonist Regulation of the Cell Cycle in Breast Cancer Cells

The role of estrogen in the growth of breast cancer was recognised over a century ago when it was shown that ovariectomy in premenopausal women with breast cancer resulted in tumor regression (Beatson 1896). Subsequent research showed that estrogen exerted its proliferative effects through a specific receptor (estrogen receptor-ER) and was essential for the initiation and progression of mammary cancer in experimental animals. This and other observations, such as the correlation between ER status of the tumor and a positive response to endocrine therapy, led to the development of estrogen antagonists (antiestrogens) for the treatment of breast cancer (Lerner and Jordan 1990). Tamoxifen, the antiestrogen most commonly employed in the treatment of hormone sensitive breast cancer, significantly decreases the rates of both disease recurrence and death (Early Breast Cancer Trialists’ Collaborative Group 1992; Early Breast Cancer Trialists’ Collaborative Group 1998; Fisher et al. 2001). However, tamoxifen therapy is limited by the frequent development of cellular resistance. In addition, synthetic nonsteroidal antiestrogens like tamoxifen possess both estrogen agonist and antagonist activity and as such have the potential to induce proliferative side effects in other reproductive organs such as the endometrium (MacGregor and Jordan 1998).

Estrogen Regulation of Cell Cycle Progression

Estrogens (Es) are essential for the normal development and physiological function of the female reproductive tract and secondary sex organs including the mammary gland. The development and progression of cancer in adult E target tissues is also dependent on E which has led to the effective use of E antagonists, particularly tamoxifen, in the treatment and potential prevention of breast cancer. The involvement of E in these major physiological and pathological processes is thought to be mediated via the potent mitogenic activity of Es. While these properties of Es have been long appreciated, it is only recently that the links between E action and the cell cycle machinery have begun to be dissected.