Qun Zhoua,c, Torsten Hoppb, Suzanne A. W. Fuquab and Patricia S. Steega, d
aWomen's Cancers Section, Laboratory of Pathology, Division of Clinical Sciences, National Cancer Institute, Bethesda, MD 20892
bBaylor Breast Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.
cCurrent Address: Genzyme Corp., Structure Protein Chemistry, PO Box 9322, Framingham, MA 01701-9322.
dTo whom correspondence should be addressed at Building 10, Room 2A33, NIH, Bethesda MD 20892.
Introduction.
This review will focus on the roles of cyclin D1 in the development of human breast cancer, until the point of tumor invasion. Why is this window of breast cancer progression important? Should we understand it on a molecular and signal transduction basis, the information can be used for the rational design of breast cancer prevention measures. Our current prevention armimentarium includes Tamoxifen (1) and Raloxifene trials are ongoing (2), but neither agent addresses the estrogen receptor (ER) negative tumor subpopulation. Recent prevention trials with retinoids showed potential promise for the premenopausal subpopulation, and the effect of retinoids in the context of cyclin D1 expression and function are discussed herein.
Many molecular alterations occur in premalignant or ductal carcinoma in situ (DCIS) human breast lesions, which represent indicators of high risk or may actually be precursor lesions. One of the most consistent is cyclin D1 overexpression, at the level of ductal carcinoma in situ (DCIS). This review attempts to integrate disparate data concerning cyclin D1's cell cycle promotion role within this window of cancer development. Moreover, we focus on novel, non- G1 cell cycle progression roles recently discovered for cyclin D1, such as estrogen response element transcriptional activation and induction of apoptosis, and their possible function in premalignancy. Excellent reviews of cyclin D1 biochemistry and its expression and function in invasive breast cancer and other cancer types can be found elsewhere (3-8).
Human premalignant and DCIS breast lesions.
The study of human biopsy premalignant and DCIS lesions, their molecular characteristics and the implications of these data for breast oncogenesis, is generally underappreciated in breast cancer research. Classes of lesions have been defined on histologic and cytologic features by pathologists; careful follow-up of patients with these lesions has provided risk estimates for their eventual development of invasive breast cancer. Proliferative disease without atypia includes moderate or florid hyperplasias of the usual type, intraductal pappilomas and sclerosing adenosis. In studies of DuPont and Page (9) these lesions were observed in 26% of benign breast biopsies and were associated with a mildly elevated risk for subsequent development of invasive breast cancer (relative risk 1.6, augmented to 2.1 with a family history). Atypical hyperplasias (ADH), found in 4% of benign biopsies, have some of the cytological and architectural features of carcinoma in situ, but lack the complete criteria for such a diagnosis. ADH is associated with a higher risk for development of invasive breast cancer (relative risk 4.4, augmented to 8.9 with positive family history). All of the risks listed above extend to either the ipsilateral or contralateral (i.e., either) breast. DCIS encompasses a heterogeneous group of lesions that have been classified several ways, but describe tumor cells confined within the basement membrane surrounding the duct/lobular system (rev. in (10)). Approximately 14% of breast cancers diagnosed in the US are DCIS (11). A DCIS biopsy confers an 8- to 10-fold increase in risk for the development of invasive breast cancer, predominately ipsilateral (same breast), and the high grade or comedo form of DCIS is associated with the highest risk. Confirmation and extension of these important trends have been reported by several laboratories (12-18).
Does "risk" as defined above mean causality? What is the evidence that any of the classes described are actual precursors of invasive breast cancer? The most definitive study examined allelic deletion or loss-of-heterozygosity (LOH) at multiple loci in a panel of premalignant lesions (19). A portion of the cohort contained premalignant lesions accompanied by invasive breast cancer. For those lesions, the simultaneous occurrence of LOH in the premalignant lesion and the invasive carcinoma would stand as supportive evidence that one eminated from the other. Indeed, of the lesions exhibiting greater than one LOH event, 37% of typical hyperplasias, 45% of ADH, 77% of low grade (noncomedo) DCIS and 80% of high grade (comedo) DCIS shared an LOH event with a synchronous invasive breast cancer. These data suggest that a proportion of each class, increasing with increasing risk assignment, may represent a true precursor lesion. In the subset of cases with multiple typical hyperplasia lesions in a cancerous breast, only one or two hyperplasias shared LOH with the synchronous cancer, indicating that the percentage of true precursor lesions may be much smaller. Similar results were recently reported by Amari et al. (20). A note of caution, however, comes from the work of Moinfar et al. (21), who examined LOH in DCIS and distant mesenchymal cells in the stroma. 73% of the cases displayed at least one LOH event in both the epithelial and mesenchymal components. Unless one postulates a common origin of stromal and DCIS cells, these data suggest the possibility that synchrogenicity of LOH events may be observable by chance alone, or due to gross structural chromosomal similarities such that mutational events find similar regions of chromosomes in different cells. Better estimates of causality may be obtained when we observe concordance of LOH at many loci. For DCIS, a study using comparative genomic hybridization has fulfilled these criteria, albeit with a low sample number (22). Of six DCIS cases with concurrent invasive cancer, 5/6 showed identical CGH profiles in the in situ and invasive components. This study raises another note of caution: DCIS associated with an invasive component exhibited different levels of alterations at 11q13 and 14q loci than "pure" (no invasive component) DCIS. Thus, data obtained with lesions in a field of invasive cancer may not apply en toto to "pure" lesions, the latter of which might represent the more relevant target of prevention research.
Most premalignant lesions are diagnosed as incidental findings in a biopsy and no further treatment is suggested. At the level of DCIS, patients have multiple treatment options. Mortality from DCIS, in the period of 1984-1989, was 0.7% within five years and 1.9% within 10 years (11). The NSABP has shown that lumpectomy (conservative surgery) and radiation has comparable survival statistics to mastectomy (23, 24). More recently the NSABP treated women with DCIS with lumpectomy and radiation and then randomized them to Tamoxifen or placebo, and reported a benefit of Tamoxifen on local recurrence rates (25). A meta analysis comparing multiple clinical trials, however, yielded a recurrence rate of 22% for conservative surgery alone, 8.9% for conservative surgery plus radiation, and 1.4% for mastectomy (26). The efficacy of radiation therapy was also correlated with the margin width of normal tissue surrounding the DCIS (27). As with many other aspects of breast cancer treatment, incontrovertible answers are generally not available to date.
Cyclin D expression in premalignant lesions and DCIS.
We first reported a cohort study of cyclin D mRNA expression using in situ hybridization on human premalignant and DCIS breast lesions (28). The probe shared homology to cyclins D1-D3. Approximately 80% of DCIS exhibited grains of hybridization higher than those over matched normal ductal/lobular units in the margin of the specimen. A similar trend was not observed for cyclin A. Lesions conferring less risk for the patient's development of invasive cancer, including ADH, typical hyperplasia, sclerosing adenosis and radial scar, exhibited cyclin D overexpression in 18% of cases. Quantitation of grains revealed that the minimum overexpression observed was approximately a 2-3 fold increase in grain intensity; many lesions were double this.
Cyclin D1 protein overexpression in DCIS, as compared to normal ducts, has now been reported by four groups using immunohistochemistry (29-32)(Table 1). As is typical with immunohistochemistry, the methods used for quantitating positivity varied, including staining intensity and percentage of positive cells. Another trend that is agreed upon is the presence of cyclin D1 gene amplification in a proportion of cyclin D1 overexpression cases, and protein overexpression without gene amplification in others. Simpson et al (31) performed a thorough evaluation of this question. Amplification was observed in 18% of the DCIS specimens, and was preferentially found in high grade lesions. The mean percentage of amplified cells in the lesions examined was 35-46%, depending on grade. Similar trends were observed by Vos et al (29). Given the demonstration of at least two different mechanisms for cyclin D1 protein overexpression in DCIS, it can be predicted that the selective pressure for this alteration is high. Based on the percentage of cells exhibiting cyclin D1 amplification, it is unlikely that this is an initiating event in breast cancer.
Additional trends are noted. Less frequent cyclin D1 overexpression was consistently observed in ADH. Both our study using in situ hybridization detection of mRNA levels (28) and the Gillett et al (32) study using immunohistochemistry found the major breakpoint for increased expression to lie between ADH and DCIS (Table 1). Given the fact that typical hyperplasia and ADH represent proliferative lesions, the data segragate cyclin D1 overexpression and certain forms of proliferation in vivo. Vos et al (29) used a different cutpoint in immunohistochemistry, where the presence of 5% positively stained nuclei signaled a positive tumor. Under this system DCIS exhibited greater cyclin S1 postivitity than ADH. In no study did cyclin D1 overexpression significantly correlate with grade of DCIS, defined by several different scoring systems. In the study of Vos et al (29), cyclin D1 overexpression was concentrated among ER+ tumors (P=0.02). The study of Gillette et al (32) did not report raw data, but the same trend correlation of cyclin D1 and ER expression just missed statistical significance (p=0.06).
Many molecular events are altered in DCIS, including growth factor receptors, angiogenesis, apoptosis, and other signaling events. What makes the cyclin D1 data of notice? Many of the altered events are principally found in high grade or comedo DCIS, which confers the highest risk for invasive cancer and is most frequently thought to be a true precursor. As a non-exhaustive list of examples, comedo or high grade DCIS (as opposed to noncomedo or low grade DCIS) exhibits increased Her-2/erb-B-2 expression (33), dense microvessel patterns indicative of high angiogenesis (34) (35, 36), VEGF angiogenesis factor production (37, 38), p21 expression (39), p53 expression (40-44), aneuploidy (35), apoptosis (45), reduced nm23 (46). The overexpression of cyclin D1 at a different risk stratification, at the boundary of ADH and any form of DCIS, suggests a different role. At this boundary risk estimates for the development of invasive breast cancer shift from both breasts to ipsilateral, suggesting that the risk stratification of DCIS may represent direct causal influences.
Cyclin D1 function in G1 cell cycle progression, and beyond.
Cyclin D1 is best studied as a regulatory subunit for two cyclin dependent kinases, cdk4 and cdk6, in the G1 phase of the cell cycle (rev. in (6, 47)). The G1 phase preceeds S phase in which DNA is replicated. Cyclin D1 expression levels are thought to act as a sensor for various mitogenic stimuli, rising to a point where sufficient cdk4 is activated and G1 progresses. The best studied target of cyclin D-cdk4 is Rb, which releases the E2F transcription factor upon phosphorylation, permitting expression of many proliferation related genes. Other RB like "pocket" proteins include p130 and p107 and may represent cyclin D1-cdk4 targets. One of the E2F induced genes is cyclin E, a regulatory subunit for cdk2, whose activation is required to complete G1. Other Rb functions include the release of histone deacetylase ( rev. in (48)), which can influence the transcription of critical genes through chromatin structural remodeling.
The action of the cyclin D-cdk complex is also influenced by levels of cyclin dependent kinase inhibitors (CKI), of which there are two general classes (47). The ink4 family, consisting of p16, p15, p18 and p19, share a common anykin repeat domain, and compete with cyclin D1 for binding to the cdk. The Cip/Kip inhibitors, consisting of p21, p27 and p57, bind all G1 cyclin-cdk complexes, although reports of preference for cdk2 exist. An important point with regard to the Cip/Kip family is that their binding to cyclin D1-cdk4 may serve to titrate out "free" Cip/Kip inhibitors, thereby facilitating cyclin E-cdk2 function. p21 and p27 binding to the cyclin D1-cdk4 complex serves additional functions, as it has been reported to be required for enzymatic activation (49, 50).
Tumor growth is more than proliferation via the cell cycle. In a brilliant review, Hanahan and Weinberg (51) described six essential traits of tumor formation, self sufficiency in growth signals, insensitivity to anti-growth signals, evading apoptosis, sustained angiogenesis, limitless replicative potential and tumor invasion and metastasis. Cyclin D1 overexpression may contribute to two of these functions: Increased levels of cyclin D1 are thought to activate cdk4 (whose expression is not thought to be limiting), and high cyclin D1 levels may produce sufficient cdk4 activity that G1 progression occurs independently of mitogenic stimulation (47). High cyclin D1 levels may also titrate out free Cip/kip inhibitors, thus making the cell insensitive to their action.
It is important to understand that cyclin D1 can and does participate in pathways outside of G1 cdk activation. Some of these pathways are similar to cyclin D1 (via cdk4) binding of Cip/kips, serving a titration or sequestration function. These binding partners include Rb (52) and Proliferating cell nuclear antigen (PCNA, a subunit of DNA polymerase)(53). Other reports link cyclin D1 to distinct biological phenotypes involved in cancer, such as gene amplification (54, 55), transcription (56, 57), apoptosis and estrogen responsive gene transcription, the latter two of which will be discussed below.
One way in which the role of cyclin D1 overexpression can be studied in breast oncogenesis is the use of transgenic mice. Overexpression of cyclin D1 via a MMTV-LTR in the mammary gland produced hyperplasias in all lines and 8/12 mice produced mammary adenocarcinomas with a mean latency of 551 days, considered long for this promoter (58). The long latency suggests the participation of other genetic events. The differences in the phenotypic consequences of cyclin D1 overexpression in transgenic animals and human cohort studies/human cell line transfection studies is distinct in several respects: First, cyclin D1 transgenic mice develop mammary tumors, while no cyclin D1 transfection study using an immortal, nontumorigenic human breast cell line has shown in vivo tumorigeneicity. Second, cyclin D1 transgenics develop hyperplasias. Cyclin D1 overexpression was infrequently associated with typical or atypical hyperplasia in human cohort studies. These observations underscore the importance of study of human model systems and lesion cohorts, and impress upon us that a mouse is not a woman.
Cyclin D1 transfection into nontumorigenic breast cell lines: Growth.
In human cells, the simplest way to understand the biochemistry and cell biology of cyclin D1 has been to transfect it into the most "normal" breast cell lines that can be obtained. A comparison of five transfection studies, four of which used human breast cell lines, is presented on Table 2 (59-63). Where densitometry was reported, overexpression of cyclin D1 was in the 5-10 fold range. This level was comparable to the degree of cyclin D mRNA overexpression observed in a DCIS tumor cohort (28). At this point, the similarities between the studies end.
For a protein with a well studied function of binding and activating a cyclin dependent kinase in G1 progression, the effect of cyclin D1 overexpression on any aspect of growth is unclear. Anchorage-dependent proliferation was measured by counting cell numbers, doubling times, cell cycle progression and/or saturation densities. In two studies aspects of proliferation were decreased (Table 2); in both studies multiple measurements were reported (saturation density, cell cycle progression), ensuring the validity of the trend reported. Increased proliferation was observed in two other studies, generally two-fold or less over a 5-7 day assay. We reported a minimal increase in proliferation on day 5, and a consequent decrease on day 9 of culture in cyclin D1 transfectants (62). The anchorage-independent growth of cyclin D1 transfectants was increased in one study, decreased in two, and undetectable in one (Table 2). Tumorigenicity in vivo was reported in two studies, and in no case did cyclin D1 overexpression augment tumorigenesis (59, 62).
Resolution of the five disparate studies is difficult. One explanation for the differences observed lies in data from Han et al (60) and Zhou et al. (62). Overexpression of cyclin D1 in murine HC11 cells resulted in the concurrent overexpression of other signal pathway members including cyclins A and E, cdk2, and p27. How to calculate the overall impact of these positive and negative proliferation regulators is a difficult question. The increased p27 was proposed to contribute to the reduced anchorage-dependent and –independent proliferation rates (60).
Based on these detailed observations in the HC11 line, the MCF10A control and cyclin D1 transfectants were screened for alterations in multiple signal transduction pathway proteins; cyclin D1 expression was elevated without concurrent detectable changes (62). Figure 1 diagrams the effects of cyclin D1 overexpression on aspects of growth in vitro in this model system. A minimal increase, followed by the minimal decrease in anchorage dependent growth in vitro was observed for the cyclin D1 transfectants. In contrast to anchorage-dependent growth data, a significant augmentation of anchorage-independent growth in soft agar or methylcellulose cultures by the MCF10A cyclin D1 transfectants was observed (62). The latter cultures permitted harvest of the cells for analysis. The cyclin D1 transfectants under anchorage independent culture conditions exhibited increased G1-S cell cycle progression. The control- and cyclin D1 transfectants exhibited comparable cdk4 activities, but the latter showed significantly increased cdk2 activities which function late in G1. Upon comparison of protein expression patterns under anchorage-dependent and –independent conditions, we observed decreased p21 inhibitor expression by all of the MCF10A cell lines when placed in anchorage-independent conditions. Thus two changes, increased cyclin D1, which could bind p21 in a complex, and overall lower p21 levels, may have resulted in its titration to a point where cyclin A-cdk2 activity was increased in anchorage-independent growth.
Other potential contributors to the variable data may lie in the nature of the cell lines used. Growth requires multiple pathways in addition to cyclin-cdk-inhibitors (rev. in (51)), and these pathways require interacting proteins for their completion. Thus the competency of other necessary signaling pathways may differ among the cell lines reported, contributing to the variability observed. Two conclusions are evident: (1) When carefully controlled conditions are used such that other G1 proteins are expressed at constant levels, cyclin D1 overexpression contributes to a significant increase in anchorage-independent growth, which is a hallmark of breast oncogenesis. (2) The data indicate that proliferation is a tightly controlled event, and that cyclin D1 overexpression amidst the heterogeneous cellular milleu does not consistently override these controls in vitro or in vivo.
Cyclin D1 and transcriptional activation: The estrogen receptor connection.
One of the underappreciated surprises in the cyclin D1 literature concerns its ability to transactivate estrogen receptor (ER) response elements, independent of its cdk activating functions. Zwijsen et al (64) initially reported that cyclin D1 enhanced transcription of estrogen response elements (EREs) in reporter constructs. Neither cdk4, nor cyclins D2, D3,E, A, B1 or B2 exhibited a similar activity. Transactivation of EREs was observed without estrogen, although the addition of hormone induced a synergistic effect. No effect was observed on transactivation of another member of the steroid family, the glucocorticoid receptor. Cyclin D1 bound the EF portion of the unliganded ER, which apparently facilitated enhanced ER binding to the ERE. The EF region of the ER is known to bind heat shock proteins and other transcriptional machinery proteins, suggesting their involvement. This mechanism was proposed to enable cells to bypass the need for estrogen, and may contribute to estrogen independent breast oncogenesis.
On the heels of this remarkable paper came an equally intelligent independent confirmation and extension of the data, from Neumann et al (65). A cyclin D1-ER complex, devoid of cdk4 and estrogen, transactivated EREs. Three areas of difference were reported by the two groups, which may be translationally important: a) the synergistic effect of estrogen is not agreed upon; b) Neumann et al (65) observed an inhibitory effect of tamoxifen and ICI 182,780 on cyclin D1-ER transactivation, while Zwijsen et al. (64) did not; c) the ability of cyclins D2 and D3 to participate was debated. With regard to point b, the ability of tamoxifen to inhibit this transactivation, to the extent that it occurs in vivo, is of paramount importance. If the cyclin D1-ER transactivational activity occurs in vivo and is independent of tamoxifen, it may constitute an important mechanism of tamoxifen resistance.
Two additional papers have attempted to shed light on the mechanism of cyclin D1-ER interaction, and suggest that recruitment of co-activating proteins may be important. Zwijsen et al. (66) reported that cyclin D1 binds to the SRC-1 family of transcriptional co-activators and factilitates their recruitment to ER. Distinct from this group, McMahon et al. (67) reported that cyclin D1 bound the p300/CREB-binding protein associated protein (P/CAF), a histone acetyltransferase, and facilitated its interaction with ER. It appears that cyclin D1 stimulates transactivation of other DNA motifs as well. Inoue and Sherr (56) reported that cyclin D1 binds the DMP1 transcription factor and antagonizes its ability to transactivate proliferation related gene expression. Again, this interaction was independent of cdks. A cyclin D1 complex with TAF(II)250 was reported (57), which may regulate Sp1 mediated transcription. Clearly, the limits of cyclin D1 transactivational potential have not been defined, in terms of the proteins bound, the transcriptional motifs involved, and the phenotypic consequences.
Cyclin D1 induced ERE transactivation in the MCF10A model system.
MCF10A cells are very poor in ER (68). Our RT-PCR data using control- and cyclin D1 transfected MCF-10A clones detected ER-a at roughly comparable levels under normal, serum free and hormone supplemented culture conditions. ER-b mRNA was at the limit of detection (data not shown). Two clonal cell lines, the C3 control transfectant and the D2 cyclin D1 transfectant of MCF-10A (62), were cotransfected with a vitellogenin (vit)-ERE(2)-tk-Luciferase reporter construct and a b-galactosidase construct as a normalization control (Figure 2). Transactivation of an ERE was minimal in the control transfectant in the absence of estradiol, and increased three fold in response to hormone. The cyclin D1 transfectant exhibited a slight but interesting increase in unstimulated transactivation, and was five-fold stimulated by exogenous estradiol.
A variation of the MCF10A model system was employed to elevate the ER expression. In the experiment shown in Figure 3 one control transfected clone (C3) and three cyclin D1 MCF10A transfected clones (D1-D3) (62) were cotransfected with the vit-ERE-tk-Luciferase reporter construct as well as a pC-ER estrogen receptor expression construct, and reporter activity analysed. Now at higher levels of ER expression, a 2-4 fold increased transactivation of ERE is apparent in the cyclin D1 transfectants in the absence of hormone. A further increase in response to estradiol was observed in all clonal lines. Notably, addition of Tamoxifen reduced the ERE transactivation of the cyclin D1 transfectants to a level at or below its unstimulated activity, but this level remained above that of the unstimulated control transfectant. These trends will require confirmation and extension with different levels of ER expression, concentrations of hormone and antagonists, etc. However, they suggest the possibility that cyclin D1 overexpression in an ER poor or adequate environment may contribute to ERE transactivation in nontumorigenic breast cells.
Cyclin D1 transfection into nontumorigenic breast cell lines: Apoptosis.
Precedent for a causal connection between cyclin D1 expression and apoptosis has been published in neural and other systems (69-71). Cyclin D1 stimulation of apoptosis has been confirmed in multiple immortal, nontumorigenic breast line transfections (Table 2). Han et al. (60) reported that apoptosis was 4-8 fold higher in the cyclin D1 transfectants after 3d of normal culture conditions; serum deprivation lead to 30-fold higher induction of apoptosis in the cyclin D1 transfectants. The cyclin D1 transfectants of both lines exhibited increased apoptosis in response to other apoptosis inducing drugs including the PKC inhibitors CGP41251, Ro31-8220, calphostin C, and the DNA synthesis inhibitor hydroxyurea. It must be remembered that other G1 cell cycle proteins varied in the HC11 model system, and that the apoptotic phenotype cannot be assigned solely to cyclin D1 therein.
We arrived at a similar conclusion in the MCF10A model system by examining the context in which patients with DCIS are treated. Clinical trials indicated that, for patients with DCIS, lumpectomy and radiation resulted in local recurrence rates that favored lumpectomy alone (23, 24). The effect of radiation was examined on the major cyclin D1 phenotype observed in MCF-10A cells, anchorage independent growth (Figure 4). The cyclin D1 transfectants were several- to ten-fold more sensitive to 1-8 Gy doses of radiation, in terms of inhibition of colonization, than were control transfectants (72). Doses of 1-2 Gy are repeatedly administered in breast cancer treatment (73). The effect of repeated radiation doses on colonization has not yet been determined. Harvest of cells from methylcellulose anchorage independent cultures indicated that 8 Gy irradiated cultures failed to exhibit the traditional G1-S cell cycle progression. A prominent G2 cell cycle expression characteristic of irradiated cells was evident in both control- and cyclin D1 transfectants. Flow cytometric analysis of Annexin V for apoptosis, however, revealed a specific increase in the irradiated cyclin D1 transfectants (72).
Given the correlation of cyclin D1 overexpression and stimulation of apoptosis, the role of specific apoptosis inducers in colonization was investigated. Two families of apoptosis inducers were tested, the Apo-2L/TRAIL family (74, 75), and TNF-a family. Apo-2L/TRAIL, at concentrations of 50-200 ng/ml, preferentially inhibited the colonization of cyclin D1 transfectants. TNF-a exerted an equivalent inhibitory effect on the colonization of both control and cyclin D1 transfectants, providing the first evidence of specificity in this pathway. It was therefore reasoned that receptor-ligand interactions may be involved. RT-PCR amplification of the Apo2L ligand and receptors from methylcellulose cultures was performed (72). Apo-2L ligand production was similar in all cultures examined, and only minor differences were observed in the expression of decoy receptors, DcR1 and DcR2. Of the two functional receptors for Apo2L, the DR5 receptor was virtually undetectable in unirradiated cyclin D1 transfectants, but present in controls. Thus, the increased colonization of cyclin D1 transfectants may not only relate to increased G1 progression and ERE transactivation, but may also represent a lack of apoptosis via DR5. Upon irradiation, DR5 expression was increased in both control- and cyclin D1 transfectants, but the amount of increase was quantitatively higher in the cyclin D1 transfectants, possibly rendering them more sensitive to Apo2L.
An intriguing question is to what degree the Apo2L pathway mediates the effects of radiation in early breast cancer? In our model system, we added anti-Apo2L to colonization cultures prior to semi-solification and irradiation to test this question. Anti-Apo2L, but not a control antibody, increased the colonization of cyclin D1 transfectants upon irradiation: For instance, at clinically relevant doses of radiation, cyclin D1 transfectants produced 347 colonies/culture, which was decreased by 1 Gy to 288 (83% of control); anti-Apo2L restored colonization to 356 (103% of control). At higher, but relevant doses the effect was partial: 2 Gy decreased colonization to 205 (59% of control) and anti-Apo2L raised this value to 285 (82% control). The data suggest that the Apo-2L pathway may be one contributor to radiation mediated cell death.
Several interesting questions eminate from these data. First, given the heterogeneity seen in transfection model systems and DCIS cohort studies, what is the relevance of these data to actual breast DCIS? A cohort study of the Apo2L receptors and ligand would be instructive in this regard when antibodies functional in formalin fixed, paraffin embedded tissue are available. For those patients who recur locally after conservative surgery and radiation for DCIS, were their tumors deficient in cyclin D1 expression? A very recent paper studying early breast cancer provides a first clue: A case-control study of 98 patients with and without local recurrence after lumpectomy and radiation therapy were investigated for cyclin D1 expresssion in the primary tumor. Low cyclin D1 correlated with ipsilateral breast tumor recurrence (p = 0.001) (76).
What is the relevance of the apoptotic phenotype in DCIS?
If DCIS is viewed under the microscope as an overabundance of cells within the breast duct, what is the significance of cyclin D1 stimulation of apoptosis? This phenoytpe appears counter-intuitive to the accumulation of tumor cells, and one wonders whether the transfection model system data is relevant in vivo.
Several cohort studies have identified apoptosis in DCIS (45, 77). But to what extent does it meaningfully contribute to the development and progression of breast cancer? A remarkable study of apoptosis and proliferation, measured by careful morphological criteria side-by-side in a breast tumor cohort, sheds light on this question. Mommers et al. (78) quantitated the percentage of apoptotic (A) and mitotic (M) cells in 72 premalignant/DCIS lesions and 103 invasive breast carcinomas. Starting with the median values, several trends are evident: Mitosis in low grade DCIS (0.5) was comparable to that of lesions conferring a lower risk (ADH was 0.7). High grade DCIS exhibited higher mitosis (2.9). Apoptosis in the DCIS lesions was prominent, 3.8 in low grade DCIS, leading to a M/A of 0.13, and 23.7 in high grade DCIS, with a M/A of 0.2. Thus, by morphological criteria, apoptosis is a frequent and potentially significant feature of DCIS. How do cells then accumulate into the specimens we observe in pathology labs? The actual ratios in this study are probably unimportant as they represent processes that may take different amounts of time to complete. Thus, none of the ratios suggest a net loss of cells, but a level of one process vs. the other. One possibility is that this is where the long times for breast cancer development may be important. If proliferation is just slightly more frequent than apoptosis, cell accumulation may take years and additional molecular alterations. Two additional points from this paper are noteworthy: First, it is with invasion, that proliferation outdistances apoptosis: the M/A ratio of invasive breast carcinomas was 0.57 for well differentiated and 0.52 for poorly differentiated invasive carcinomas. Zaugg and Bodis (77) observed a similar trend in a review of the literature. Second, the medians listed are accompanied by wide variations from specimen to specimen, indicating tremendous phenotypic and biochemical heterogeneity that must be reconciled. If apoptosis is present in breast premalignancy and slows down the accumulation of cells, we may indeed have a long window of opportunity to use apoptosis related prevention strategies on women at high risk. Besides the comparitors already described from this paper, another statistic supports this point: Apoptosis was significant in other classes of breast premalignancy: 6.5 in ADH and 3.7 in typical hyperplasia.
Prevention Strategies:
Several translational strategies emerge from the preceding data. First, laboratories and companies are attempting to interfere with the cyclin-cdk association/activation to block cell cycle progression in invasive breast cancer. If nontoxic, such strategies can be considered for prevention. A variety of peptides mimicking p16 and E2F have been reported (79-81). A new drug, Flavopiridol, is currently in Phase II trial. Its major effect is thought to be cdk inhibition (82). Limiting toxicities in Phase I trials were diarrhea, suggesting that at lower doses it could be well tolerated if effective. It should be noted that mechanisms designed to interfere with cyclin-cdk activation may ignore other functions of cyclin D1 such as its transcriptional and apoptotic roles, and thus miss several potentially important phenotypes.
Efforts to down-regulate cyclin D1 expression are also underway, and would be expected to impact the wider range of cyclin D1 induced phenotypes. We and others published in vitro data suggesting that retinoids reduce cyclin D1 expression in human breast carcinoma cell lines (83). Retinoids affect other phenotypes germane to prevention (84, 85). Their use as a potential breast cancer prevention agent was tested in one Italian trial using fenretinide, a less toxic analog, with second breast malignancy as an endpoint (86). While fenretinide was ineffective at prevention of ipsilateral or contralateral breast cancer overall, subsequent analysis of the pre- and postmenopausal subgroups revealed striking differences: For premenopausal women the adjusted hazard ratio for contralateral cancer was 0.66 (95% CI 0.41-1.07); in postmenopausal women fenretinide correlated with an increased hazard. Thus an interplay between retinoid biochemical pathways and the hormonal milleau was suggested. The signaling mechanism of retinoids is complex, and that of fenretinide is incompletely known (85, 87, 88). The expression patterns of the major retinoid receptors, RARs and RXRs in breast premalignant and DCIS lesions have been reported (89-91), but differing methodologies prevent a simple collation of the data. Other retinoid receptor ligands also show promise against breast cancer in model systems. Retinoid-Tamoxifen combinations have been tested in model systems as well. In the rat NMU system, a synergistic inhibitory effect was observed (92), while our data in breast carcinoma cell lines showed no synergy in inhibiting cyclin D1 expression (83). Pilot trials of this combination in a prevention setting have been conducted (93).
Until the cyclin D1-apoptosis connection was published, all prevention efforts focused on eliminating cyclin D1 expression. However, if cyclin D1 overexpression in premalignancy enhances the induction of apoptosis, can we use tolerable apoptotic stimuli to eliminate the premalignant cells from the ductal system? Our data with Apo-2/TRAIL speak to this possibility (72). While many apoptosis agents are thought to elicit broad based toxicity, decoy receptors for Apo-2L/TRAIL, which bind ligand but do not signal, are found on many normal cells and may protect them from ligand induced apoptosis. While Apo-2L/TRAIL was well tolerated in primate studies, a recent report of hepatic toxicity using a tagged form indicates the need for further study (94). If a tolerable form is found, the possibility of local delivery through cannulated ducts may not be in the star wars future, and would further minimize general toxicity.
A trend in chemoprevention research is the identification of intermediate endpoints or biomarkers, which can serve as endpoints for the conduct of quicker prevention trials ( rev. in (95, 96)). The emerging literature on the multiple functions of cyclin D1 questions its value as a marker of progression. Indeed, when viewed as a paradigm, one wonders whether most markers except those that are clear initiating oncogenes would fall into the same category with comprehensive study.
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Table 1. Cyclin D overexpression in DCIS: Summary of cohort studies.
| Reporta: | Overexpression Criteria: | % Postive Tumors: | % Positive DCIS b: | |||
| ADH | DCIS | High | Int. | Low | ||
| Weinstat-Saslow | mRNA grain intensity | 18 | 80 | 87 | 76 | |
| Simpson | IHC % + cells | 50 | 39 | 54 | 57 | |
| Gillett | IHC Intensity in majority of cells | 11 | 64 | 22 | 30 | 50 |
| Vos | IHC % + cells and intensity | 21 | 9 | 29 | 19 | |
| Alle | IHC > 5% + cells | 39 | 46 | 48 | 43 | |
aReferences (28-32) bTumor grade |
||||||
Table 2. Comparison of five studies of cyclin D1 overexpression in immortalized, nontumorigenic breast epithelial cells a.
| Studyb: | 1 | 2 | 3 | 4 | 5 |
| Cell Line: | Murine HC11 | Human HBL100 | Human HB4a | MCF10A | Human MCF10A |
| Achorage Dependent Growth: | |||||
| Cell Number: | Inc.c | Inc. | Inc. & Dec. | ||
| Saturation Density: | Dec. | Dec. | |||
| Doubling Time: | Inc. | Dec. | |||
| G0-G1: | Inc. | Dec. | Dec. | = | |
| Achorage Independent Growth: | |||||
| Dec. | Dec. | Undet. | Inc. | ||
| Differentiation: | |||||
| Inc. | |||||
| Apoptosis: | |||||
| Inc. | Inc. | Inc. | |||
| Expression: | |||||
| Cyclin D1 | Inc. 8x | Inc. 5-7x | Inc. | Inc. | Inc. 5-10x |
| Cyclin D2 | = | Undet. | |||
| Cyclin D3 | = | = | |||
| Cyclin A | Inc. | = | = | ||
| Cyclin E | Inc. | = | |||
| Cdk2 | Inc. | = | |||
| Cdk4 | = | = | |||
| Cdk6 | = | ||||
| P21 | Undet. | = | |||
| P27 | Inc. | Undet. | |||
| P16 | Undet. | Undet. | |||
| P53 | = | ||||
Figure Legends:
Figure 1. Schematic diagram of anchorage-dependent and –independent growth of control- and cyclin D1 transfectants of the MCF10A human breast cell line. Increased cyclin D1 in anchorage-dependent growth failed to alter either cdk4 or cdk2 activity as compared to controls. In anchorage-independent growth, decreased p21 inhibitor expression was observed in control- and cyclin D1 transfectants. The combination of increased cyclin D1 and decreased p21 resulted in enhanced cdk2 activity and increased colonization.
Figure 2:MCF10A cells stably expressing cyclin D1 exhibit an increased response to estrogen on a vitellogenin luciferase reporter.
Clonal cell lines stably transfected with vector alone (C3) or a cyclin D1 expression vector (D2) were transiently transfected with the vitellogenin reporter gene and tested for transcriptional activation in the absence (vehicle only) or presence of estradiol. Transfections were normalized for efficiency by cotransfecting an expression plasmid containing b-galactosidase. A representative assay is shown in which triplicate transfections were performed.
Figure 3: Expression of ERa increases basal activity of the vitellogenin reporter gene in MCF-10A cells stably transfected with cyclin D1. Clonal cell lines stably transfected with vector alone (C3) or a cyclin D1 expression vector (D2, D3, D4) were transiently cotransfected with an expression plasmid containing the human ERa and the vitellogenin luciferase reporter gene. Transcriptional activation was tested in the presence of vehicle only, estradiol, or tamoxifen. Transfections were normalized for efficiency by cotransfecting an expression plasmid containing b-galactosidase. A representative assay is shown in which triplicate transfections were performed.
Figure 4. Preferential inhibition of cyclin D1 transfectant colonization by radiation. Control- (C1, C2, C3) or cyclin D1 transfected (D1, D2, D3) clones of MCF-10A were cultured in soft agar and irradiated with the indicated Gy of g-irradiation immediately after plating. The number of colonies two weeks later were counted. Above each bar is the percentage of unirradiated colonization for that transfectant (72).
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