Chromosomal Instability A New Paradigm for Estrogeninduced Oncogenesis

Chemo Secrets From a Breast Cancer Survivor

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Jonathan J. Li, Jeffrey Salisbury, and Sara Antonia Li Introduction

Human sporadic breast cancer (BC) comprises >90% of all BC cases whereas familial BC is less than 10% (1). Despite its likely multifactorial origin, there is now pervasive evidence from epidemiological and animal studies, developed over the past several decades, that the causation of human sporadic BC primarily involves female sex hormones, particularly estrogens (Es) (1-8). This view is consistent with long standing epidemiological data relating extended exposure to Es and elevated BC risk, such as early first menarche, late age at menopause, nulliparity, late age at full-term pregnancy, and absence of lactation (5, 6). These BC risk factors are all related to pre-menopausal women. Moreover, all ofthe well-established BC risk factors are associated with elevated circulating E levels. Even lesser risk factors such as obesity and alcohol ingestion are known to significantly increase serum E concentrations in women (9, 10). These earlier studies are buttressed by results of the recent Breast Cancer Prevention Trial in which tamoxifen (TAM) treatment markedly reduced (44-55%) BC risk in women considered at increased risk for the disease (11). Since it is evident that Es are crucial to our understanding of sporadic BC etiology, it is surprising that so little is known about the involvement of Es in oncogenic processes in target tissues such as the breast, other than its ability to elicit cell proliferation. Our laboratory has utilized two seemingly disparate animal models in which malignant tumors are induced solely by Es in the absence of any other exogenous intervening carcinogenic or promotional agent (12-15). Based on our recent findings, it has been concluded that the early detection of chromosomal instability (CIN) and aneuploidy, in early lesions and primary neoplasms in both systems, is a hallmark characteristic of E-induced oncogenesis.

General Considerations

Animal models are most useful when they closely resemble the biologic and molecular aspects of the human disease they intend to study. Although the two experimental models to be discussed have many fundamental differences, they have in common the ability of certain cells within each of these tissues (i.e., kidney, breast) to undergo neoplastic transformation in response to E treatment alone. Moreover, the neoplasms induced by Es in the castrated hamster kidney and in the intact female ACI rat mammary gland (MG) are both prevented by concomitant anti-estrogen treatment (Table l) (15, 16). These data strongly indicate that estrogen receptor is central in mediating the oncogenic response of Es.

Although the minimum oncogenic dose of E to elicit tumors in the kidney is not known, typically, serum 17|3-estradioI (E2) levels of 1.6-2.4 ng/ml are used to induce a 100% tumor incidence. In contrast, only 60-130 pg/ml serum E2 levels are sufficient to result in a 100% mammary tumor (MT) incidence in female ACI rats (15). The lower serum E2 concentrations are well within the physiological range of serum E2 levels reported in cycling female rats (17-88 pg/ml) (17). On the other hand, serum E2 levels in normal cycling women have shown to be in the range of 40-360 pg/ml (18). Importantly, in normal breast and primary BC tissues, the E2 concentrations were found to be extremely low (Table 1) (19-22) and approximate that seen in the hamster kidney during chronic E treatment.

Table 1. Serum and Tissue E2 Concentrations During E-induced Oncogenesis.

Serum

Tissue

Tissue site

(pg/ml)

(pg/mg protein)

Hamster EUTK

1670-2480

4.6

ACI Rat MT

60- 130

Human Breast

40 - 350

5.7

Human BC

8.9

Ectopic Uterine Tumors in the Kidney (EUTK)

While the tumors in the hamster kidney have been the foremost animal model studied in E-induced oncogenesis, there are a number of unusual aspects to this system which may be now understood. Unlike the breast, prevention of these tumors can be effectively blocked by the concomitant treatment with either progesterone or androgen (16). This finding provides a clue as to the cell of origin of these tumors arising in the kidney. Since the hamster reproductive and urinary systems arise from a common germinal ridge of multi-potential stem cells, we have postulated that some of these germinal/stem cells, normally destined to the uterus, migrate and establish themselves in the cortico-medullary area of the kidney, the earliest region formed in the developing kidney (23). These ectopically located "uterine" germ cells remain dormant unless exposed to a sustained level ofE. This contention is supported by the following findings: 1. Resemblance of early renal interstitial lesions to blastema, with positive staining of mesenchymal markers (e.g. vimentin, desmin) but only a trace of cytokeratin (24, 25). The cytokeratin expression markedly rises during tumor progression. 2. Only a subset ofthese renal interstitial stem cells in the cortico-medullary region express progesterone receptor (PR) after only two to three weeks ofE treatment (Figure 1) (23). 3. After one mo, this subset of interstitial stem cells co-express ERa (Figure 2) (23). 4. The lack of expression in these E-sensitive renal interstitial stem cells, in early tumorous lesions, and primary tumors is consistent with the established selective expression and proliferative role of ERa in uterine tissue (26). 5. The ability of P to completely block these now designated ectopic uterine stem cell tumors in the kidney (EUTK) is consistent with the opposing action of P in the uterus. 6. The essentially identical isoform profiles of and PR in primary EUTK to those of corresponding receptor profiles in the hamster uterus (23). 7. The specific high proliferative activity in this same subset of interstitial stem cells in response to E and the overexpression (OE) of early E response genes (12) further supports our contention. An attractive aspect ofthis experimental model is that EUTK occur in the absence of any sequential intervening morphological stages, but develop as a continuous progression of a subset of E-sensitive interstitial stem cells leading to tumor formation.

Table 2. Prevention of E-induced Oncogenesis of Hamster EULTK and ACI Rat MT by Tamoxifen.

Species

Treatment

No.

% Animals

Tumor

Animals

w/Tumors

Multiplicity/Animal

Untreated

8

0

0

Hamster1

tam

8

0

0

e2

10

100

16.7 ± 1.1

e2 + tam

8

0

0

Untreated

10

0

0

aci Rat2

tam

8

0

0

e2

12

100

15.6 ± 1.6

e2+tam

8

0

0

1 Castrated hamsters were treated with a 20-mg pellet of E (DES or E2) alone or in combination with a 20-mg pellet of TAM, every 3.0 mo for 8 mo.

2 Intact female ACI rats were treated with a single 20-mg pellet containing 3 mg of E2 + 17 mg of cholesterol alone or in combination with a single 20-mg pellet of TAM for a 6.0-mo period.

1 Castrated hamsters were treated with a 20-mg pellet of E (DES or E2) alone or in combination with a 20-mg pellet of TAM, every 3.0 mo for 8 mo.

2 Intact female ACI rats were treated with a single 20-mg pellet containing 3 mg of E2 + 17 mg of cholesterol alone or in combination with a single 20-mg pellet of TAM for a 6.0-mo period.

Figure 1. Immunohistochemical detection ofE-induced PR expression in castrated male hamster after 1.0, 2.0, 3.0, and 5.0 months of E treatment. Note the PR+ interstitial cells (arrows) at the cortical-medullary junction in the animals treated for 1.0 mo, PR+ cells in a nascent tumor foci after 2.0 mo, in an early renal tumor foci after 3.0 mo, and in a moderate size EUTK foci, after 5.0 mo (200x).

Figure 1. Immunohistochemical detection ofE-induced PR expression in castrated male hamster after 1.0, 2.0, 3.0, and 5.0 months of E treatment. Note the PR+ interstitial cells (arrows) at the cortical-medullary junction in the animals treated for 1.0 mo, PR+ cells in a nascent tumor foci after 2.0 mo, in an early renal tumor foci after 3.0 mo, and in a moderate size EUTK foci, after 5.0 mo (200x).

Figure 2. ERa expression during E-induced oncogenesis. Normal hamster kidney cortical tubule cells (arrows) exhibited ERa+ ERa+ expression in normal proximal tubular cells are downregulated upon E-treatment. Thereafter, individual and small groups of ERa+ interstitial cells are seen after 3.0-mo, and moderate size renal tumor foci after 4.0-mo E-treatment (250x). In addition, a small tumor foci is shown stained for PCNA.

Figure 2. ERa expression during E-induced oncogenesis. Normal hamster kidney cortical tubule cells (arrows) exhibited ERa+ ERa+ expression in normal proximal tubular cells are downregulated upon E-treatment. Thereafter, individual and small groups of ERa+ interstitial cells are seen after 3.0-mo, and moderate size renal tumor foci after 4.0-mo E-treatment (250x). In addition, a small tumor foci is shown stained for PCNA.

Mammary Tumors in Female ACI Rats

Numerous rat strains exhibit variable susceptibility to E-induced MTs including ACI, Noble, and Long Evans (60-100% tumor incidence) and to a lesser extent,

August, Wistar-Wag, and SD (36-42%) (14, 15, 27-32). Recently, we have shown that >40% MTs may be induced in female SD rats after only 5.0-mo of estrone treatment (El-Bayoumy, K and Li, JJ, unpublished data). An attractive feature of the E-induced female ACI rat model to study human sporadic BC causation is the ability ofthis strain to elicit 100% multiple MTs employing serum levels of only 60-180 pg/ml (Figure 4) (15). Moreover, we have established that <60 pg/ml of E2 was also effective in producing essentially a 100% MT incidence (Li, SA, unpublished data). An E2 dose/response relationship between MT multiplicity and incidence was evident. Below 35 pg/ml ofE2, no MTs were detected within a 6.0-mo treatment period. Additionally, above an E2 serum level of200 pg/ml, there was a reduction in MT multiplicity and incidence (Figure.3). These results indicate that within a relatively narrow range of serum E2 concentrations (all at low E2 pg/ml). MT multiplicity and incidence may be enhanced. Below and above this serum range, MTs are either absent or inhibited, respectively. These data are consistent with epidemiological findings that only modest elevations in serum levels may increase human BC risk and high levels of E2, such as found during pregnancy, may reduce BC risk (33, 34).

Months

Figure 3. Serum levels determined at monthly intervals in female ACI rats treated with either 1, 2, and 3 mg E2. The data represent the mean of four individual samples measured in duplicate. 100% MT incidence was elicited at a E2 serum concentration range between 60-180 pg/ml.

Synthetic chemical carcinogens (dimethylbenz(a)anthracene, DMBA and nitrosomethylurea, NMU) induced MT models have dominated experimental BC research for nearly a half century. Recent studies in Long Island, NY, however, provide compelling evidence that environmental carcinogens, including polycyclic aromatic hydrocarbons, do not have an appreciable role in BC etiology (35,36). In a blinded study, employing nuclear image cytometry (NIC), we have shown that two synthetic chemical carcinogens (i.e., DMBA and NMU) and one environmental carcinogen, 6-nitrochrysene (6-NC) yielded primarily diploid (> 85%) MTs in female rats (Table 3) (14). In contrast, both DCIS and primary MTs induced in either female ACI or Noble rats by E treatment alone resulted in MTs which were highly aneuploid (> 84%). These results provide very strong evidence that the induction of experimental breast tumors by potent mammary chemical carcinogens occurs by distinctly different molecular mechanisms utilizing different pathways compared to those breast tumors induced solely by Es, and it is the latter mammary neoplasms which more closely resemble those seen in human DCIS and invasive ductal BCs (37, 38).

Table 3. NIC Assessment of Aneuploid Frequency in MTs Induced by Hormones or Chemical Carcinogens in a Variety of Rat Strains.

Strain

% Aneuploid Cells1 CIS MGT

HORMONES

17ß-E2

ACI

84.5 (6)

90.9 (9)

17ß-E2+TP

Noble

85.2 (5)

89.3 (5)

CHEMICAL CARCINOGENS

DMBA

BuF/N

14.6 (5)

NMU

BuF/N

12.8 (5)

6-NC

SD

10.5 (5)

1 Number in parentheses indicates number of individual scans (x 100 cells). Steroid Hormone Receptors (ERa, PR)

1 Number in parentheses indicates number of individual scans (x 100 cells). Steroid Hormone Receptors (ERa, PR)

Generally, similar hormone receptor responses to sustained E exposure were obtained in the kidneys of male hamsters and mammary glands of female ACI rats (15, 23). In kidneys of untreated control hamsters, a predominate 50-kDa ERa variant and a slightly lower expression of a 64-kDa ERa isoform was found (Figure 4). In contrast, untreated normal mammary glands contained only a single major 56-kDa ERa variant (Figure 4). Following chronic treatment with E2, the 64-kDa variant level was mainly elevated in hamster kidneys. With further treatment, a 58-kDa isoform appeared after 4.0- to 5.0-mo and was present in all primary EUTK examined whereas the 64-kDa isoform was evidently loss (Figure 4). In addition to the presence of both 50- and 58-kDa ERa variants, both the hamster uterus and EUTK samples invariably expressed the 66-kDa form, presumably the full length ERa. Based on the immunohistochemical analyses in renal tissue sections, changes in these isoforms following E exposure were evidently confined to the E sensitive renal interstitial cells which subsequently undergo neoplastic development and multiply. Similarly, after low dose E2

treatment, a number of ERa forms were detected in female ACI rat mammary glands. In addition to the full length 66-kDa ERa form, the major ERa variants were 56- and 47-kDa (Figure 4). Moreover, two lesser ERa variants, a 55- and 72-kDa, were also seen as well as a new 54-kDa ERa form. The dominant form of ERa in MTs, however, was this 54-kDa ERa. This latter finding may be important in mediating E-dependent MT growth advantage. E elevated PR both in treated male hamster kidneys and in female ACI rat mammary glands and in their respective primary tumors (Figure 4). In primary EUTK, PR-B, -A, and-C expressions were markedly increased whereas in primary ACI rat MTs, only PR-A and -C exhibited strong expression following sustained E exposure.

Figure 4. Western blot analysis of ERa and PR expression during E-induced hamster EUTK oncogenesis. IMK, Intact male hamster kidney, E-1, E-3, E-5, E treatment for 1.0, 3.0, and 5.0 mo, respectively. T, Tumor, HU, Hamster uterus. MC, control mammary gland, MTx, Mammary gland treated with TAM alone, MTX + E or in combination with E2. MT„ MT2, MT3, Individual MT samples. UC, ACI rat uterus control

Figure 4. Western blot analysis of ERa and PR expression during E-induced hamster EUTK oncogenesis. IMK, Intact male hamster kidney, E-1, E-3, E-5, E treatment for 1.0, 3.0, and 5.0 mo, respectively. T, Tumor, HU, Hamster uterus. MC, control mammary gland, MTx, Mammary gland treated with TAM alone, MTX + E or in combination with E2. MT„ MT2, MT3, Individual MT samples. UC, ACI rat uterus control c-Myc Gene Overexpression and Amplification

In addition to a similarity in steroid receptor responses, characteristic of E-dependent cells, both developing E-induced hamster EUTK and ACI MTs (i.e., DCISs) overexpress c-myc and MYC protein (12, 14, 39). This upstream c-myc/Myc response to E treatment is a characteristic response of this hormone on its target tissues. Recently, it has been shown that OE of c-myc elicits CIN (40, 41). However, it is likely, that downstream cell cycle genes, mediated by c-myc OE, are more intimately involved in eliciting the loss of mitotic stability resulting in CIN and subsequent aneuploidy. Southern blot analyses were performed on primary hamster EUTK and ACI rat MTs taken from individual E2-treated animals (Figure 5) (14, 39). In EUTK, 67% (8/12) exhibited c-myc amplification (range 2.4-3.6).

A 66% (6/9) amplification of c-myc (range 3.4-6.9) was also found in ACI rat MTs. In untreated control hamster kidneys and ACI rat mammary glands, the mean densitometric level of c-myc had a range between 0.7-1.5. The amplification of c-myc in E2-induced EUTK and ACI rat MTs is due, in part, to a consistent gain in the number of copies of chromosome 6 (hamster) and 7 (rat), where the c-myc gene resides (Figure 6).

Figure 5. Representative southern blot analyses of c-myc expression. A. Hamster. Three control age-matched castrated male kidneys (CrC3) and five EUTKs (TrT5) isolated from individual animals after 7 mo E-treatment. B. ACI rat. Three control age-matched mammary glands from intact female ACI rats (Ci-C3) and 5 MT (Tr T5) isolated from individual animals after 6.0 mo E2 treatment.

Figure 5. Representative southern blot analyses of c-myc expression. A. Hamster. Three control age-matched castrated male kidneys (CrC3) and five EUTKs (TrT5) isolated from individual animals after 7 mo E-treatment. B. ACI rat. Three control age-matched mammary glands from intact female ACI rats (Ci-C3) and 5 MT (Tr T5) isolated from individual animals after 6.0 mo E2 treatment.

Figure 6. Representative Giemsa G-banded karyotypes from A. Hamster EUTK induced by E after 8 mo. Note the trisomy (consistent gain) in chromosome 6 where c-myc resides in the hamster. B. E-induced MT from a 5.5 mo treated female ACI rat. Note the tetrasomy (consistent gain) in chromosome 7 where c-myc gene resides in the rat.

Figure 6. Representative Giemsa G-banded karyotypes from A. Hamster EUTK induced by E after 8 mo. Note the trisomy (consistent gain) in chromosome 6 where c-myc resides in the hamster. B. E-induced MT from a 5.5 mo treated female ACI rat. Note the tetrasomy (consistent gain) in chromosome 7 where c-myc gene resides in the rat.

Centrosome Amplification

The centrosome is the major microtubule-organizing center. Centrosomes are essential to the control of spindle bipolarity, spindle positioning, and cytokinesis. Abnormalities in centrosome number, common in human DCISs and primary invasive ductal BCs (42-44), can interfere with bipolar spindle formation and chromosome segregation, thus leading to CIN and aneuploidy. Normal hamster renal tissues exhibit regular comet-like staining for pericentrin (red) which occurs apically and low levels of centrin staining (Figure 7). EUTK sections show large aggregates of pericentrin and elevated levels of centrin staining (green), typical of centrosome amplification (Figure 7). Centrosome amplification was also commonly detected in primary ACI rat MTs (Lingle, W., Salisbury, J, Li, JJ, and Li SA, unpublished data). It is concluded from these findings that centrosome defects may be a characteristic feature of solely estrogen-induced oncogenic processes and a primary causative event leading to E2-induced tumor formation.

Figure 7. Normal control and EUTK specimens stained with a cocktail of antibodies against pericentrin (rabbit Ig) and centrin (mouse monoclonal ascites) followed by the appropriate secondary antibodies (Alexa 568 - red for pericentrin and Alexa 488 - green for centrin) and the DNA intercalating dye DAPI. Sections were observed using a 63X 1.2 NA obj ecti ve and recorded digitally on a Zeiss 510 confocal microscope. The images represent maximum projections of five 0.2 ^m optical sections. The schematic representations (lower figures) illustrate nuclear (blue), pericentrin (red), and centrin (green) signals. Bar = 10 (im.

Figure 7. Normal control and EUTK specimens stained with a cocktail of antibodies against pericentrin (rabbit Ig) and centrin (mouse monoclonal ascites) followed by the appropriate secondary antibodies (Alexa 568 - red for pericentrin and Alexa 488 - green for centrin) and the DNA intercalating dye DAPI. Sections were observed using a 63X 1.2 NA obj ecti ve and recorded digitally on a Zeiss 510 confocal microscope. The images represent maximum projections of five 0.2 ^m optical sections. The schematic representations (lower figures) illustrate nuclear (blue), pericentrin (red), and centrin (green) signals. Bar = 10 (im.

Chromosomal Instability and Aneuploidy

In addition to the nonrandom occurrence of trisomies and tetrasomies in chromosome 6 and 7 in E2-induced hamster EUTK and ACI rat MTs, respectively, where the c-myc gene resides (Figure 6), other chromosomes were also consistently gained or lost (14, 45). Nonrandom gains in chromosomes 3, 6, 20, and 21 were detected in primary hamster EUTK employing a stringent criterion. Consistent chromosome losses, however, were not found. In E2-induced ACI rat MTs, chromosomes 11, 13, 19, and 20 exhibited high frequencies of trisomies and to a lesser extent tetrasomies and a monosomy in chromosome 12 using the same criterion. In these E2-induced neoplasms, consistent or nonrandom numerical chromosome gains or losses were considered to have a frequency of occurrence >30%. Recurrent numerical chromosome alterations (>20 to <29.9%) were also detected in both primary neoplasms, as well as random chromosome changes were seen. A major challenge is to discern of the mechanisms whereby consistent, recurrent, and random numerical chromosomal alterations are generated together.

Summary and Conclusions

The cellular and molecular resemblance of primary hamster EUTK and

ACI rat MTs to human DCIS and invasive ductal BC (iDC) is striking (Table 4).

Table 4. Similarities in Salient Molecular Parameters Between Primary Human Ductal Breast Carcinomas and E-induced Tumors.

Hamster Human ACI Rat

Table 4. Similarities in Salient Molecular Parameters Between Primary Human Ductal Breast Carcinomas and E-induced Tumors.

Hamster Human ACI Rat

EarlyFoci

EUTK

DCIS

IDC

DCIS

IDC

(%)

{%)

(%)

(%)

<%)

(%>

ER+

97

100

60-84

73

100

100

ER'+PRf

100

100

66

71

100

100

Aneuploid

94

too

55-78

85-92

84

91

MYC Protein OE

100

100

100

71-100

100

c-myc Gene Amp]

67

12-86

66

Cyclin D1 Protein OE

100

72

43

100

Cyclin D1 mRNA OE

--

76-87

83

100

Centrosome Ampi

100

100

80

100

AurA Protein OE

100

94

100

In particular, the presence ofERa and PR and the histopathology ofthe ACI rat MT are analogous to human ductal BC. Other similarities to human ductal BC and these solely E-induced neoplasms are: MYC protein OE, c-myc amplification, and cyclin D1 OE (46-49). Most remarkable, however, is the coincident high frequency of centrosome amplification and aneuploidy in these solely E-induced rodent neoplasms, and human DCIS and primary invasive ductal BCs (Table 4) (4245). The OE of Aurora A kinase [Aur A, breast tumor amplified kinase (BTAK)] was also detected in both primary hamster EUTK and ACI rat MTs (Table 4). While no direct association between Aur A OE, centrosome amplification, and CIN have been shown, they always occur together (50). Aur A OE has been implicated in eliciting these latter events (51-53).

Based on our current knowledge, the scheme in Figure 7 is proposed for E-induced oncogenesis in the two mammalian models studied. E interacts with its receptor (E Ra) in susceptible cells to elicit c-myc/MYC OE. The OE of MYC turns on specific down stream cell cycle-related genes (i.e., cyclins Dl, D2, and E). It is also possible that E-ERa may directly activate the cyclin D gene family. The persistent OE of these cell cycle related proteins lead to elevated levels of centrosomal proteins (i.e., pericentrin, centrin, y tubulin), and centrosome-associated kinases (Aur A) located in the lattice network/spindle regions of centrosomes. Thus, disturbances in centrosome regulation result in defects in duplication/separation, resulting in centrosome amplification, considered a mutational event. Importantly, our findings for the first time directly link E-action to the deregulation of the cell cycle, Aur A OE, CIN, and aneuploidy as causative events leading to solely E-induced tumor development in two in-vivo systems. It is noted that these aforementioned similarities in molecular characteristics between E2-induced hamster EUTK and ACI rat MTs and their respective early lesions occur despite the absence of P in the E-induced hamster EUTK model, and the presence of this hormone in the E-induced ACI MT system.

Figure 8. Scheme proposed for the development of EUTK in the hamster kidney and ACI MT-induced solely by chronic E exposure. E binds to its overexpressed ERa as a result ofE treatment. The E-ERa complex transactivate c-myc leading to its OE and subsequent amplification. Also, E-ERa interactions lead to a rise in cyclin E, cyclin D2, and MDM2. The cyclin*cdk complexes and other regulatory proteins (MDM2, DHFR) bind to specific centrosome proteins located in the lattice network of centrosomes. The concerted interactions of these complexes cause chromosomal instability and eventual malignant tumor formation.

Figure 8. Scheme proposed for the development of EUTK in the hamster kidney and ACI MT-induced solely by chronic E exposure. E binds to its overexpressed ERa as a result ofE treatment. The E-ERa complex transactivate c-myc leading to its OE and subsequent amplification. Also, E-ERa interactions lead to a rise in cyclin E, cyclin D2, and MDM2. The cyclin*cdk complexes and other regulatory proteins (MDM2, DHFR) bind to specific centrosome proteins located in the lattice network of centrosomes. The concerted interactions of these complexes cause chromosomal instability and eventual malignant tumor formation.

Acknowledgements

This investigation was supported by NIH Grants CA58030 and 33519. We wish to thank Dan Papa, Ph.D., and S. John Weroha for their able technical assistance in these studies.

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