Rainer B. Lanz ^1, *, Steven S. Chua ¹, Niall Barron ², Bettina M. Söder ¹, Francesco DeMayo ¹, and Bert W. O'Malley ¹

¹ Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030,
² National Cell and Tissue Culture Center, Dublin City University, Glasnevin, Dublin 9, Ireland

* Corresponding author. Mailing address: Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Phone: (713) 798-6478. Fax: (713) 790-1275.
E-mail:    rlanz@bcm.tmc.edu

Steroid receptor RNA activator (SRA) is an RNA that coactivates steroid hormone receptor-mediated transcription in vitro. Its expression is strongly up-regulated in many human tumors of the breast, uterus, and ovary, suggesting a potential role in pathogenesis. To assess SRA function in vivo, a transgenic-mouse model was generated to enable robust human SRA expression by using the transcriptional activity of the mouse mammary tumor virus long terminal repeat. Transgenic SRA was expressed in the nuclei of luminal epithelial cells of the mammary gland and tissues of the male accessory sex glands. Distinctive evidence for SRA function in vivo was obtained from the elevated levels of estrogen-controlled expression of progesterone receptor in transgenic mammary glands. Although overexpression of SRA showed strong promoting activities on cellular proliferation and differentiation, no alterations progressed to malignancy. Epithelial hyperplasia was accompanied by increased apoptosis, and preneoplastic lesions were cleared by focal degenerative
transformations. In bitransgenic mice, SRA also antagonized ras-induced tumor formation. This work indicates that although coactivation of steroid-dependent transcription by SRA is accompanied by a proliferative response, overexpression is not in itself sufficient to induce turmorigenesis. Our results underline an intricate relationship between the different physiological roles of steroid receptors in conjunction with the RNA activator in the regulation of development, tissue homeostasis, and reproduction. 

The observation that preneoplastic lesions in older SRA-transgenic animals did not develop to malignancies suggested that overexpression of SRA alone was not sufficient to convert the morphological alterations to a malignant phenotype. To further test our deduction, we interbred SRA-transgenic mice with the MMTV-ras
transgenic line, which produces tumors at a high rate. The lesions are palpable in MMTV-ras mice, allowing their growth rate to be easily monitored (38). To create comparable mouse models, we first interbred MMTV-ras mice from Charles River Laboratories (44) into the FVB background used for the generation of MMTV-SRA lines. MMTV-ras-transgenic FVB animals showed a variety of proliferative disturbances that were similar to the originally reported phenotypes (38). Male animals developed predominantly nonmalignant Harderian gland hyperplasia with severe exophthalmos and alopecia around the eyes secondary to glandular enlargement, along with parotid salivary gland lesions (not shown). Female animals developed spontaneous mammary tumors (adenocarcinomas and malignant lymphomas) and parotid adenocarcinomas at about 3
months of age. Mammary gland whole-mount analyses of ras-transgenic mice revealed a spatially limited ductal penetration of the fat pad with a few ductal systems forming focal hyperplastic nodules at the proximity of the primary ducts (Fig. 7A, micrographs a and b), which at an early stage of tumorigenesis were of watery consistency and resembled translucent blisters (inset in panel b).

The analysis of the ras/SRA bitransgenic animals revealed some surprising results. Of the 18 ras/SRA bitransgenic animals analyzed by necropsy, only 1 had developed salivary gland lesions and 8 (44%) showed proliferative disturbances of the mammary glands (Fig. 7B).

Figure 7b. Analysis of the ras transgenic and ras/SRA bitransgenic animals.

.	.	ras	.	ras/SRA	.
.	.	#	%	#	%
I. Necroscopy:	n =	27	100	18	100
.	normal by gross anatomy	1	4	1	6
.	mammary gland  lesions	25	93	8	44
.	salivary gland lesions	12	44	1	6
.	.	.	.	.	.
II. Whole-mounts:	n =	20	100	17	100
.	normal by whole- mount analysis	3	15	1	5
.	little ductal growth	15	75	6	35
.	ductal penetration	2	10	10	60

FIG. 7. SRA Expression obstructs ras-mediated tumorigenesis.

 (B) The table compares mammary gland and salivary gland tumor frequencies in MMTV-ras transgenic
(ras) and MMTV-ras/SRA bitransgenic (ras/SR) mice as analyzed by necropsy (I) and whole-mounts (II). #, number of analyzed mice; %, percentage; little ductal growth, no ductal development beyond the
lymph node (as illustrated in panel A); ductal penetration, normal ductal outgrowth filling the entire fad pad; other abbreviations as indicated.

These results indicate that SRA function interfered with ras-induced tumor formation, which is an interesting aspect requiring further investigation. Most importantly with respect to our initial objectives, however, is the notion that overexpression of SRA in murine mammary tissue, although a factor in increased cellular proliferation and apoptosis, was not in itself sufficient to induce turmorigenesis.
...
To summarize, MMTV-controlled expression of human SRA resulted in various effects on murine organ development but was, in itself, not sufficient to induce turmorigenesis. Although abundant SRA in general showed strong promoting activities on proliferation and differentiation, it also enhanced apoptosis. Little to no cellular pleomorphism was observed, and preneoplastic lesions were cleared by focal degenerative
transformations.

Discussion:

The strong up-regulation of SRA in various tumors of the human breast, uterus, and ovary, together with its relatively low expression in normal tissues, suggests a role for the RNA activator as a biological marker of steroid-dependent tumors. While additional studies and in situ analysis are necessary to validate SRA as predictive diagnostic marker, its expression profile in breast tissues is in agreement with previous findings
(26). In addition, because SRA was expressed at high levels regardless of tumor type, a role in early turmorigenesis is implicated. To assess the pathogenic potential of SRA in vivo, an MMTV-SRA-transgenic mouse model was generated and analyzed. All SRA-transgenic lines produced tumor-free animals that exhibited a life span comparable to that of wild-type animals, indicating that overexpression of SRA is not in itself sufficient to induce tumors.

Still, SRA-transgenic mice displayed a plethora of phenotypes, which have to be viewed in relation to the various functions of SRs expressed in the target tissues. Few SR-transgenic models exist, and the physiological roles of SRs have been based largely on the characterization of mouse models genetically ablated for a specific receptor function. Because of the intricate circuitry of steroid actions, coordinated receptor function is often disrupted in these animals. In this respect, MMTV-SRA-transgenic mice display overall elevated SR activities and therefore provide a model that accounts for coordinated functions of the receptors and their isoforms.

The proliferative phenotypes observed in mammary glands of SRA-transgenic mice resemble the physiological changes reported for PR-A-transgenic mice (37). Although the levels of PR isoforms are altered in these mice, their mammary gland development primarily reflects increased responsiveness to progestins. By comparative analysis, the morphological changes observed in SRA-transgenic mammary glands may be partially attributed to SRA-potentiated PR activity. While SRA may affect mammary gland development through PR transactivation, PR expression itself is also modulated by ovarian estrogen, whose levels in serum are elevated during puberty and pregnancy. Proper individual mechanistic roles for progesterone and estrogen in vivo have yet to be established, but the supposition from the targeted deletion of PR (22) and the ERa knockout (1, 9) models emphasizes the specific role of estrogen rather than progesterone in mammary gland ductal elongation during puberty (40). At this particular stage of mammary development, SRA-transgenic mammary glands showed significantly elevated levels of PR expression (Fig. 6B). Because this expression profile subsided as the virgins matured and because it correlates with ovarian estrogen signaling in mammary ductal outgrowth, our results suggested the presence of SRA-enhanced ER transactivation of the PR gene and provided us with the first distinctive evidence for an SRA function in vivo.

PR, as both a target for SRA function and a key molecule to transmit the actions of endocrine mammogens, is expressed at the highest level in the mammary epithelial structures of the peripubertal (5-weak-old) mouse (16). The expression profile of PR coincides with the appearance of ductal epithelial proliferation and multilocular fat in SRA-transgenic mice at this stage of mammary gland development. Mammary fat tissue plays a crucial role in epithelial growth and lobulo-alveolar morphogenesis of the mammary parenchyma (11, 32, 33). While it was long assumed that the mammary fat was composed exclusively of energy-storing white adipose tissue, energy-dissipating brown adipose tissue (BAT) (17) has recently been reported to occupy temporal and spatial compartments in the mammary gland stroma (13, 25). While promoting concurrent PR functions, SRA overexpression also enhanced the appearance of BAT at this time in mammary gland development, suggesting that the dedifferentiation of adipose tissue is related to the paracrine signaling (3) that is part of the progesterone-induced proliferative signals. This hypothesis is supported by the enhanced complexity of mammary ductal branching observed in a transgenic mouse model depleted of BAT. In this model, brown fat, in contrast to white adipose tissue, negatively regulated the differentiation of transplanted mammary epithelial cells during peripubertal ductal outgrowth (13). This observation suggests that the abnormal branching morphology observed in SRA-transgenic virgins may be the consequence of the temporal exposure of the young ductal system to excessive BAT. In addition, while the developmental and environmental stimuli responsible for adaptive thermogenesis in the mammary gland are still theoretical (13, 25), the dramatic temporal increase in BAT in mammary glands of young SRA-transgenic mice may suggest a paracrine function for the RNA activator in this process.

Under pathological conditions, apoptosis results in the re-gression of neoplastic tissue, as well as substantial cell loss and retardation of cancer growth (2). In SRA-transgenic mice, augmented apoptosis was observed to counteract strong mitotic activities and, assisted by inflammatory responses, to clear metaplastic tissues and ductal structures. The mechanism by which SRA mediates the balance between proliferation and apoptosis is not known. However, it is important to reiterate the plasticity of the mammary gland (27, 43). This allows for
speculation about an intrinsic attempt by the mammary gland to ensure the specification and spatial organization of ductal and alveolar structures. PR plays an important role in this process (16, 36), which is tested and reestablished by recurring parturition. The observation that early first pregnancy reduces the risk of breast cancer supports this notion (41). Early parity-induced protection against mammary cancer involves p53-mediated signaling (39). Investigation of SRA-associated molecules resulted in the identification of a protein that specifically binds to RNA substructure STR1 of SRA (19; A. Redfem, D. Beveridge, R. B. Lanz, L. Stuart, B. O’Malley, and P. Leedman, Proc. Endocr. Soc. 84th Annu. Meet., abstr. OR46-6, 2002). This protein was functionally linked to the p53 and NF- B pathways and, as such, provides a molecular link between SRA function and apoptosis, inflammation, and parity-induced tumor protection (39). Still, further experiments are necessary to delineate the specific roles of SRA and SR in mammary gland tissue homeostasis.

The significantly lower tumor rates observed in ras/SRA-bitransgenic mice provided more evidence of the antitumorigenic potential of SRA, a presumptive function of the RNA coactivator that will have to be confirmed by using other bi-transgenic mouse models. In contrast to the SRA-transgenic models, no phenotypic abnormalities have been observed by the targeted deletion of the SRA gene (unpublished result), suggesting that a functionally redundant molecule may compensate for the phenotype. SRA overexpression, however, triggered proliferation, inflammation, and apoptosis in estrogen, progesterone, and testosterone-sensitive tissues of male and female mice, suggesting a physiological role for the RNA activator in the establishment of tissue homeostasis in steroidal tissues. Our results raise the possibility that in contrast to our initial conclusions, the up-regulation of SRA in many human tumors of steroid-dependent tissue may reflect a cellular effort to antagonize excessive proliferation. Additional studies will eventually elucidate the mechanisms involved. It is possible that tumor progression is controlled by the specific composition of ribonucleoprotein complexes containing SRA, whose expression level determines whether transcriptional coactivators or corepressors are incorporated. This model is consistent with to the reported role of SRA in attenuation of SR transactivation in conjunction with SHARP (35). Even so, our analyses of SRA mouse models suggest that the promotion of proliferating functions used to achieve established tissue structures occurs in conjunction with mechanisms to prevent tumor formation.

ACKNOWLEDGMENTS:

We thank J. Lydon, D. Medina, and J. Rosen for helpful discussion; E. Hayes and F. Moormehei for technical assistance; W. J. Muller for the Mmtv-Sv40-Bssk plasmid; and P. Ismail and K. Schillinger for help with PR-immunohistochemistry and the manuscript; respectively. This work was supported by grants from the Texas Higher Education Advanced Technology Program (ATP 004949-0154-1999), from The CONCERN Foundation, SPORE P50CA58183 Breast Cancer, and the Edward Mallinckrodt Jr. Foundation to R.B.L. and by an HD-NIH grant to B.W.O.

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