Genomic signatures of pregnancy-associated breast cancer epithelia and stroma and their regulation by estrogens and progesterone.

Pregnancy-associated breast cancers (PABC) generally present at advanced stages and have a poor prognosis. The reasons are unclear but we hypothesized that the continuous high levels of estrogens and progesterone were involved. We have now carried out a detailed analysis of PABC compared to tumors of age-matched nonpregnant (non-PABC) women. Malignant epithelia and tumor-associated stroma of PABC and non-PABC were isolated by laser capture microdissection and gene expression profiled. Additionally, normal breast epithelia and stroma adjacent to the two tumor types were analyzed. Lastly, subsets of previously identified E- and P-regulated genes were defined in all tissues. We find that PABC signatures cluster with established breast cancer subtypes. Major hormone-regulated genes whose expression correlated with epithelia of PABC dealt with regulation of cell proliferation, metabolism, and tumor aggressiveness, including genes used to predict tumor recurrence. Compared to normal epithelia, a significant number of genes associated with cell cycle processes were enriched in PABC, many of which are hormone regulated. Thus, compared to normal epithelia, many of the genes that were differentially expressed in epithelia of PABC were distinct from those differentially expressed in non-PABC. With regard to the tumor microenvironment, immune-related genes were enriched in tumor-associated stroma of PABC. Compared to normal stroma, PABC-associated stroma overexpressed immune response genes, while genes involved in angiogenesis and extracellular matrix deposition were more commonly downregulated. This suggests that the heightened aggressiveness of PABC may involve a predisposition to metastasis through extracellular matrix degradation, plus angiogenesis independence. Moreover, genes encoding cell proliferative factors, signaling, immunomodulators and cell death, were hormone regulated in stroma. In sum, these analyses demonstrate complex patterns of enrichment and hormonal regulation of genes in PABC and suggest that it may have a distinct biological nature.

Estrogen regulated gene expression in response to neoadjuvant endocrine therapy of breast cancers: tamoxifen agonist effects dominate in the presence of an aromatase inhibitor.

Estrogens (E) and estrogen receptors (ER) are implicated in breast cancer growth and are targets of hormonal therapies. Such therapies commonly use aromatase inhibitors (AI) to block E production, or antiestrogens like tamoxifen (TAM), which targets ER. Here we compare genes in pre-and post-treatment tumor pairs of patients with ER+ tumors, that were treated preoperatively with the AI exemestane alone, or with exemestane plus TAM. The accompanying manuscript shows that tumors from patients treated with AI + TAM responded less well than tumors treated with AI alone. The present manuscript defines the E-signaling mechanisms underlying these differences, and describes genetic differences between hormone responsive versus intrinsically resistant ER+ tumors. Gene expression profiling was performed on paired tumor biopsies of individual patients before treatment, and after 4 months of treatment with AI or AI + TAM. Separately, E and TAM regulated genes were defined using a human breast cancer xenograft model. We demonstrate: (1) that AI alone alters global gene expression approximately 5 times more than AI + TAM, and is 11 times more effective in modifying expression of E regulated genes; (2) among E regulated genes, there is little overlap between AI and AI + TAM treatment groups. AI + TAM preferentially induce genes, like androgen receptors, expressing TAM "E-like" agonist activity, or genes uniquely regulated by TAM. (3) A pre-treatment 25 gene signature of ER+ tumors may predict response or intrinsic resistance to endocrine therapies. We conclude that in the presence of exemestane, the agonist properties of TAM are paradoxically exposed, diminishing the effectiveness of combination therapy.

Molecular signatures of neoadjuvant endocrine therapy for breast cancer: characteristics of response or intrinsic resistance.

Approximately 30% of patients with estrogen receptor (ER) positive breast cancers exhibit de novo or intrinsic resistance to endocrine therapies. The purpose of this study was to define genes that distinguish ER+ resistant from ER+ responsive tumors, prior to the start of hormone therapies. Previously untreated post-menopausal patients with ER+ breast cancers were treated for 4 months in a neoadjuvant setting with the aromatase inhibitor exemestane alone, or in combination with the antiestrogen tamoxifen. Matched pre- and post-treatment tumor samples from the same patient, were analyzed by gene expression profiling and were correlated with response to treatment. Genes associated with tumor shrinkage achieved by estrogen blockade therapy were identified, as were genes associated with resistance to treatment. Prediction Analysis of Microarrays (PAM) identified 50 genes that can predict response or intrinsic resistance to neoadjuvant endocrine therapy of ER+ tumors, 8 of which have been previously implicated as useful biomarkers in breast cancer. In summary, we identify genes associated with response to endocrine therapy that may distinguish ER+, hormone responsive breast cancers, from ER+ tumors that exhibit intrinsic or de novo resistance. We suggest that the estrogen signaling pathway is aberrant in ER+ tumors with intrinsic resistance. Lastly, the studies show upregulation of a "lipogenic pathway" in non-responsive ER+ tumors that may serve as a marker of intrinsic resistance. This pathway may represent an alternative target for therapeutic intervention.

Contaminating cells alter gene signatures in whole organ versus laser capture microdissected tumors: a comparison of experimental breast cancers and their lymph node metastases.

Genome-wide expression profiling has expedited our molecular understanding of the different subtypes of breast cancers, as well as defined the differences among genes expressed in primary tumors and their metastases. Laser-capture microdissection (LCM) coupled to gene expression analysis allows us to understand how specific cell types contribute to the total cancer gene expression signature. Expression profiling was used to define genes that contribute to breast cancer spread into and/or growth within draining lymph nodes (LN). Whole tumor xenografts and their matched whole LN metastases were compared to LCM captured cancer cells from the same tumors and matched LN metastases. One-thousand nine-hundred thirty genes were identified by the whole organ method alone, and 1,281 genes by the LCM method alone. However, less than 1% (30 genes) of genes that changed between tumors and LN metastases were common to both methods. Several of these genes have previously been implicated in cancer aggressiveness. Our data show that whole-organ and LCM based gene expression profiling yield distinctly different lists of metastasis-promoting genes. Contamination of the tumor cells, and cross reactivity of mouse RNA to human-specific chips may explain these differences, and suggests that LCM-derived data may be more accurate.

Estrogen insensitivity in a model of estrogen receptor positive breast cancer lymph node metastasis.

The lymphatic system is a common avenue for the spread of breast cancer cells and dissemination through it occurs at least as frequently as hematogenous metastasis. Approximately 75% of primary breast cancers are estrogen receptor (ER) positive and the majority of these maintain receptor expression as lymph node (LN) metastases. However, it is unknown if ER function is equivalent in cancer cells growing in the breast and in the LNs. We have developed a model to assess estrogen responsiveness in ER(+) breast tumors and LN metastases. Fluorescent ER(+) MCF-7 tumors were grown in ovariectomized nude mice supplemented with estradiol. Once axillary LN metastasis arose, estradiol was withdrawn (EWD), for 1 or 4 weeks, or continued, to assess estradiol responsiveness. On EWD, proliferation rates fell similarly in tumors and LN metastases. However, estradiol-dependent ER down-regulation and progesterone receptor induction were deficient in LN metastases, indicating that ER-dependent transcriptional function was altered in the LN. Cancer cells from estradiol-treated and EWD primary tumors and matched LN metastases were isolated by laser capture microdissection. Global gene expression profiling identified transcripts that were regulated by the tissue microenvironment, by hormones, or by both. Interestingly, numerous genes that were estradiol regulated in tumors lost estradiol sensitivity or were regulated in the opposite direction by estradiol in LN metastases. We propose that the LN microenvironment alters estradiol signaling and may contribute to local antiestrogen resistance.

Progesterone receptors (PR)-B and -A regulate transcription by different mechanisms: AF-3 exerts regulatory control over coactivator binding to PR-B.

The two, nearly identical, isoforms of human progesterone receptors (PR), PR-B and -A, share activation functions (AF) 1 and 2, yet they possess markedly different transcriptional profiles, with PR-B being much stronger transactivators. Their differences map to a unique AF3 in the B-upstream segment (BUS), at the far N terminus of PR-B, which is missing in PR-A. Combined mutation of two LXXLL motifs plus tryptophan 140 in BUS, to yield PR-BdL140, completely destroys PR-B activity, because strong AF3 synergism with downstream AF1 and AF2 is eliminated. This synergism involves cooperative interactions among receptor multimers bound at tandem hormone response elements and is transferable to AFs of other nuclear receptors. Other PR-B functions-N-/C-terminal interactions, steroid receptor coactivator-1 coactivation, ligand-dependent down-regulation-also require an intact BUS. All three are autonomous in PR-A, and map to N-terminal regions common to both PR. This suggests that the N-terminal structure adopted by the two PR is different, and that for PR-B, this is controlled by BUS. Indeed, gene expression profiling of breast cancer cells stably expressing PR-B, PR-BdL140, or PR-A shows that mutation of AF3 destroys PR-B-dependent gene transcription without converting PR-B into PR-A. In sum, AF3 in BUS plays a critical modulatory role in PR-B, and in doing so, defines a mechanism for PR-B function that is fundamentally distinct from that of PR-A.

Estradiol regulates different genes in human breast tumor xenografts compared with the identical cells in culture.

In breast cancers, estrogen receptor (ER) levels are highly correlated with response to endocrine therapies. We sought to define mechanisms of estrogen (E) signaling in a solid breast tumor model using gene expression profiling. ER(+) T47D-Y human breast cancer cells were grown as xenografts in ovariectomized nude mice under four conditions: 1) 17beta-estradiol for 8 wk (E); 2) without E for 8 wk (control); 3) E for 7 wk followed by 1 wk of E withdrawal (Ewd); or 4) E for 8 wk plus tamoxifen for the last week. E-regulated genes were defined as those that differed significantly between control and E and/or between E and Ewd or control and Ewd. These protocols generated 188 in vivo E-regulated genes that showed two major patterns of regulation. Approximately 46% returned to basal states after Ewd (class I genes); 53% did not (class II genes). In addition, more than 70% of class II-regulated genes also failed to reverse in response to tamoxifen. These genes may be interesting for the study of hormone-resistance issues. A subset of in vivo E-regulated genes appears on lists of clinical ER discriminator genes. These may be useful therapeutic targets or markers of E activity. Comparison of in vivo E-regulated genes with those regulated in identical cells in vitro after 6 and 24 h of E treatment demonstrate only 11% overlap. This indicates the extent to which gene expression profiles are uniquely dependent on hormone-treatment times and the cellular microenvironment.

Progestins initiate a luminal to myoepithelial switch in estrogen-dependent human breast tumors without altering growth.

Although long-term clinical use of progestins is associated with an increased incidence of breast cancers, their role in established cancers is unclear. Estrogens are considered to be the main mitogens in the majority of breast cancers. Whether progesterone affects proliferation and/or differentiation is under debate. To assess the role of progesterone in established breast cancers, we used T47D human breast cancer cells that are estrogen receptor (ER) positive and either progesterone receptor (PR) negative or positive for PRA, PRB, or both. These cells were grown as strictly estrogen-dependent solid tumors in ovariectomized female nude mice. Progesterone or medroxyprogesterone acetate (MPA) alone did not support tumor growth, nor did progesterone or MPA given simultaneously with estrogen significantly alter estrogen-dependent tumor growth. However, treatment of mice bearing ER+PR+ but not ER+PR- tumors with either progesterone or MPA increased expression of the myoepithelial cytokeratins (CK) 5 and 6 in a subpopulation of tumor cells. These CK5+/CK6+ cells had decreased expression of luminal epithelial CK8, CK18, and CK19. We conclude that progestins exert differentiative effects on tumors characterized by transition of a cell subpopulation from luminal to myoepithelial. This may not be beneficial, however, because such a phenotype is associated with poor prognosis.

Rat strain specific attenuation of estrogen action in the anterior pituitary gland by dietary energy restriction.

The purpose of this study was to compare the effects of a 40% restriction of dietary energy consumption, relative to that consumed by rats allowed to feed ad libitum, on the ability of 17beta-estradiol (E2) to induce pituitary tumorigenesis in two inbred rat strains, ACI and Copenhagen (COP), which are very closely related genetically. Ovary-intact ACI and COP rats were fed either a control or an energy-restricted diet beginning at 8 wk of age. Continuous treatment with E2, released from subcutaneous Silastic tubing implants, was initiated at 9 wk of age and the animals were killed 12 wk later. Estrogen-induced pituitary tumorigenesis is associated with rapid induction of lactotroph hyperplasia, increased pituitary mass, and hyperprolactinemia. E2 significantly increased pituitary mass and circulating prolactin (PRL) in both ACI and COP rats, and this response was significantly greater in ACI rats relative to COP. Dietary energy restriction did not inhibit E2-induced pituitary growth in the ACI rat. By contrast, E2-induced pituitary growth in COP rats was attenuated by dietary energy restriction, as evidenced by quantification of pituitary mass, pituitary weight to body weight ratio, circulating PRL, and pituitary cell proliferation. This study indicates that sensitivity to the inhibitory actions of dietary energy restriction on E2-induced pituitary tumorigenesis is genetically determined.

Dietary energy restriction inhibits estrogen-induced mammary, but not pituitary, tumorigenesis in the ACI rat.

Because of the suggested role of energy consumption and the well-documented role of estrogens in the etiology of breast cancer, we have examined the effect of a 40% restriction of dietary energy consumption on the ability of administered 17beta-estradiol (E2) to induce mammary tumorigenesis in female ACI rats. Experiments herein test the hypothesis that at least part of the inhibitory effect of energy restriction on mammary tumorigenesis is exerted downstream of potential effects of dietary manipulation on the production of estrogens by the ovaries. Ovary-intact ACI rats were fed a control or a 40% energy-restricted diet and were either treated continuously with E2 from subcutaneous Silastic tubing implants or received no hormone treatment. Mammary cancers rapidly developed in E2-treated rats fed the control diet; within 216 days of initiation of E2 treatment 100% of the population at risk exhibited palpable mammary tumors. Dietary energy restriction markedly inhibited E2-induced mammary tumorigenesis, as evidenced by significant reductions in cancer incidence and tumor burden as well as a significant increase in the latency to the appearance of the first palpable cancer. The inhibitory actions of dietary energy restriction on E2-induced mammary tumorigenesis were associated with an inhibition of E2-stimulated mammary cell proliferation. However, this inhibition was insufficient to block induction of lobuloalveolar hyperplasia or appearance of focal regions of atypical epithelial hyperplasia. These data suggest that dietary energy restriction inhibits E2-induced mammary cancer by attenuating or retarding the progression of atypical hyperplasia to carcinoma. Expression of progesterone receptor (PR) was up-regulated within the focal regions of atypical hyperplasia and the carcinomas induced by E2, regardless of whether the rats were fed the control or energy-restricted diet. However, circulating progesterone was reduced by dietary energy restriction, suggesting a possible mechanism for inhibition of mammary tumorigenesis. Dietary energy restriction did not inhibit the ability of administered E2 to induce prolactin (PRL)-producing pituitary tumors and associated hyperprolactinemia, indicating that the inhibitory effects of dietary energy restriction on mammary tumorigenesis are tissue specific and independent of circulating E2 and PRL.