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Method of using estrogen-related receptor gamma (ERRgamma) status to determine prognosis and treatment strategy for breast cancer, method of using ERRgamma as a therapeutic target for treating breast cancer, method of using ERRgamma to diagnose breast cancer, and method of using ERRgamma to identify individuals predisposed to breast cancer

Abstrict

The present invention provides that ERR.gamma. is both a breast cancer biomarker and a breast cancer treatment target. A high ERR.gamma. level in breast cancer cells indicates good prognosis. A high level of ERR.gamma. in breast tissue suspected of being cancerous is indicative of breast cancer. Analyzing ERR.gamma. status and optionally along with the status of ER.alpha. can help breast cancer patients make treatment choices. Furthermore, breast cancer can be treated by decreasing ERR.gamma. activity in breast cancer cells.

Claims

We claim:

1. A method for determining prognosis of breast cancer comprising the step of determining the expression level of estrogen-related receptor .gamma. (ERR.gamma.) in breast cancer cells wherein a low level indicates poor prognosis and a high level indicates good prognosis.

2. A method for identifying breast cancer patients that may benefit from tamoxifen comprising the step of determining ERR.gamma. status in breast cancer cells of a patient wherein a high ERR.gamma. expression level indicates that the patient is likely to respond to tamoxifen and a low ERR.gamma. expression level indicates that the patient is less likely to respond to tamoxifen.

3. A method for determining whether a breast cancer patient will respond to hormonal blockade therapy comprising the step of: determining the status of ERR.gamma. and estrogen receptor (ER) .alpha. in breast cancer cells of the patient, wherein if the breast cancer cells are ER.alpha. positive and express ERR.gamma. at a high level, the patient is likely to respond to hormonal blockade therapy, and if the breast cancer cells are ER.alpha. positive and express ERR.gamma. at low level, the patient is less likely to respond to hormonal blockade therapy.

4. A method for determining whether a breast cancer patient will respond to ErbB4-based therapy comprising the step of: determining the status of ERR.gamma. and ErbB4 in breast cancer cells of the patient, wherein if the breast cancer cells express ErbB4 at high level and ERR.gamma. at a high level, the patient is likely to respond to ErbB4-based therapy, and if the breast cancer cells express ErbB4 at high level and ERR.gamma. at low level, the patient is less likely to respond to ErbB4-based therapy.

5. A method for diagnosing breast cancer comprising the steps of: measuring the ERR.gamma. expression level of breast cells suspected of being cancerous in a human subject; and comparing the expression level to a reference ERR.gamma. level wherein a higher ERR.gamma. expression level than the reference level indicates breast cancer.

6. The method of claim 5, wherein the reference ERR.gamma. level is the ERR.gamma. level of normal breast epithelial cells of the human subject obtained during a breast cancer-free period.

7. The method of claim 5, wherein the reference ERR.gamma. level is the ERR.gamma. level of the non-cancerous breast epithelial cells of the human subject obtained from a disease-free region of the breast.

8. The method of claim 5, wherein the reference ERR.gamma. level is the normal range of ERR.gamma. levels established by analyzing ERR.gamma. levels of normal breast epithelial cells from a large number of human subjects.

9. A method for treating breast cancer comprising the step of decreasing ERR.gamma. activity in breast cancer cells.

10. The method of claim 9, wherein decreasing ERR.gamma. activity is achieved by decreasing the amount of ERR.gamma. protein.

11. The method of claim 9, wherein decreasing ERR.gamma. activity is achieved by treating breast cancer cells with an ERR.gamma. antagonist.

12. A method for identifying an agent that modulates ERR.gamma. expression, the method comprising the steps of: treating a group of cells that express ERR.gamma. with a test agent; determining the level of ERR.gamma. expression of the cells; and comparing the level of ERR.gamma. expression in the cells to that of a group of control cells that are not exposed to the test agent.

13. A method for identifying an agent that modulates ERR.gamma. activity in a cell, the method comprising the steps of: treating a group of cells that express ERR.gamma. with a test agent; determining ERR.gamma. activity in the cells; and comparing ERR.gamma. activity in the cells to that of a group of control cells that are not exposed to the test agent.

14. A method for identifying an agonist or an antagonist of ERR.gamma., the method comprising the steps of: exposing ERR.gamma. to a test agent; determining whether the agent binds to ERR.gamma.; treating a group of cells that express ERR.gamma. with a test agent determined to bind to ERR.gamma.; determining ERR.gamma. activity in the cells; and comparing ERR.gamma. activity in the cells to that of a group of control cells that are not exposed to the test agent.

15. A method for identifying a human subject that is predisposed to developing breast cancer comprising the step of determining whether a mutation exists in the ERR.gamma. gene.

16. A method for identifying a human subject that is predisposed to developing breast cancer comprising the step of determining whether ERR.gamma. is expressed at a high level in breast cells of the human subject.

Description

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. provisional application Serial No. 60/408,400, filed on Sep. 5, 2002.

BACKGROUND OF THE INVENTION

[0003] Breast cancer afflicts one in eight women in the United States over their lifetime (1). ER.alpha. (NR3A1, (2)) mediates estrogen responsiveness (reviewed in (3)) and plays crucial roles in the etiology of breast cancer (reviewed in (4)). It has been developed into the single most important genetic biomarker and target for breast cancer therapy. ER.alpha. is present at detectable levels by ligand-binding and immunohistochemical assays in approximately 75% of clinical breast cancers. Selection of patients with ER.alpha.-positive breast tumors increases endocrine-based therapy response rates from about one-third in unselected patients to about one-half in patients with ER.alpha.-positive tumors (5). Since expression of PgR is dependent upon ER.alpha. activity, further selection of patients with ER.alpha.- and PgR-positive tumors enhances the breast cancer hormonal therapy response rate to nearly 80% (5). Although ER.beta. (NR3A2 (2)) also mediates responses to estrogens (reviewed in (3)), its roles in breast cancer are not as well understood. Reports have linked ER.alpha. expression with low tumor aggressiveness (6) and higher levels of proliferation markers in the absence of ER.alpha. (7).

[0004] Members of the ErbB family of transmembrane tyrosine kinase receptors have been implicated in the pathogenesis of breast cancer. The members include EGFR (also HER1; ErbB1), ErbB2 (HER2; Neu), ErbB3 (HER3) and ErbB4 (HER4) (reviewed in (8)). ErbB members stimulate signal transduction pathways that involve MAPK. In response to initial binding of EGF-like peptide hormones, ErbB members form homodimers and heterodimers in various combinations to recruit distinct effector proteins (reviewed in (9)). Although ErbB2 has not been demonstrated to interact directly with peptide hormones, it serves as a common regulatory heterodimer subunit with other ligand-bound ErbB members (reviewed in (10, 11)). Unlike the other ErbB members, ErbB3 lacks intrinsic kinase activity and, therefore, is required to heterodimerize with other ErbB members to participate in signaling (12).

[0005] Independent overexpression of either EGFR (reviewed in (13)) or ErbB2 (reviewed in (14)) associates with ER-negative tumor status, indicates aggressive tumor behavior, and predicts poor prognosis. Moreover, patients whose tumors coexpress both EGFR and ErbB2 exhibit a worse outcome than patients with tumors that overexpress only one of these genes (15, 16). Overexpression of ErbB2, most often due to gene amplification, occurs in approximately 15-30% of all breast cancers ((17), reviewed in (14)). Some (18-23), but not all reports (24, 25), have implicated ErbB2 in the development of resistance to antiestrogens.

[0006] ErbB2 has been targeted for development of the successful clinical agent Herceptin (trastuzumab), a recombinant humanized monoclonal antibody directed against this receptor's ectodomain (reviewed in (26)). Herceptin has been shown to be a suitable option as a first-line single-agent therapy (27), but will likely prove most beneficial as an adjuvant (28, 29). Clinical trials are currently underway to evaluate the combination of Herceptin with antiestrogens as a rational approach to treating ER.alpha.-positive/ErbB2-overexpressing tumors (23). In the near future, Herceptin will also likely be evaluated in combination with the small molecule EGFR tyrosine kinase inhibitor ZD 1829 (Iressa), since this ATP-mimetic has been shown to almost completely block transphosphorylation of ErbB2 via heterodimerization with EGFR in intact cells (30) and inhibits the growth of breast cancer cell lines overexpressing both EGFR and ErbB2 (31). Hence, a combination of ZD 1829 and Herceptin may be particularly beneficial to those patients whose tumors coexpress EGFR and ErbB2.

[0007] The ability of ErbB3 and ErbB4 to predict clinical course is not as clearly recognized as that of EGFR and ErbB2. ErbB3 has been observed at higher levels in breast tumors than normal tissues, showing associations with unfavorable prognostic indicators including ErbB2 expression (32), lymph node-positive status (33), and tumor size. However, it also associated with ER.alpha.-positive status, a favorable marker of hormonal sensitivity (34). In contrast, ErbB4 has shown associations with only positive indicators. ErbB4 overexpression has been associated with ER.alpha.-positive status (34, 35), more differentiated histotypes (36), a more favorable outcome (16) and it may oppose the negative effects of ErbB2 on clinical course (16).

[0008] Despite the utility of ERs and ErbB members as indicators of clinical course, there remains a great need to identify additional breast cancer biomarkers. A family of potential candidate biomarkers includes the orphan nuclear receptors ERR.alpha. (37-39), ERR.beta. (37, 40), and ERR.gamma. (40-42) (NR3B1, NR3B2, and NR3B3, respectively (2)). These orphan receptors share significant amino acid sequence identity with ER.alpha. and ER.beta.. They also exhibit similar but distinct biochemical and transcriptional activities as the ERs. Each of the ERRs has been demonstrated to bind and activate transcription via consensus palindromic EREs (43-46) in addition to ERREs (39, 42, 44, 47-50), which are composed of an ERE half-site with a 5' extension of 3 base pairs. However, whereas ERs are ligand-activated transcription factors, the ERRs do not bind natural estrogens (37, 51). Instead, the ERRs may serve as constitutive regulators, interacting with transcriptional coactivators in vitro in the absence of ligands (45, 50, 52). Bulky amino acid side chains in the ligand binding pockets of ERRs substitute for the analogous ligand-induced interactions observed in ER.alpha. (52, 53). However, the ligand-binding pockets of the ERRs still allow binding of the synthetic estrogen diethylstilbestrol, but as an antagonist because it also disrupts coactivator interactions with ERRs (51). Similarly, the selective estrogen receptor modulator (SERM) 4-hydroxytamoxifen selectively antagonizes ERR.gamma. in cell-based assays (46, 52, 54).

[0009] The transcriptional activity of each ERR depends upon the promoter and the particular cell line in which it is assayed as well as the presence of ERs. (42-46, 48-51, 53-62) For example, whereas ERR.alpha. stimulates ERE-dependent transcription in the absence of ER.alpha. in HeLa cells, it down-modulates E.sub.2-stimulated transcription in ER.alpha.-positive human mammary carcinoma MCF-7 cells via an active mechanism of repression. (43) ERR.alpha. can also modulate transcription of at least some genes that are estrogen responsive and/or implicated in breast cancer such as pS2 (55), aromatase (59), osteopontin (57, 58) and lactoferrin (56, 61). Thus, the ERRs likely play important roles in at least some breast cancers by modulating or substituting for ER-dependent activities.

BRIEF SUMMARY OF THE INVENTION

[0010] The present invention discloses that ERR.gamma. is both a breast cancer biomarker and a breast cancer treatment target. In one embodiment, the present invention is a method of determining prognosis of breast cancer by determining the level of ERR.gamma. in the breast cancer cells. A low level of ERR.gamma. indicates a poor prognosis and a high level of ERR.gamma. indicates a good prognosis.

[0011] In another embodiment, the present invention is a method of categorizing breast cancer patients for treatment purposes by determining the status of ERR.gamma. either alone or in conjunction with ER.alpha. and/or ErbB4 in breast cancer cells.

[0012] In another embodiment, the present invention is a method of determining whether an individual is at increased risk or predisposed to developing breast cancer by screening for overexpression and/or mutations in the ERR.gamma. gene.

[0013] In another embodiment, the present invention is a method to assist in diagnosing breast cancer by measuring ERR.gamma. expression in the breast cells which are suspected of being cancerous.

[0014] In another embodiment, the present invention is a method of treating breast cancer by decreasing ERR.gamma. activity.

[0015] It is an object of the present invention to identify more biomarkers and treatment targets for breast cancer.

[0016] It is an advantage of the present invention that the biomarker identified helps diagnose, predict prognosis and select treatment strategies for breast cancer.

[0017] Other objects, advantages, and features of the present invention will become apparent from the following detailed description of the invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0018] FIG. 1 shows gene expression distributions of ER and ERR family members (A) and ErbB family members (B). Expression levels are depicted as the 95% confidence intervals of the medians of log.sub.2-transformed values.

[0019] FIG. 2 shows ER family member mRNA levels (A for ER.alpha. levels and B for ER.beta. levels) in normal MECs, breast tumors, and tumors segregated by ER-LB and PgR-LB status. The numbered solid horizontal bars indicate the median level within each group. The dashed horizontal bars in the tumor groups indicate the level 10-fold above the upper limit of the range of expression for the normal MECs. The solid symbols indicate tumors expressing mRNA at levels greater or less than the entire range of expression observed in the normal MECs. Statistical significance was determined by the non-parametric Kruskal-Wallis ANOVA.

[0020] FIG. 3 shows ErbB family member mRNA levels (A for EGFR levels, B for ErbB2 levels, C for ErbB3 levels and D for ErbB4 levels) in normal MECs, breast tumors, and tumors segregated by ER-LB and PgR-LB status. Note the different scales used within (A). The numbered solid horizontal bars indicate the median level within each group. The dashed horizontal bars in the tumor groups indicate the level 10-fold above or below the upper or lower limit, respectively, of the range of expression for the normal MECs. The solid symbols indicate tumors expressing mRNA at levels greater or less than the entire range of expression observed in the normal MECs. Triangles in (A) indicate tumors expressing EGFR mRNA at levels greater or less than one standard deviation surrounding the mean for the tumor group. Statistical significance was determined by the non-parametric Kruskal-Wallis ANOVA.

[0021] FIG. 4 shows ERR family member mRNA levels (A for ERRS levels, B for ERRS levels and C for ERR.gamma. levels) in normal MECs, breast tumors, and tumors segregated by ER-LB and PgR-LB status. The numbered solid horizontal bars indicate the median level within each group. The dashed horizontal bars in the tumor groups indicate the level 10-fold above or below the upper or lower limit, respectively, of the range of expression for the normal MECs. The solid symbols indicate tumors expressing mRNA at levels greater or less than the entire range of expression observed in the normal MECs. Triangles in (A) indicate tumors expressing ERR.alpha. mRNA at levels greater or less than one standard deviation surrounding the mean for the tumor group. Statistical significance was determined by the non-parametric Kruskal-Wallis ANOVA.

[0022] FIG. 5 shows ER.alpha. and ERR.alpha. mRNA levels within the same tissue sample. Significance was assessed by 1-way ANOVA with repeated measures on log.sub.2-transformed values.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The Example below shows that in breast cancer cells, high ERR.gamma. levels associate with ER-LB-positive and PgR-LB-positive status and with DNA diploid status. In breast cancer cells, ERR.gamma. levels also associate with levels of the positive prognosis indicator ErbB4. Thus, ERR.gamma. can be a biomarker for prognosis and for sensitivity to hormonal blockade therapy (such as tamoxifen therapy) in that high levels of ERR.gamma. indicates good prognosis and high likelihood of being sensitive to hormonal blockade therapy. The Example below also shows that breast cancer cells have higher ERR.gamma. levels than normal mammary epithelial cells indicating that ERR.gamma. plays a role in breast cancer development. Thus, ERR.gamma. may serve as a diagnostic marker and a treatment target. Furthermore, overexpression of ERR.gamma. or mutations in ERR.gamma. may predispose a human being for breast cancer development. Therefore, screening for ERR.gamma. overexpression and mutations may help identify individuals for further breast cancer examination. Without intending to be limited by theory, we believe that high levels of ERR.gamma. can lead to the development of breast cancer, but breast cancer with a high level of ERR.gamma. has a good prognosis because it is likely sensitive to hormonal blockade therapy.

[0024] In the specification and claims, the term "status" of ERR.gamma., ER.alpha. and ErbB4 refers to the "expression status" of the genes. The status can be determined either at the mRNA level or at the protein level, and either qualitatively (expressed or not expressed) or quantitatively (the level of expression). There are many methods known to a skilled artisan that can be used in the present invention to determine the status of ERR.gamma. and ER.alpha.. As an example, the status of ER.alpha. was determined by the ligand binding assays in the Example below.

[0025] In one aspect, the present invention is a method for determining breast cancer prognosis by analyzing the status of ERR.gamma. in the breast cancer tissue. A high ERR.gamma. level in breast cancer cells indicates good prognosis and a low ERR.gamma. level in breast cancer cells indicates poor prognosis. This means that after diagnosis, breast cancer patients with a high ERR.gamma. level have a greater likelihood of responding to treatment such as hormonal blockade than those with low ERR.gamma. levels. As used in the specification and claims, a high or low level of ERR.gamma. in breast cancer cells is measured against a median level of ERR.gamma. obtained from breast cancer tissues of multiple patients. The more the breast cancer tissue samples of different breast cancer patients are used to establish the median level, the more accurate the median level is. Preferably, the median level is obtained from analyzing breast cancer tissues of at least 25 patients. The high or low level of ERR.gamma. in breast cancer cells can also be measured against the median or average level of ERR.gamma. in normal breast cells obtained from multiple human subjects. Preferably, normal breast cells of at least 25 human subjects are used to establish the median or average level of ERR.gamma.. The high or low level of ERR.gamma. in breast cancer cells can also be measured against other standards that a skilled artisan can readily establish. For example, ERR.gamma. levels of normal breast epithelial cell lines and breast cancer cell lines can be used as standards.

[0026] In another aspect, the present invention is a method for categorizing breast cancer patients for treatment purposes. The method involves determining the status of ERR.gamma. in breast cancer cells. Because 4-OH tamoxifen is an antagonist of ERR.gamma., if the breast cancer cells of a patient exhibit high levels of ERR.gamma., the patient has a greater likelihood of responding to tamoxifen therapy. Tamoxifen therapy should be provided to such a patient.

[0027] In a preferred embodiment of a method of categorizing breast cancer patients for treatment purposes, the ERR.gamma. status is determined in conjunction with ER.alpha. status. If the breast cancer cells of a patient are ER.alpha.-positive and exhibit high levels of ERR.gamma., the patient is likely to respond to hormonal blockade therapy. Hormonal blockade therapy includes the use of anti-estrogens, aromatase inhibitors and other agents that can block the production or activity of estrogen.

[0028] If the breast cancer cells of a patient are ER.alpha.-positive and exhibit low levels of ERR.gamma., the patient may still respond to hormonal blockade therapy but the likelihood is less than those patients who have ER.alpha.-positive cancers that also exhibit a high level of ERR.gamma. expression. Thus, hormonal blockade therapy remains an option for patients with ER.alpha.-positive and low level ERR.gamma. cancers. However, other therapies should be considered as well for these patients.

[0029] If the breast cancer cells of a patient are ER.alpha.-negative and have high levels of ERR.gamma., the patient may still respond to tamoxifen, although the likelihood is less than those patients who have ER.alpha.-positive cancers that also exhibit a high level of ERR.gamma. expression. The reason that such a patient may respond to hormonal blockade therapy is that the antiestrogen 4-hydroxytamoxifen binds to and antagonizes the activity of ERR.gamma.. Therefore, a high level of ERR.gamma. expression in cancers of patients that are also ER.alpha.-negative identifies a subset of patients that may benefit from tamoxifen therapy, but otherwise would not have been good candidates for tamoxifen therapy. Nonetheless, while tamoxifen therapy remains an option for patients with ER.alpha.-negative cancers that also exhibit a high level of ERR.gamma. expression, other breast cancer therapies should be considered as well.

[0030] If the breast cancer cells of a patient are ER.alpha.-negative and have low levels of ERR.gamma., the patient is not likely to respond to hormonal blockade therapy. Other breast cancer therapies should be provided for the patient.

[0031] In another embodiment of a method of categorizing breast cancer patients for treatment purposes, the ERR.gamma. status is determined in conjunction with ErbB4 status. In the Example described below, ERR.gamma. levels in breast cancer cells correlate with ErbB4 levels. We hypothesize that ERR.gamma. is a downstream target of ErbB4 signal transduction. By analogy with ErbB2, there is the potential that anti-tumor agents will be developed that target ErbB4. For instance, the anti-ErbB2 monoclonal antibody Herceptin has been developed and is currently in use in the clinic as a therapeutic agent. ErbB4-based therapy includes the use of agents that either directly inhibit ErbB4 activity (anti-ErbB4 antibodies and ErbB4-specific kinase inhibitors) or indirectly inhibit ErbB4 activity through heterodimerization with other ErbB members. If ErbB4-based therapies are developed for the treatment of breast cancer, when the breast cancer cells of a patient have a high ErbB4 level and a high ERR.gamma. level, the patient is likely to respond to the ErbB4-based therapy. When the breast cancer cells of a patient have a high ErbB4 level and a low ERR.gamma. level, the patient is less likely to respond to ErbB4-based therapy. However, breast cancer therapies other than ErbB4-based therapy and hormonal blockade therapy should be seriously considered as well.

[0032] In another aspect, the present invention is a method for diagnosing breast cancer by measuring the ERR.gamma. level in the suspected or abnormal breast cells and comparing it to a reference normal level. If someone has a lump or a locus in a breast that is suspected of being breast cancer, cells from the lump or locus can be taken and the ERR.gamma. level can be measured. The reference normal level to which the measured ERR.gamma. level is compared can be obtained from several sources. For example, the reference normal level can be the ERR.gamma. level of normal mammary epithelial cells obtained from the same human subject at the time the sample from the suspected lump or locus was taken; the reference normal level can also be the ERR.gamma. level of normal mammary epithelial cells obtained from the same human subject during a breast cancer-free period; the reference normal level can also be a normal range of ERR.gamma. levels determined by analyzing a large number of normal breast tissue samples from multiple human subjects. Finding that the cells from the lump or locus exhibit an ERR.gamma. level higher than the reference normal level is indicative of the tissue in question being cancerous. In a preferred embodiment, the ERR.gamma. level is used in combination with other breast cancer diagnostic tools such as pathological analysis of suspected tissue to diagnose breast cancer.

[0033] In another aspect, the present invention is a method of treating breast cancer by decreasing ERR.gamma. activity. For example, an antisense oligonucleotide or small inhibitor RNA (siRNA) targeted against ERR.gamma. can be used to inhibit the RNA expression of ERR.gamma.. Other agents that can decrease ERR.gamma. expression can be identified by exposing a group of cells that express ERR.gamma. to a test agent and comparing the ERR.gamma. expression of these cells at the mRNA level or at the protein level to that of control cells that are not exposed to the agent.

[0034] One can also use an ERR.gamma. antagonist to decrease ERR.gamma. activity. 4-hydroxytamoxifen is an ERR.gamma. antagonist. Other ERR.gamma. antagonists can be identified by exposing ERR.gamma. to a test agent and determining whether the test agent binds to ERR.gamma.. There are many ways that this can be done. For example, a test agent can be labeled and ERR.gamma. can be exposed to the test agent, washed and purified. If the label is detected with the purified ERR.gamma., then the test agent has bound to ERR.gamma.. Once a test agent is determined to be capable of binding ERR.gamma., whether the agent can inhibit ERR.gamma. activity can be determined. For example, a reporter gene for ERR.gamma. activity with and without an ERR.gamma. expression plasmid can be introduced into a cell. The cell can then be exposed to the agent and the reporter gene expression levels in the absence and presence of overexpressed ERR.gamma. can be determined.

[0035] In a more general manner, a cell culture reporter gene assay for ERR.gamma. can be used directly to screen for agents that can decrease ERR.gamma. activity by exposing the cells to a test agent and determining the reporter gene expression levels in the absence and presence of overexpressed ERR.gamma..

[0036] ERR.gamma. activity can also be inhibited by an ERR.gamma. antibody (either monoclonal or polyclonal), which can be readily generated by a skilled artisan. Encapsulating liposomes or other similar technology can be used to deliver anti-ERR.gamma. antibodies into cells. Alternatively, recombinant DNAs expressing an anti-ERR.gamma. antibody (such as in the form of a single-chain antibody fragment) can be introduced into cells via gene therapy methodologies.

[0037] In another aspect, the present invention is a method of identifying an individual for further breast cancer examination by screening the individual for overexpression of the ERR.gamma. gene, for mutations in the ERR.gamma. gene, or for both. An individual who overexpresses or carries a mutated ERR.gamma. gene should be monitored closely for breast cancer development. Whether ERR.gamma. is overexpressed can be determined using a reference normal level described above. Whether a mutation exists in the ERR.gamma. gene is determined using a reference ERR.gamma. sequence. For example, the National Center for Biotechnology Information (NCBI) Reference Sequence entry for ERR.gamma., NM.sub.--001438, can be used as a reference ERR.gamma. sequence. An individual who overexpresses or carries a mutated ERR.gamma. gene should be monitored closely for breast cancer development.

[0038] The invention will be more fully understood upon consideration of the following non-limiting example.

EXAMPLE

Estrogen-Related Receptor .alpha. and Estrogen-Related Receptor .gamma. Associate with Unfavorable and Favorable Biomarkers, Respectively, in Human Breast Cancer

[0039] Abstract

[0040] The importance of estrogen-related receptors (ERRs) in human breast cancer was assessed by comparing their mRNA profiles with established clinicopathologic indicators and mRNA profiles of estrogen receptors (ERs) and ErbB family members. Using real-time quantitative polymerase chain reaction assays, mRNA levels of ER.alpha., ER.beta., EGFR (epidermal growth factor receptor), ErbB2, ErbB3, ErbB4, ERR.alpha., ERR.beta., and ERR.gamma. were determined in unselected primary breast tumors (n=38) and normal mammary epithelial cells (MECs) enriched from reduction mammoplasties (n=9). ERR.alpha. was shown to be a biomarker of unfavorable clinical outcome and hormonal insensitivity. ERR.alpha. mRNA was expressed at levels greater than or similar to ER.alpha. mRNA in 24% of unselected breast tumors, and generally at higher levels than ER.alpha. in the PgR-negative tumor subgroup (1-way ANOVA with repeated measures, P=0.030). Increased ERR.alpha. levels associated with ER-negative (Fisher's exact, P=0.003) and PgR-negative tumor status (Fisher's exact, P=0.006; Kruskal-Wallis ANOVA, P=0.021). ERR.alpha. levels also correlated with expression of ErbB2 (Spearman's rho, P=0.005), an indicator of aggressive tumor behavior. Thus, ERR.alpha. was the most abundant nuclear receptor in a subset of tumors that tended to lack functional ER.alpha. and expressed ErbB2 at high levels. Consequently, ERR.alpha. may potentiate constitutive transcription of estrogen response element-containing genes independently of ER.alpha. and antiestrogens in ErbB2-positive tumors. ERR.beta.'s potential as a biomarker remains unclear: it showed a direct relationship with ER.beta. (Spearman's rho, P=0.0002) and an inverse correlation with S-phase fraction (Spearman's rho, P=0.026). Unlike ERR.alpha., ERR.gamma. was shown to be a biomarker of favorable clinical course and hormonal sensitivity. ERR.gamma. was overexpressed in 75% of the tumors, resulting in the median ERR.gamma. level being elevated in breast tumors compared to normal MECs (Kruskal-Wallis ANOVA, P=0.001). ERR.gamma. overexpression associated with hormonally responsive ER- and PgR-positive status (Fisher's exact, P=0.054 and P=0.045, respectively). Additionally, ERR.gamma. expression correlated with levels of ErbB4 (Spearman's rho, P=0.052), a likely indicator of preferred clinical course, and associated with diploid-typed tumors (Fisher's exact, P=0.042). Hence, ERR.alpha. and ERR.gamma. status can be predictive of sensitivity to hormonal blockade therapy, and ERR.alpha. status can also be predictive of ErbB2-based therapy such as Herceptin. Moreover, ERR.alpha. and ERR.gamma. are targets for therapeutic development.

[0041] Materials and Methods

[0042] Tissue Sources: Random primary breast cancer samples were obtained from the National Breast Cancer Tissue Resource SPORE at Baylor College of Medicine (Houston, Tex.). Records of previously determined clinicopathologic tumor biomarkers were maintained at the SPORE, including ER-LB (ligand binding) and PgR-LB protein levels measured by the ligand-binding assay, and S-phase fraction and DNA ploidy determined by flow cytometry. The mRNA profiling studies were conducted in a blinded manner regarding these previously determined biomarkers.

[0043] As a basis of comparison, mammary gland tissues were also profiled for mRNA expression. Because bulk mammary gland contains overwhelming amounts of adipose, it was necessary to enrich for epithelial cells prior to isolation of nucleic acid. Hence, mammary tissues from reduction mammoplasties were processed through collagenase digestion and differential centrifugation and filtration steps. These enriched MECs were kindly provided by Dr. Stephen Ethier (University of Michigan-Ann Arbor) and Dr. Michael N. Gould (University of Wisconsin-Madison). The normal MECs used here were not expanded in culture to avoid any possible changes in RNA profiles that might occur with passage. All use of human tissues was approved by the University of Wisconsin's Human Subjects Committee.

[0044] Real-time Q-PCR Assays: The mRNA abundances of ER, ErbB and ERR family members were determined by real-time Q-PCR assays. As detailed below, amplification of PCR products was continuously monitored by fluorescence facilitated by specific complex formation of SYBR Green I with double-stranded DNA (reviewed in (63)).

[0045] Total RNA was isolated from tissues using the Total RNeasy kit (Qiagen; Valencia, Calif.), treated with RNase-free DNase I (Ambion; Austin, Tex.), and again purified with the Total RNeasy kit. cDNA was synthesized by incubation of 10 .mu.g total RNA with SuperScript II reverse transcriptase (Invitrogen Life Technologies; Carlsbad, Calif.) and 50 nmoles each of oligo dT.sub.15VN (where V=A, G, or C and N=any nucleotide) and random hexamers as primers in a total reaction volume of 100 .mu.l at 45.degree. C. for 1 h. Because the quality of the mRNA purified from the tumors likely varied considerably, differences in mRNA integrity were compensated for by careful quantitation by trace radiolabel incorporation of the amount of cDNA synthesized from each sample followed by use of the same amount of cDNA in each Q-PCR assay. Quantitation of cDNA involved trace radiolabeling of a parallel cDNA synthesis reaction carried out in the presence of [.alpha.-.sup.32P]dCTP. Incorporated and total radiolabeled amounts were measured in triplicate by trichloroacetic acid precipitation and scintillation counting. Calculation of the total mass of cDNA synthesized was based upon the molar amount of nucleotides present in the reaction converted to mass and multiplied by the ratio of incorporated-to-total radiolabel. Q-PCR assays involving tissue samples employed 1 ng cDNA as template and were carried out in triplicate.

[0046] PCR primer sets were designed to promote efficient amplification by yielding products smaller than 150 bp in length. PCR products were verified by sequence analysis. The PCR primer set sequences and amplicon sizes were as follows: ER.alpha. forward primer 5'-GGAGGGCAGGGGTGAA-3' (SEQ ID NO:1) and reverse primer 5'-GGCCAGGCTGTTCTTCTTAG-3' (SEQ ID NO:2), 100-bp amplicon; ER.beta. forward primer 5'-TTCCCAGCAATGTCACTAACTT- -3' (SEQ ID NO:3) and reverse primer 5'-TTGAGGTTCCGCATACAGA-3' (SEQ ID NO:4), 137-bp amplicon; EGFR forward primer 5'-GTGACCGTTTGGGAGTTGATGA-3' (SEQ ID NO:5) and reverse primer 5'-GGCTGAGGGAGGCGTTCTC-3' (SEQ ID NO:6), 104-bp amplicon; ErbB2 forward primer 5'-GGGAAGAATGGGGTCGTCAAA-3' (SEQ ID NO:7) and reverse primer 5'-CTCCTCCCTGGGGTGTCAAGT-3' (SEQ ID NO:8), 82-bp amplicon; ErbB3 forward primer 5'-GTGGCACTCAGGGAGCATTTA-3' (SEQ ID NO:9) and reverse primer 5'-TCTGGGACTGGGGAAAAGG-3' (SEQ ID NO:10), 106-bp amplicon; ErbB4 forward primer 5'-TGCCCTACAGAGCCCCAACTA-3' (SEQ ID NO:11) and reverse primer 5'-GCTTGCGTAGGGTGCCATTAC-3' (SEQ ID NO:12), 105-bp amplicon; ERR.alpha. forward primer 5'-AAAGTGCTGGCCCATTTCTAT-3' (SEQ ID NO:13) and reverse primer 5'-CCTTGCCTCAGTCCATCAT-3' (SEQ ID NO:14), 100-bp amplicon; ERR.beta. forward primer 5'-TGCCCTACGACGACAA-3' (SEQ ID NO:15) and reverse primer 5'-ACTCCTCCTTCTCCACCTT-3' (SEQ ID NO:16), 144-bp amplicon; and ERR.gamma. forward primer 5'-GGCCATCAGAACGGACTTG-3' (SEQ ID NO:17) and reverse primer 5'-GCCCACTACCTCCCAGGATA-3' (SEQ ID NO:18), 67-bp amplicon. PCR primer sequences were designed using Oligo 5.0 software (National Biosciences; Plymouth, Minn.) and synthesized at the University of Wisconsin-Biotechnology Center (Madison, Wis.).

[0047] Serial dilution standard curves of each specific PCR product were included in every experiment and allowed calculation of transcript copy numbers in the unknown samples by regression analysis. The PCR product standards were in the form of ssDNA to better emulate cDNA in the unknown samples. The ssDNA standards were produced by linear amplification, using only the reverse primer (corresponding to the non-coding DNA strand), instead of by exponential amplification with two primers. The amount of each template required for the standard curves was determined in a similar manner as described above by trace radiolabeling with [.alpha.-.sup.32P]dCTP incorporation during the PCR amplification process. The mass of PCR product synthesized was converted to copy numbers according to the molecular weight of the specific amplicon's size in base pairs. All standard curves covered eight orders-of-magnitude and were assayed in triplicate.

[0048] Q-PCR assays were carried out in a total volume of 20 .mu.l with 10 .mu.l of 0.1 ng/.mu.l cDNA. SYBR Green I (Molecular Probes; Eugene, Oreg.) was diluted in anhydrous DMSO at 1:2,500, then added to the enzyme reaction buffer to obtain a final concentration of 1:50,000 SYBR green I and 5% DMSO. To normalize fluorescence intensity between samples, the enzyme reaction buffer contained 180 nM passive reference dye ROX (Molecular Probes). The final concentrations of the remaining constituents were as follows: 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl.sub.2, 50 .mu.M each dNTP, 500 nM each forward and reverse primer, and 0.025 units per .mu.l HotStar Taq DNA polymerase (Qiagen). The thermal cycling parameters were 1 cycle of 95.degree. C. for 10 min, and 40 cycles of 96.degree. C. denaturation for 15 s followed by 60.degree. C. annealing/extension for 1 min. Q-PCR assays were carried out with an ABI 7700 sequence detection system (Applied Biosystems, Foster City, Calif.).

[0049] ER and PgR by Ligand-binding Assays: ER and PgR content of the breast tumors were previously determined in a central laboratory. The standard multipoint, dextran-coated charcoal assay was modified as previously described (64) to incorporate .sup.125I-labeled estradiol and .sup.3H-labeled R5020 in a single assay, allowing the simultaneous determination of both ER and PgR. ER-LB levels greater than or equal to 3 fmol/mg protein were considered positive, and PgR-LB levels greater than or equal to 5 fmol/mg protein were considered positive.

[0050] DNA Ploidy and S-phase Fraction by Flow Cytometry: Flow cytometry was performed as previously described to determine DNA ploidy and S-phase fraction (64, 65). S-phase fractions were estimated using the MODFIT program (Verity House Software, Inc., Topsham, Me.). S-phase fractions less than 6% were considered low; S-phase fractions greater than 10% were considered high; and values between 6 and 10% were considered intermediate.

[0051] Statistics: Differences in abundance among separate mRNA species grouped by biological function were evaluated as independent variables using the 95% confidence intervals of the medians of log.sub.2-transformed values (FIG. 1). Changes in the abundance of a single mRNA species between tissue groups were tested by the non-parametric Kruskal-Wallis ANOVA (FIG. 2, FIG. 3, and FIG. 4). Associations between aberrant mRNA levels and clinicopathological biomarkers in the breast tumors were evaluated by Fisher's exact tests (Table 1). To analyze aberrant tumor expression relative to MECs, high and low expression in the breast tumors was defined as mRNA levels above or below, respectively, the range of expression in the normal MECs. Similarly, very high and very low expression in the tumors was defined as 10-fold above or below, respectively, the range of expression in normal MECs. Additionally, to analyze aberrant tumor expression relative to only other tumors and not MECs in the cases of EGFR and ERR.alpha., typical expression was defined as being within a standard deviation and aberrant expression as greater than a standard deviation away from the mean tumor level. Differences in expression between ER.alpha. and ERR.alpha. mRNA levels within the same tissue sample were assessed by 1-way ANOVA with repeated measures on log.sub.2-transformed data (FIG. 5). To discern whether ER.alpha. and ERR.alpha. were expressed at approximately equivalent levels within tumors, the ratio of their levels was stratified according to those found in normal MECs; ratios within a standard deviation of the average ratio in normal MECs were defined as equivalent. Pairwise relationships among gene expression levels and clinicopathologic factors were tested by the non-parametric rank correlation method, Spearman's rho analysis (Table 2). Spearman rank correlations involving ER-LB assays, PgR-LB assays, S-phase fraction and DNA ploidy used raw values on continuous scales instead of simple status assessments. All of the analyses described above were performed using SAS version 8.2 from SAS Institute, Inc. (Cary, N. C.).

[0052] Results and Discussion

[0053] Statistical Considerations: The sample size in this study was modest: 38 tumors and 9 normal MEC preparations. However, statistically significant results observed with this modest sample size can indicate truly important relationships and differences. Notably, gene expression was accurately measured, even when at low levels, because of the use of real-time Q-PCR, thereby allowing much finer stratification of tissue samples than would have been possible by less quantitative methods (e.g., IHC or ligand-binding assays). Consequently, these more refined stratifications allowed improved statistical considerations given the modest sample size.

[0054] ER.alpha. exhibited significantly higher mRNA levels than the other evaluated nuclear receptors in approximately three-fourths of the tumors (FIG. 1A, compare FIG. 2A with FIG. 2B, FIG. 4, and FIG. 5). The median ER.alpha. mRNA level was 14-fold higher in breast carcinomas compared to normal MECs (Kruskal-Wallis ANOVA, P=0.002; FIG. 2A) and expressed at high or very high levels in 74% (28 of 38) of the breast tumors (FIG. 2A). These results exemplify the critical role ER.alpha. plays in the majority of breast cancers. The median ER.alpha. mRNA level was 34-fold greater in ER-LB-positive and 31-fold greater in PgR-LB-positive tumors relative to negative tumors (Kruskal-Wallis ANOVA, P<0.0001 and P=0.0001, respectively; FIG. 2A). Tumors that overexpressed ER.alpha. mRNA segregated with ER-LB and PgR-LB-positive status (Fisher's exact, P<0.0001 and P<0.0001, respectively; Table 1). Further, ER.alpha. mRNA levels strongly correlated with ER-LB (p.sub.s=0.86, P<0.0001; Table 2) and PgR-LB protein levels (p.sub.s=0.68, P<0.0001; Table 2) in the tumors as evaluated using the raw ligand-binding values over a continuous scale. These expected relationships validated the real-time Q-PCR assays and conformed well with established findings of others regarding both typical percentage of ER-LB-positive tumors and elevated levels of ER.alpha. in these tumors (67).

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