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Senior Member
Join Date: Jun 2006
Location: Bradenton,FL
Posts: 977
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New findings on Tamoxifen resistance...
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Regulation of ERBB2 by Estrogen Receptor−PAX2 Determines Response to Tamoxifen
Nature. 2008 Nov 12;[Epub ahead of print], A Hurtado, KA Holmes, TR Geistlinger, IR Hutcheson, RI Nicholson, M Brown, J Jiang, WJ Howat, S Ali, JS Carroll
Estrogen-receptor (ER)−positive breast cancers that overexpress the ERBB2/HER2 oncogene confer a poor prognosis. Genetic mapping of ER-binding sites indicates that ER-gene promoter binding (and therefore transcription) requires the presence of a pioneer transcription factor. Using genome-wide ER chromatin immunoprecipitation (ChIP)–on-chip analyses of ER-positive MCF-7 breast cancer cells, researchers identified a novel ER-binding site with homology to paired box (PAX) transcription factors that was located in the intragenic region of the ERBB2 proto-oncogene. Preliminary evidence suggests that PAX has a role in hormone signaling. PAX2 is expressed in certain breast cancer cells and was recently shown to be involved in tamoxifen regulation in endometrial cancer. Tamoxifen resistance in breast cancer is common. Tamoxifen-resistant tumors are characterized by ERBB2 overexpression; in addition, as many as half of ER-positive, ERBB2-overexpressing cells develop acquired resistance to tamoxifen. Tamoxifen-resistant breast cancers also exhibit increased levels of a steroid receptor co-activator, amplified in breast cancer–1 (AIB-1) factor (also known as SRC-3), which promotes tumorigenesis. AIB-1 expression is essential for ERBB2-driven oncogenesis.
Researchers from the Cambridge Research Institute (UK), Dana-Farber Cancer Institute/Harvard Medical School (Boston), Cardiff University (UK), and Imperial College (London) conducted a series of in vitro experiments in several breast cancer cell lines to investigate the role of PAX2 binding to the novel ER-binding site within ERBB2. PAX2 was recruited to this site after treatments with estrogen and tamoxifen. Because ERBB2 can be suppressed by both estrogen and tamoxifen, the researchers hypothesized that PAX2 may function as an ER-associated transcription repressor. An additional hypothesis was that the newly discovered ER-binding site within ERBB2 may be a cis-regulatory element involved in ER repression. The researchers also postulated that the antiproliferative effects of tamoxifen repress ERBB2 and that this dysregulation of ERBB2 may result in acquired tamoxifen resistance.
The results of the study showed that inhibition of PAX2 binding to the cis-regulatory element represents a crucial mechanism for tamoxifen resistance in breast cancer cells. In addition, competitive displacement by AIB-1 confers acquired resistance to tamoxifen. Therefore, the balance between PAX2 transcriptional repression and AIB-1 activation of the cis-regulatory element determines the level of ERBB2 expression, and thus response to tamoxifen, in breast cancer cells.
Co-immunoprecipitation experiments showed that dimerization of ER and PAX2 occurs after treatment with tamoxifen. Re-ChIP experiments were used to confirm that ER and PAX2 simultaneously occupy the same ER-binding site within the ERBB2 gene. To investigate the role of PAX2 in estrogen- and tamoxifen-mediated ERBB2 repression in ER-positive MCF-7 tumor cells, gene silencing of PAX2 was performed using short interfering (si) RNAs. In contrast to the results of control experiments, in which the introduction of both estrogen and tamoxifen repressed ERBB2, ERBB2 expression increased when PAX2 was inhibited. In addition, tumor cells proliferated in the experimental, but not the control, cells. The experiment was repeated using pretreatment with Herceptin (ie, anti-ERBB2 antibody). The result was a reversal of cell proliferation, suggesting that PAX2 inhibition leads to increased ERBB2 expression. The experiments were subsequently validated using ZR75-1 cells.
The researchers next evaluated the potential for competitive binding of AIB-1, and thus displacement of PAX2 binding, to the newly identified cis-regulatory element of ERBB2. In the presence of both tamoxifen and estrogen, PAX2 inhibition via (si)RNAs resulted in recruitment of AIB-1 to the ERBB2-binding site and increased ERBB2 expression. The results suggested that AIB-1 expression competes with PAX2 for binding to the cis-regulatory element. Thus, PAX2 repressor binding is inhibited and ERBB2 expression increases. Therefore, tumor growth occurs in the presence of tamoxifen because the antiproliferative effects of tamoxifen are reversed. The results were subsequently validated in TD47D cells.
The next experiment showed that ERBB2 expression was reduced by 40% in wild-type MCF-7 cells that were treated with tamoxifen. In contrast, a tamoxifen-resistant MCF-7 cell line (Tam-R) showed elevated ERBB2 expression. Although ER and AIB-1 levels were similar in both cultures, PAX2 levels were lower in the Tam-R cells, suggesting that ERBB2 overexpression occurs in the absence of PAX2.
Finally, immunohistochemical detection of PAX2 was used to confirm these findings in 109 ER-positive samples from women with breast cancer. Recurrence-free survival (RFS) was significantly better in the 68 women whose tumors were PAX2 positive than in the 41 women whose tumors were PAX2 negative (P < .0001). Moreover, among women with PAX2-positive tumors, those whose tumors were also AIB-1 negative had better RFS than did those whose tumors were AIB-1 positive. In addition, the PAX2-positive, AIB-1–negative tumors had decreased ERBB2 levels. Cox regression analysis showed that PAX2 and AIB-1 levels were inversely associated with relapse (P < .03).
In summary, the researchers showed that PAX2 is a key regulator of tamoxifen-induced repression of ERBB2 gene expression in breast cancer cells. PAX2 and AIB-1 competitively bind to a cis-regulatory element located within the ERBB2 gene, and the balance between these 2 components within breast tumor cells determines the efficacy of tamoxifen by means of up-regulation or down-regulation of ERBB2. The study results suggest a mechanistic association between ER- and ERBB2-driven breast cancers. "
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