Identification of gene regulation patterns underlying both oestrogen- and tamoxifen-stimulated cell growth through global gene expression profiling in breast cancer cells

Abstract

Purpose A c-Src inhibitor blocks estrogen (E2)-induced stress and converts E2 responses from inducing apoptosis to growth stimulation in E2-deprived breast cancer cells. A reprogrammed cell line, MCF-7:PF, results in a functional estrogen receptor (ER). We addressed the question of whether the selective ER modulator 4-hydroxytamoxifen (4-OHT) could target ER to prevent E2-stimulated growth in MCF-7:PF cells. Methods Expression of mRNA was measured through real-time RT-PCR. Global gene expression profile was analyzed through microarray. Transcriptome profiles were screened by RNA-sequencing. Results Unexpectedly, both 4-OHT and E2 stimulated cell growth in a concentration-dependent manner. Expression profiling showed a remarkable overlap in genes regulated in the same direction by E2 and 4-OHT. Pathway enrichment analysis of the 280 genes commonly deregulated in MCF-7:PF cells by 4-OHT and E2 revealed functions mainly related to membrane, cytoplasm, and metabolic processes. Further analysis of 98 genes up-regulated by both 4-OHT and E2 uncovered a significant enrichment in genes associated with membrane remodeling, cytoskeleton reorganization, cytoplasmic adapter proteins, cytoplasm organelles proteins, and related processes. 4-OHT was more potent than E2 in up-regulating some membrane remodeling molecules, such as EHD2, FHL2, HOMER3 and RHOF. In contrast, 4-OHT acted as an antagonist to inhibit expression of the majority of enriched membrane-associated genes in wild-type MCF-7 cells. Conclusions Long-term selection pressure has changed the cell population responses to 4-OHT. Membrane-associated signaling is critical for 4-OHT-stimulated cell growth in MCF-7:PF cells. This study provides a rationale for the further investigation of target therapy for tamoxifen resistant patients.

The function of membrane-associated molecules in acquired resistance to antiestrogens in breast cancer

“…It therefore appears that ER remains fully functional in the acquired tamoxifen resistance. In support with these findings, global gene expression microarray in back-to-back article reveals a remarkable overlap in genes deregulated in the same direction by E2 and 4-OHT in MCF-7: PF cells [14] . The deregulation of these genes by E2 or 4-OHT is able to be blocked by the pure antiestrogen ICI [14] .…”

“…In support with these findings, global gene expression microarray in back-to-back article reveals a remarkable overlap in genes deregulated in the same direction by E2 and 4-OHT in MCF-7: PF cells [14] . The deregulation of these genes by E2 or 4-OHT is able to be blocked by the pure antiestrogen ICI [14] . In contrast to E2 that activates classical ER-target genes, SERMs continue to act as effective antiestrogens to inhibit classical ER-target genes, even at the time of growth stimulation [11,14] .…”

“…In addition, the well known focal adhesion molecules FAK and p130CAS are activated by 4-OHT which participate in the non-genomic pathway of ER [11,18] . Our pathway enrichment analysis also reveals a significant enrichment in a variety of genes associated with actin cytoskeleton remodeling, adaptor proteins, and other membrane-related functions activated by 4-OHT or E2 in MCF-7: PF cells [14] . For example, both E2 and 4-OHT up-regulated EH-domain containing 2 (EHD2) [14,19] , four and a half LIM domains 2 (FHL2) [14,20] , homer homolog 3 (HOMER3) [14,21] , and Ras homolog family member F (RHOF) [14,22] .…”

“…The deregulation of these genes by E2 or 4-OHT is able to be blocked by the pure antiestrogen ICI [14] . In contrast to E2 that activates classical ER-target genes, SERMs continue to act as effective antiestrogens to inhibit classical ER-target genes, even at the time of growth stimulation [11,14] . This result is supported by our previous finding in vivo [15] that growth of tamoxifen or fulvestrant resistant tumors does not rely on classical ER transcriptional pathways, which is evidenced by suppression of E2-responsive genes [15] .…”

“…Inhibition of tyrosine kinase activities of IGF-1R or c-Src is able to modulation the expression of these molecules [14] , demonstrating functional interactions exist among these membrane function-associated molecules and growth pathways [14] . Quite interestingly, in contrast to the overlapping gene regulation by E2 or 4-OHT in MCF-7:PF cells [14] , the gene regulation pattern of 4-OHT is distinct from that of E2 in wild-type MCF-7 cells [14] , in which 4-OHT acts primarily to antagonize these membrane function-associated molecules [14] . These findings suggest that the constant nuclear pressure by SERMs causes broad activation of membrane-associated signaling to aid breast cancer cell survival and creates new surviving cell populations during the selection process required for acquired SERM resistance.…”