Epidermal Growth Factor Receptor (EGFR)
Introduction: Basal-like breast cancers (BLBCs) are very aggressive, and present serious clinical challenges as there are currently no targeted therapies available. We determined the regulatory role of Y-box binding protein-1 (YB-1) on epidermal growth factor receptor (EGFR) overexpression in BLBC, and the therapeutic potential of inhibiting EGFR. We pursued this in light of our recent work showing that YB-1 induces the expression of EGFR, a new BLBC marker.
Methods: Primary tumour tissues were evaluated for YB1 protein expression by immunostaining tissue microarrays, while copy number changes were assessed by comparative genomic hybridization (CGH). The ability of YB-1 to regulate EGFR was evaluated using luciferase reporter, chromatin immunoprecipitation (ChIP) and gel shift assays. The impact of Iressa on monolayer cell growth was measured using an ArrayScan VTI high-throughput analyser to count cell number, and colony formation in soft agar was used to measure anchorage-independent growth.
Results: YB-1 (27/37 or 73% of cases, P = 3.899 x 10) and EGFR (20/37 or 57.1% of cases, P = 9.206 x 10) are expressed in most cases of BLBC. However, they are not typically amplified in primary BLBC, suggesting overexpression owing to transcriptional activation. In support of this, we demonstrate that YB-1 promotes EGFR reporter activity. YB-1 specifically binds the EGFR promoter at two different YB-1-responsive elements (YREs) located at -940 and -968 using ChIP and gel shift assays in a manner that is dependent on the phosphorylation of S102 on YB-1. Inhibiting EGFR with Iressa suppressed the growth of SUM149 cells by ~40% in monolayer, independent of mutations in the receptor. More importantly anchorage-independent growth of BLBC cell lines was inhibited with combinations of Iressa and YB-1 suppression.
Conclusion: We have identified for the first time a causal link for the expression of EGFR in BLBC through the induction by YB-1 where it binds specifically to two distinguished YREs. Finally, inhibition of EGFR in combination with suppression of YB-1 presents a potential opportunity for therapy in BLBC.
Identifying molecular targets for aggressive types of breast cancer is a milestone in the pursuit of individualized therapies. Gene-expression profiling of primary tumours has led to the following subcategories: luminal A, luminal B, the human epidermal growth factor receptor 2 (HER2) and the basal-like subtypes. Our attention was drawn to the basal-like subtype, because these tumours do not respond to available targeted therapies and patients often die within two years of diagnosis. Approximately 16% of all breast cancers are basal-like; this corresponds to 46,400 women among the ~290,000 women in North America who will be diagnosed with breast cancer each year. What sets these tumours apart is that unlike many breast cancers, basal-like tumours do not express the estrogen receptor (ER) or progesterone receptor (PR), nor do they have amplified HER2. In the clinic, these tumours are often referred to as 'triple negative'. Women with triple negative tumours are not eligible for treatments that target ER (tamoxifen, aromatase inhibitors) or HER2 (trastuzumab). Instead they are treated with conventional chemotherapies, which have limited efficacy and many side effects. Therefore, it is critically important to identify alternative therapeutic strategies for basal-like breast cancer (BLBC).
We recently found that the transcription factor, Y-box binding protein-1 (YB-1), protein is commonly expressed in ER-negative breast cancers, and loss of this receptor is one of the hallmarks of BLBC. More recently, YB-1 was pulled out of a screen from the BLBC cell line SUM149 in an attempt to identify genes that promote malignant transformation and tumour cell growth. It has also been shown recently that epidermal growth factor receptor (EGFR) is highly expressed in approximately 50% of BLBCs. Interestingly, YB-1 was originally isolated as a transcription factor that bound to enhancer sites on the EGFR gene, a finding that could explain, at least in part, why it promotes the growth of breast tumour cells. In keeping with this possibility, Berquin et al. expressed YB-1 in mammary epithelial cells and observed a concomitant induction of EGFR. We demonstrated in MCF-7 (ER-positive breast cancer cells) that overexpression of YB-1 leads to an increase in the levels of EGFR mRNA and protein. This depends on the phosphorylation of YB-1 at S102. The YB-1 S102 site is located in the DNA-binding domain, suggesting that the effect on EGFR expression was likely to be through transcriptional regulation. We demonstrated that Akt binds directly to YB-1 and phosphorylates the S102 site, an observation that was subsequently confirmed in NIH3T3 cells. We now believe that Akt is one of several kinases capable of phosphorylating the S102 site of YB-1. In support of this idea, inhibition of the kinase mTOR with rapamycin also inhibits YB-1 phosphorylation. To understand this further, we demonstrated that YB-1 binds directly to the EGFR promoter within the first 1 kb of the transcription start site, and this occurs in a phosphorylation-dependent manner. Consistent with these preclinical developments, we found that YB-1 is strongly correlated with EGFR in primary breast tumours by screening a tissue microarray of ~490 cases. More recently, we have confirmed this observation in a cohort of ~2,222 primary breast tumours. In this study, YB-1 and EGFR are once again tightly correlated (P = 1.414 x 10; data not shown).
As both YB-1 and EGFR are expressed in BLBC, we questioned whether there was a relationship between these two genes in this particular subtype of breast cancer. First, we determined whether the overexpression was caused by gene amplification, and then further dissected the regulatory relationship between the two. Finally, we addressed whether inhibiting EGFR with Iressa (also referred to as ZD1839 or gefitinib) would slow the growth of BLBC.
Abstract and Introduction
Abstract
Introduction: Basal-like breast cancers (BLBCs) are very aggressive, and present serious clinical challenges as there are currently no targeted therapies available. We determined the regulatory role of Y-box binding protein-1 (YB-1) on epidermal growth factor receptor (EGFR) overexpression in BLBC, and the therapeutic potential of inhibiting EGFR. We pursued this in light of our recent work showing that YB-1 induces the expression of EGFR, a new BLBC marker.
Methods: Primary tumour tissues were evaluated for YB1 protein expression by immunostaining tissue microarrays, while copy number changes were assessed by comparative genomic hybridization (CGH). The ability of YB-1 to regulate EGFR was evaluated using luciferase reporter, chromatin immunoprecipitation (ChIP) and gel shift assays. The impact of Iressa on monolayer cell growth was measured using an ArrayScan VTI high-throughput analyser to count cell number, and colony formation in soft agar was used to measure anchorage-independent growth.
Results: YB-1 (27/37 or 73% of cases, P = 3.899 x 10) and EGFR (20/37 or 57.1% of cases, P = 9.206 x 10) are expressed in most cases of BLBC. However, they are not typically amplified in primary BLBC, suggesting overexpression owing to transcriptional activation. In support of this, we demonstrate that YB-1 promotes EGFR reporter activity. YB-1 specifically binds the EGFR promoter at two different YB-1-responsive elements (YREs) located at -940 and -968 using ChIP and gel shift assays in a manner that is dependent on the phosphorylation of S102 on YB-1. Inhibiting EGFR with Iressa suppressed the growth of SUM149 cells by ~40% in monolayer, independent of mutations in the receptor. More importantly anchorage-independent growth of BLBC cell lines was inhibited with combinations of Iressa and YB-1 suppression.
Conclusion: We have identified for the first time a causal link for the expression of EGFR in BLBC through the induction by YB-1 where it binds specifically to two distinguished YREs. Finally, inhibition of EGFR in combination with suppression of YB-1 presents a potential opportunity for therapy in BLBC.
Introduction
Identifying molecular targets for aggressive types of breast cancer is a milestone in the pursuit of individualized therapies. Gene-expression profiling of primary tumours has led to the following subcategories: luminal A, luminal B, the human epidermal growth factor receptor 2 (HER2) and the basal-like subtypes. Our attention was drawn to the basal-like subtype, because these tumours do not respond to available targeted therapies and patients often die within two years of diagnosis. Approximately 16% of all breast cancers are basal-like; this corresponds to 46,400 women among the ~290,000 women in North America who will be diagnosed with breast cancer each year. What sets these tumours apart is that unlike many breast cancers, basal-like tumours do not express the estrogen receptor (ER) or progesterone receptor (PR), nor do they have amplified HER2. In the clinic, these tumours are often referred to as 'triple negative'. Women with triple negative tumours are not eligible for treatments that target ER (tamoxifen, aromatase inhibitors) or HER2 (trastuzumab). Instead they are treated with conventional chemotherapies, which have limited efficacy and many side effects. Therefore, it is critically important to identify alternative therapeutic strategies for basal-like breast cancer (BLBC).
We recently found that the transcription factor, Y-box binding protein-1 (YB-1), protein is commonly expressed in ER-negative breast cancers, and loss of this receptor is one of the hallmarks of BLBC. More recently, YB-1 was pulled out of a screen from the BLBC cell line SUM149 in an attempt to identify genes that promote malignant transformation and tumour cell growth. It has also been shown recently that epidermal growth factor receptor (EGFR) is highly expressed in approximately 50% of BLBCs. Interestingly, YB-1 was originally isolated as a transcription factor that bound to enhancer sites on the EGFR gene, a finding that could explain, at least in part, why it promotes the growth of breast tumour cells. In keeping with this possibility, Berquin et al. expressed YB-1 in mammary epithelial cells and observed a concomitant induction of EGFR. We demonstrated in MCF-7 (ER-positive breast cancer cells) that overexpression of YB-1 leads to an increase in the levels of EGFR mRNA and protein. This depends on the phosphorylation of YB-1 at S102. The YB-1 S102 site is located in the DNA-binding domain, suggesting that the effect on EGFR expression was likely to be through transcriptional regulation. We demonstrated that Akt binds directly to YB-1 and phosphorylates the S102 site, an observation that was subsequently confirmed in NIH3T3 cells. We now believe that Akt is one of several kinases capable of phosphorylating the S102 site of YB-1. In support of this idea, inhibition of the kinase mTOR with rapamycin also inhibits YB-1 phosphorylation. To understand this further, we demonstrated that YB-1 binds directly to the EGFR promoter within the first 1 kb of the transcription start site, and this occurs in a phosphorylation-dependent manner. Consistent with these preclinical developments, we found that YB-1 is strongly correlated with EGFR in primary breast tumours by screening a tissue microarray of ~490 cases. More recently, we have confirmed this observation in a cohort of ~2,222 primary breast tumours. In this study, YB-1 and EGFR are once again tightly correlated (P = 1.414 x 10; data not shown).
As both YB-1 and EGFR are expressed in BLBC, we questioned whether there was a relationship between these two genes in this particular subtype of breast cancer. First, we determined whether the overexpression was caused by gene amplification, and then further dissected the regulatory relationship between the two. Finally, we addressed whether inhibiting EGFR with Iressa (also referred to as ZD1839 or gefitinib) would slow the growth of BLBC.