Judith Clancy Keen

Cytoplasmic accumulation of the RNA binding protein HuR is central to tamoxifen resistance in estrogen receptor positive breast cancer cells

With prolonged exposure, a majority of estrogen receptor positive cancers develop resistance to tamoxifen and subsequent therapies including selective estrogen receptor modulators (SERMs) and aromatase inhibitors (AIs). While much is known about overexpression of key growth promoting receptors including EGF, erbB2/Her2 and IGF receptors and subsequent activation of MAPK signaling associated with resistance, the underlying mechanism in the development of resistance still remains unknown. We found that inhibition of JNK, a member of the MAPK family, decreases cytoplasmic accumulation of the RNA binding protein HuR. This data combined with previous reports that erbB2/Her2 and IGF-IR signals through JNK, led us to hypothesize that cytoplasmic accumulation of HuR may be a key contributor to development of tamoxifen resistance. Therefore, we tested the effect of HuR expression on tamoxifen responsiveness in both tamoxifen sensitive MCF7 and tamoxifen resistant BT474 cell lines. We found that decreasing the cytoplasmic HuR levels in the cells increases tamoxifen responsiveness in both cell lines. Conversely, the overexpression of HuR establishes tamoxifen resistance in MCF7 cells. Therefore, our data indicate that HuR is central to tamoxifen resistance. Interestingly, we found that acute exposure (24 and 48 h) of MCF7 cells to tamoxifen increased cytoplasmic levels of HuR and concomitantly it’s ligand pp32, suggesting a novel molecular mechanism of resistance and acute response to tamoxifen through increased stability of mRNA transcripts that code for drug-resistant transcripts. Indeed, evaluation of primary breast tumors revealed a correlation between tumor grade, tamoxifen responsiveness and cytoplasmic HuR status. Therefore, inhibition of the cytoplasmic accumulation of HuR concomitantly with the administration of current therapeutics may be a successful treatment strategy. Our data describe a novel mechanism for the development of tamoxifen resistance and is the first study to identify an RNA binding protein as a key mediator of resistance in breast cancer cells

The RNA-binding protein HuR regulates GATA3 mRNA stability in human breast cancer cell lines

Meta-analyses of microarray data indicate that GATA3 is co-expressed with estrogen receptor alpha (ER) in breast cancer cells. While the significance of this remains unclear, it is thought that GATA3 may serve as a prognostic indicator in breast tumors and may play a role in ER signaling. Recently, reciprocal regulation of GATA3 and ER transcription was demonstrated, suggesting that control of their expression is intertwined. We sought to determine whether GATA3 and ER expression was also coordinately regulated at other levels. Unlike ER, GATA3 was not under epigenetic control and was not re-expressed in the presence of DNMT or HDAC inhibitors in ER/GATA3-negative cells. However, like ER, these inhibitors decreased GATA3 expression in ER/GATA3-positive cell lines. We have previously reported that ER mRNA stability is increased through binding of the RNA-binding protein HuR/ELAV1 to the 3′untranslated region (UTR) and that DNMT and HDAC inhibitors reduce ER expression by altering this interaction. Biotin pull-down assays using a biotinylated GATA3 RNA probe confirmed that HuR also binds to the GATA3 3′UTR. Inhibition of HuR using siRNA probes decreased GATA3 mRNA, mRNA stability and protein expression, indicating that HuR plays a role in regulating GATA3 expression. Inhibition of either HuR or GATA3 reduced cell growth of MCF7 cells. Based on our findings, it is clear that coordinate regulation of ER and GATA3 occurs, however differences do exist. These findings may aid in identification of new targets that control cell growth of breast cancer cells.

Inhibition of de novo purine synthesis in human prostate cells results in ATP depletion, AMPK activation and induces senescence.

4‐[2‐(2‐Amino‐4‐oxo‐4,6,7,8‐tetrahydro‐3H‐pyrimidino[5,4,6][1,4]thiazin‐6‐yl)‐(S)‐ethyl]‐2,5‐thienoylamino‐l‐glutamic acid (AG2034), is a classical antifolate shown to be an excellent inhibitor of glycinamide ribonucleotide formyltransferase (GARFT), ultimately inhibiting de novo purine synthesis. We examined some metabolic effects of this drug in prostate cancer cells, LNCaP, versus non‐tumorigenic prostatic epithelial cells, RWPE‐1.

The RNA binding protein HuR/ELAV1 regulates the estrogen receptor and epidermal growth factor receptor beta expression in BT 474 cells.

Abstract #3076 The RNA binding protein HuR has been shown to increase the mRNA stability of the majority of transcripts to which in binds. Recent data has linked the cytoplasmic distribution of HuR to the tumorigenesis process in many cancer types. HuR levels were found to be higher in more metastatic, higher grade breast tumors, suggesting that it may play a role for stabilization of growth-promoting transcripts in breast cancer cells. Previously, our lab has shown that the DNA methyltransferase inhibitor 5-aza- 29deoxycytidine (AZA) and histone deacetylase inhibitor Trichostatin A (TSA) decreased the cytoplasmic accumulation of HuR in MDA-MB-231 and MCF-7 cells. Therefore, to examine the role of HuR in the more tumorigenic, estrogen receptor (ER) positive, Tamoxifen resistant BT 474 cells were treated with AZA/TSA or siRNA probes specifically targeting HuR expression. The BT 474 cells had a baseline increase in the cytoplasmic distribution of HuR as compared to MCF-7 and MDA-MB-231 cells. The half-life of ER mRNA was also increased. Following AZA/TSA treatment for 24-96 hours, cytoplasmic HuR was noticeably lower after 48 hours of treatment. Subsequent mRNA levels of both ER and epidermal growth factor beta (erbB2/HER2) were also found to drop with treatment. siRNA inhibition of HuR had a similar effect on the mRNA levels. Biotin pulldown assays using probes specific for the ER 39 UTR showed HuR binding was decreased with AZA/TSA treatment. These were similar findings as previously reported in MCF7 cells. Since ER and erbB2 are central to hormonal therapy, Tamoxifen sensitivity was assessed using BT 474 cells transfected with siRNA probes specifically targeting HuR expression. Cellular viability following Tamoxifen treatment was significantly decreased when HuR expression was inhibited and Tamoxifen sensitivity was restored. Similar studies investigating the role of HuR in herceptin resistance are currently ongoing. Taken together, our data suggests that HuR plays a key role in both de novo and acquired Tamoxifen resistance. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3076.

Abstract 3078: CD44 mRNA stability is regulated by RNA binding protein HuR in ovarian cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL CD44, a major hyaluronic acid (HA) receptor, is involved in cell migration, metastasis and tumor progression. While in normal ovarian epithelium CD44 is not expressed, ovarian carcinoma cells express multiple CD44 isoforms as the tumor progresses and eventually CD44 expression is abrogated in advanced ovarian cancer metastatic sites. We as well as others have observed that the levels of CD44 mRNA transcript not always correspond to the CD44 protein expression levels. This observation suggests that CD44 is post-transcriptionally regulated. To test this hypothesis we first analyzed the CD44 3’ untranslated region (3’ UTR) sequence and found numerous AU-rich elements (ARE) in this region. Some of these sequences are known HuR binding site consensus sequences. HuR is an RNA binding protein that is known to stabilize transcripts. HuR is expressed in the nucleus and is transported to the cytoplasm where it functions as an mRNA stabilizer. We performed mRNA half-life experiments that showed a two-fold reduction in the stability of CD44 transcript in siHuR transfected ovarian cancer cells. Consistent with this result, treatment of these cells with inhibitors of DNA methylation and histone deacetylation (i.e., 5-azacytidine and Trichostatin A, respectively) known to diminish the presence of HuR in the cytoplasm, also decreased the stability of CD44 mRNA. Western blot analysis showed the expected reduction in cytoplasmic HuR as well as down-regulation in CD44 protein expression. A direct interaction of HuR with the CD44 3’ UTR was detected by biotin pulldown assays. These results indicate that HuR posttranscriptionally stabilizes the expression of CD44 in the cytoplasm. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3078. doi:10.1158/1538-7445.AM2011-3078

Abstract 4063: Estrogen receptor alpha functions as an RNA binding protein to regulate GATA3 protein levels: a novel role for ER

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The role of estrogen receptor alpha (ER) as a transcription factor has been well established. ER transcriptionally activates genes by binding to DNA directly within the gene promoter region or by binding to DNA indirectly through protein-protein dependent mechanisms. Many ER-responsive genes that are activated through ER-mediated transcriptional activation have been identified, including GATA3. While the role of GATA3 in breast cancer still remains largely unknown, numerous reports have shown that ER and GATA3 are coordinately expressed in breast cancer and that ER may play a role in regulating GATA3 expression. A recent report by Eeckhoute et al (2007) has shown that ER transcriptionally activated GATA3 expression in T47D breast cancer cells. Using MCF7 cells, we have recently identified a novel role for ER as an RNA binding protein that binds to GATA3 mRNA. Interestingly, silencing of ER using siRNA probes slightly increased GATA3 mRNA levels, however GATA3 protein levels decreased significantly. This decrease in protein was not due to improper export of GATA3 mRNA from the nucleus to the cytoplasm where protein translation occurs, as GATA3 mRNA expression was equivalent in both the nuclear and cytoplasmic compartments. This suggested that the decrease in protein expression was not due to impaired GATA3 mRNA transport but could be due to impaired protein translation. Since silencing of ER had dramatically different effects on RNA and protein levels, this data suggested that ER protein may function as an RNA binding protein that binds to the GATA3 mRNA to regulate its protein translation. Using biotin pulldown techniques, we found that ER does function as an RNA binding protein by binding directly to a site within a 125 nucleotide region of the GATA3 3‘UTR. This role of ER as an RNA binding protein is consistent with the data of Lanz et al (1999, 2000) that demonstrated that ER protein can bind to an RNA transcript, the steroid receptor co-activator, SRA. When bound to SRA, however, ER did not alter the RNA levels or protein expression of SRA but did result in increased activation of ER. Therefore, our data is the first to identify a novel role for ER as a RNA binding protein that can regulate the protein expression of the RNA transcript to which it is bound. Altering such ER-RNA interactions could potentially provide a new therapeutic avenue for the treatment of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4063.

A role for GATA-3 in control of estrogen receptor alpha expression.

Abstract #3050 Expression of the GATA-3 binding protein correlates with estrogen receptor (ER) alpha expression in primary and cultured breast cancer cells. While the significance of this correlation and role of GATA-3 in breast cancer remains unclear, GATA-3 may serve as a good prognostic indicator in breast tumors. Recently, a reciprocal interaction between GATA-3 and ER has been demonstrated. GATA-3 may regulate ER levels through binding to an enhancer region upstream of the ER gene while ER can associate with and regulate GATA-3 expression. Meta-analyses of several cancer microarrays indicate that GATA-3 is co-expressed with ER and other ER responsive genes, further suggesting a role of GATA-3 in the ER signaling pathway. Our laboratory has been investigating the role of the RNA binding protein HuR/ELAV1 in control of mRNA stability in breast cancer cells. We have previously reported that ER mRNA stability is increased through binding of HuR to the 39UTR. Microarray analysis of those transcripts associated with HuR in MCF7 cells showed that GATA-3 mRNA associates with HuR compared to an IgG control. Biotin pulldown assays using a biotinylated GATA-3 RNA probe confirmed that HuR binds to the GATA-3 39untranslated region (UTR). Inhibition of HuR using siRNA probes decreased GATA-3 mRNA and protein expression, further indicating a role for HuR in regulation of GATA-3 mRNA stability. siRNA mediated inhibition of GATA-3 expression significantly reduced ER mRNA and protein levels. Surprisingly, we found that GATA-3 bound to the ER mRNA by binding to two out of four regions within the ER 39UTR, suggesting that while GATA-3 may play a role in upregulation of ER mRNA transcription through the enhancer interaction, GATA-3 may stabilize ER mRNA once transcribed by binding to the ER 39UTR. Studies are ongoing to fully characterize this role of GATA-3 in control of ER mRNA stability. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3050.