Christiana S. Kappler

Regulation of Ncx1 Gene Expression in the Normal and Hypertrophic Heart

Abstract:  The Na+/Ca2+ exchanger (NCX1) is crucial in the regulation of [Ca2+]i in the cardiac myocyte. The exchanger is upregulated in cardiac hypertrophy, ischemia, and failure. This upregulation can have an effect on Ca2+ transients and possibly contribute to diastolic dysfunction and an increased risk of arrhythmias. Studies from both in vivo and in vitro model systems have provided an initial skeleton of the potential signaling pathways that regulate the exchanger during development, growth, and hypertrophy. The Ncx1 gene is upregulated in response to α‐adrenergic stimulation. We have shown that this is via p38α activation of transcription factors binding to the Ncx1 promotor at the –80 CArG element. Interestingly, most of the elements, including the CArG element, which we have demonstrated to be important for regulation of Ncx1 expression are in the proximal 184 bp of the promotor. Using a transgenic mouse, we have shown that the proximal 184 bp is sufficient for expression of reporter genes in adult cardiomyocytes and for the correct spatiotemporal pattern of Ncx1 expression in development but not for upregulation in response to pressure overload.

Regulation of Ncx1 expression: Identification of regulatory elements mediating cardiac-specific expression and up-regulation

The Na+-Ca2+ exchanger (NCX1) is up-regulated in hypertrophy and is often found up-regulated in end-stage heart failure. Studies have shown that the change in its expression contributes to contractile dysfunction. We have previously shown that the 1831-bp Ncx1 H1 (1831Ncx1) promoter directs cardiac-specific expression of the exchanger in both development and in the adult, and is sufficient for the up-regulation of Ncx1 in response to pressure overload. Here, we utilized adenoviral mediated gene transfer and transgenics to identify minimal regions and response elements that mediate Ncx1 expression in the heart. We demonstrate that the proximal 184 bp of the Ncx1 H1 (184Ncx1) promoter is sufficient for expression of reporter genes in adult cardiomyocytes and for the correct spatiotemporal pattern of Ncx1 expression in development but not for up-regulation in response to pressure overload. Mutational analysis revealed that both the -80 CArG and the -50 GATA elements were required for expression in isolated adult cardiomyocytes. Chromatin immunoprecipitation assays in adult cardiocytes demonstrate that SRF and GATA4 are associated with the proximal region of the endogenous Ncx1 promoter. Transgenic lines were established for the 1831Ncx1 promoter-luciferase containing mutations in the -80 CArG or -50 GATA element. No luciferase activity was detected during development, in the adult, or after pressure overload in any of the -80 CArG transgenic lines. The Ncx1 -50 GATA mutant promoter was sufficient for driving the normal spatiotemporal pattern of Ncx1 expression in development and for up-regulation in response to pressure overload but importantly, expression was no longer cardiac restricted. This work is the first in vivo study that demonstrates which cis elements are important for Ncx1 regulation.

KAT6A, a Chromatin Modifier from the 8p11-p12 Amplicon is a Candidate Oncogene in Luminal Breast Cancer

The chromosome 8p11-p12 amplicon is present in 12% to 15% of breast cancers, resulting in an increase in copy number and expression of several chromatin modifiers in these tumors, including KAT6A. Previous analyses in SUM-52 breast cancer cells showed amplification and overexpression of KAT6A, and subsequent RNAi screening identified KAT6A as a potential driving oncogene. KAT6A is a histone acetyltransferase previously identified as a fusion partner with CREB binding protein in acute myeloid leukemia. Knockdown of KAT6A in SUM-52 cells, a luminal breast cancer cell line harboring the amplicon, resulted in reduced growth rate compared to non-silencing controls and profound loss of clonogenic capacity both in mono-layer and in soft agar. The normal cell line MCF10A, however, did not exhibit slower growth with knockdown of KAT6A. SUM-52 cells with KAT6A knockdown formed fewer mammospheres in culture compared to controls, suggesting a possible role for KAT6A in self-renewal. Previous data from our laboratory identified FGFR2 as a driving oncogene in SUM-52 cells. The colony forming efficiency of SUM-52 KAT6A knockdown cells in the presence of FGFR inhibition was significantly reduced compared to cells with KAT6A knockdown only. These data suggest that KAT6A may be a novel oncogene in breast cancers bearing the 8p11-p12 amplicon. While there are other putative oncogenes in the amplicon, the identification of KAT6A as a driving oncogene suggests that chromatin-modifying enzymes are a key class of oncogenes in these cancers, and play an important role in the selection of this amplicon in luminal B breast cancers.

Pathways Regulating Na+/Ca2+ Exchanger Expression in the Heart

Abstract: The Na+/Ca2+ exchanger (NCX1) is regulated at the transcriptional level in cardiac hypertrophy, ischemia, and failure. Following pressure overload, activation of MAPKs coincides with the kinetics of NCX1 gene upregulation in adult cardiocytes. Using adenoviral gene delivery, we begin to identify the molecular pathways responsible for upregulation of the exchanger gene. Inhibition of ERK with the MEK inhibitor UO126, the ERK protein phosphatase MKP‐3, inhibited ERK activation, but only inhibited α‐adrenergic‐induced NCX1 upregulation by 30%. Overexpression of DN‐JNK lowered basal NCX1 expression. Overexpression of activated MKK‐3 was sufficient for α‐adrenergic‐stimulated upregulation of the reporter gene. Together, this data indicates that (1) JNK mediates basal cardiac expression of the NCX1 gene, (2) ERK and p38 play a role in α‐adrenergic‐stimulated NCX1 upregulation, and (3) p38 activation alone is sufficient for NCX1 upregulation.

Amplification of WHSC1L1 regulates expression and estrogen-independent activation of ERα in SUM-44 breast cancer cells and is associated with ERα over-expression in breast cancer

The 8p11‐p12 amplicon occurs in approximately 15% of breast cancers in aggressive luminal B‐type tumors. Previously, we identified WHSC1L1 as a driving oncogene from this region. Here, we demonstrate that over‐expression of WHSC1L1 is linked to over‐expression of ERα in SUM‐44 breast cancer cells and in primary human breast cancers. Knock‐down of WHSC1L1, particularly WHSC1L1‐short, had a dramatic effect on ESR1 mRNA and ERα protein levels. SUM‐44 cells do not require exogenous estrogen for growth in vitro; however, they are dependent on ERα expression, as ESR1 knock‐down or exposure to the selective estrogen receptor degrader fulvestrant resulted in growth inhibition. ChIP‐Seq experiments utilizing ERα antibodies demonstrated extensive ERα binding to chromatin in SUM‐44 cells under estrogen‐free conditions. ERα bound to ERE and FOXA1 motifs under estrogen‐free conditions and regulated expression of estrogen‐responsive genes. Short‐term treatment with estradiol enhanced binding of ERα to chromatin and influenced expression of many of the same genes to which ERα was bound under estrogen‐free conditions. Finally, knock‐down of WHSC1L1 in SUM‐44 cells resulted in loss of ERα binding to chromatin under estrogen‐free conditions, which was restored upon exposure to estradiol. These results indicate the SUM‐44 cells are a good model of a subset of luminal B breast cancers that have the 8p11‐p12 amplicon, over‐express WHSC1L1, and over‐express ERα, but are independent of estrogen for binding to chromatin and regulation of gene expression. Breast cancers such as these, that are dependent on ERα activity but independent of estradiol, are a major cause of breast cancer mortality.

Oncogenic signaling in amphiregulin and EGFR‐expressing PTEN‐null human breast cancer

A subset of triple negative breast cancer (TNBC) is characterized by overexpression of the epidermal growth factor receptor (EGFR) and loss of PTEN, and patients with these determinants have a poor prognosis. We used cell line models of EGFR‐positive/PTEN null TNBC to elucidate the signaling networks that drive the malignant features of these cells and cause resistance to EGFR inhibitors. In these cells, amphiregulin (AREG)‐mediated activation of EGFR results in up‐regulation of fibronectin (FN1), which is known to be a mediator of invasive capacity via interaction with integrin β1. EGFR activity in this PTEN null background also results in Wnt/beta‐catenin signaling and activation of NF‐κB. In addition, AKT is constitutively phosphorylated in these cells and is resistant to gefitinib. Expression profiling demonstrated that AREG‐activated EGFR regulates gene expression differently than EGF‐activated EGFR, and functional analysis via genome‐scale shRNA screening identified a set of genes, including PLK1 and BIRC5, that are essential for survival of SUM‐149 cells, but are uncoupled from EGFR signaling. Thus, our results demonstrate that in cells with constitutive EGFR activation and PTEN loss, critical survival genes are uncoupled from regulation by EGFR, which likely mediates resistance to EGFR inhibitors.

Chronic Administration of KB-R7943 Induces Up-regulation of Cardiac NCX1

The NCX1 (sodium-calcium exchanger) is up-regulated in human heart failure and in many animal models of heart failure. The potential benefits and risks of therapeutically blocking NCX1 in heart failure and during ischemia-reperfusion are being actively investigated. In this study, we demonstrate that prolonged administration of the NCX1 inhibitor KB-R7943 resulted in the up-regulation of Ncx1 gene expression in both isolated adult cardiomyocytes and intact mouse hearts. Ncx1 up-regulation is mediated by the activation of p38. Importantly, p38 is not activated by KB-R7943 treatment in heart tubes from Ncx1−/− mice at 9.5 days postcoitum but is activated in heart tubes from Ncx1+/+ mice. p38 activation does not appear to be in response to changes in cytosolic calcium concentration, [Ca2+]i. Interestingly, chronic KB-R7943 treatment in mice leads to the formation of an NCX1-p38 complex. Our study demonstrates for the first time that the electrogenic sarcolemma membrane cardiac NCX1 can act as a regulator of “activity-dependent signal transduction” leading to changes in gene expression.

Abstract 3297: PTEN loss enhances amphiregulin-specific signaling and gene expression in triple-negative breast cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The epidermal growth factor receptor (EGFR) is frequently overexpressed in triple-negative breast cancer, and approximately half of all basal breast cancers show autocrine activation of EGFR by amphiregulin (AREG). When AREG is the activating ligand, the dynamics of EGFR signaling are different than those of EGF-bound EGFR. AREG-ligated EGFR accumulates at the cell surface, resulting in altered receptor trafficking and downstream signaling. To further explore the role of amphiregulin in cell signaling, we performed reverse-phase protein array (RPPA) analysis on SUM149 cells, a highly tumorigenic and metastatic cell line that expresses high levels of AREG and EGFR, and MCF10A cells, a non-transformed mammary epithelial cell line cultured in the presence of either EGF or AREG. This analysis identified a number of signaling components mediated specifically by amphiregulin, including decreased caspase expression, increased Gab2 expression, elevated fibronectin levels and marked changes in integrin expression. In addition, AREG-mediated EGFR signaling resulted in increased expression and activation of c-src. Thus, a major effect of AREG signaling in both cell lines was enhanced activation of the canonical focal adhesion signaling pathway. In addition to increased expression of EGFR and AREG, SUM149 cells are known to have a genomic alteration at the PTEN locus resulting in complete abrogation of PTEN message and protein expression. RPPA analysis of signaling in SUM-149 cells confirmed this PTEN loss and, not surprisingly, revealed elevated AKT activity. In addition, several proteins in the WNT signaling pathway were altered in SUM-149 cells, including reduced expression of GSK3β, increased phosphorylation of GSK3β, and increased expression of Dvl. These results suggest enhanced Wnt-beta catenin activity in SUM-149 cells that is likely the result of enhanced AKT activity resulting from PTEN loss. Although we previously had difficulty measuring Wnt signaling in SUM-149 cells in monolayer culture, mammosphere assays performed with SUM149 cells expressing a Wnt reporter construct confirmed that Wnt signaling was highly active in mammospheres in 3D culture. We also analyzed the genes that are regulated in expression by EGFR in MCF-10A cells in the presence of EGF or AREG, and in SUM-149 cells. These results identified AREG-specific gene expression profiles in the isogenic system. In addition, SUM-149 cell-specific genes regulated by EGFR were also identified, some of which are likely to be the result of PTEN loss and enhanced AKT signaling in these cells. Finally, we found that AREG knockdown in SUM-149 cells significantly reduced the tumorigenicity of SUM149 cells in NOD-SCID mice. Analysis of signaling and gene expression in these AREG knock-down cells are consistent with the importance of AREG expression levels and of the modulation of the focal adhesion canonical pathway in tumor potential of the cells in vivo. Citation Format: Christiana Kappler, Robert Wilson, Bridget Varughese, Stephen P. Ethier. PTEN loss enhances amphiregulin-specific signaling and gene expression in triple-negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3297. doi:10.1158/1538-7445.AM2014-3297

Abstract 1339: Role of PTEN loss in basal-like 2 triple negative breast cancer

Basal-like 2 (BL2) triple negative breast cancer (TNBC) is an aggressive molecular subtype of breast cancer with a poor clinical outcome. These cancers are characterized by overexpression of epidermal growth factor receptor (EGFR), loss of PTEN protein expression, and presence of mutated TP53. Despite overexpression of EGFR, EGFR-targeted therapies have performed poorly in clinical settings. Therefore, understanding the mechanisms of resistance in these cells is critical for improved patient care. To understand the oncogenic signaling landscape in BL2 TNBC, our lab utilizes the BL2 cell lines SUM-149 and SUM-229, which overexpress EGFR activated by an amphiregulin-mediated autocrine loop, and are PTEN null. In addition, we utilize the BL1 subtype cell line MDA-MB-468, which also exhibits overexpression of EGFR and loss of PTEN. Both the BL1 and BL2 cell lines exhibit elevated levels of phosphorylated AKT (p-AKT) compared to control MCF-10A cells. However, while treatment of SUM-149 and SUM-229 cells with inhibitors targeting EGFR (Gefitinib), type-1 PI3K (BKM120, BKM), mTOR (KU-0063794, KU) and pan-AKT (MK2206, MK) did not reduce p-AKT levels, MCF-10A and MDA-MB-468 cell lines treated with the same panel of targeted inhibitors showed marked inhibition of p-AKT. In addition, re-expression of PTEN in SUM-149 and SUM-229 cells had little effect on p-AKT but dramatically reduced p-AKT in MDA-MB-468 cells. Although PTEN re-expression alone had little effect on p-AKT in SUM-149 and SUM-229 cells, these cells did display decreased AKT phosphorylation following treatment with Gefitinib, BKM, KU, and MK. There are three isoforms of AKT (AKT1, AKT2 and AKT3) of which, AKT3 has been implicated in the aggressive nature of some cancers. Previous studies in our lab showed that SUM-149 and SUM-229 cell lines have higher levels of AKT3 message and protein compared to the other AKT isoforms. AKT3 expression was also higher in the BL2 cell lines compared to BL1 and control cells. This differential expression of AKT isoforms led us to examine the impact of AKT isoform-specific signaling. Using PTEN re-expressing SUM-149 cells, we showed that PTEN re-expression resulted in decreased phosphorylation of AKT1 but not of AKT3. However, PTEN re-expression combined with Gefitinib treatment dramatically reduced phosphorylation of both AKT1 and AKT3. Interestingly, while neither re-expression of PTEN nor Gefitinib expression alone had an effect on colony-forming efficiency, PTEN re-expression coupled with Gefitinib treatment was able to reduce colony formation efficiency in both SUM-149 and SUM-229 cell lines. Taken together, these data indicate that BL2 breast cancers, with overexpression of EGFR and loss of PTEN expression, have an oncogenic signaling network involving AKT3 that contributes to the aggressive and drug-resistant phenotype observed in this subset of TNBC. Citation Format: Ericka L. Smith, Christiana S. Kappler, Stephen P. Ethier. Role of PTEN loss in basal-like 2 triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1339. doi:10.1158/1538-7445.AM2017-1339

Abstract 4596: Regulation of epidermal growth factor receptor signaling in triple-negative breast cancer

Although the basal-like 1 (BL1) and BL2 subtypes of triple negative breast cancer (TNBC) share similar genetic and biological characteristics, patients with BL2 type tumors have markedly poorer prognosis. In addition, despite the overexpression of epidermal growth factor receptor (EGFR) characteristically observed in basal-like TNBC, EGFR inhibitors have performed poorly in clinical trials. In order to understand the mechanism of resistance to EGFR inhibition in these tumors, our laboratory utilized cell line models of BL2 (SUM-149 and SUM-229) and BL1 (MDA-MB-468; MM468) TNBC, all of which overexpress EGFR, are PTEN null and exhibit elevated AKT phosphorylation (p-AKT). Compared to MCF10A and MM-468 cells, AKT phosphorylation in the SUM-149 and SUM-229 cell lines was resistant to treatment with the EGFR inhibitor gefitinib, as well as to the PI3K inhibitor BKM120 (BKM) and the mTOR inhibitor KU-0063794 (KU). Even treatment with the allosteric AKT inhibitor MK2206 (MK) only partially reduced p-AKT. RT-PCR and immunoblot analysis of AKT isoform expression showed that in SUM-149 and SUM-229 cells, AKT3 is expressed at higher levels than AKT1 or AKT2 at both the protein and message level. Because AKT3 has a higher IC50 for MK than AKT1 or AKT2, increasing doses of MK were used in an effort to abrogate AKT phosphorylation. The results showed that MK treatment at higher concentrations (5 μM) decreased p-AKT. In addition, shRNA-mediated knock-down of AKT3 sensitized the cells to inhibition of AKT by BKM, KU and MK. Interestingly, MK treatment alone had little effect on colony forming efficiency, even at higher doses, but when added in combination with gefitinib, MK dramatically reduced colony formation. We then investigated the effect of PTEN re-expression on signaling in these cell lines. PTEN re-expression alone had little effect on p-AKT in SUM-149 and SUM-229 cells, but resulted in a dramatic reduction in p-AKT in MM-468 cells. Although PTEN expression alone had little effect on p-AKT in SUM-149 and 229 cells, these cells exhibited decreased p-AKT following treatment with gefitinib and were sensitive to inhibition of AKT by MK. In addition, treatment of SUM-149/PTEN cells with gefitinib markedly reduced colony-forming efficiency. Our laboratory has previously demonstrated that in SUM-149 cells, a set of genes that are essential for survival, including PLK1, are uncoupled from regulation by EGFR. Thus, we examined the effect of AKT inhibition on expression of PLK1 and found that inhibition of AKT or re-expression of PTEN was able to recouple EGFR-mediated regulation of PLK1 expression. Our results demonstrate that PI-3 kinase and mTORC2 drive phosphorylation of AKT1 but not AKT3 in SUM-149 cells, which explains their relative resistance to these targeted drugs. Citation Format: Christiana S. Kappler, Ericka L. Smith, Stephen Ethier. Regulation of epidermal growth factor receptor signaling in triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4596.