Debra Wyatt

Cross-talk between Notch and the Estrogen Receptor in Breast Cancer Suggests Novel Therapeutic Approaches

High expression of Notch-1 and Jagged-1 mRNA correlates with poor prognosis in breast cancer. Elucidating the cross-talk between Notch and other major breast cancer pathways is necessary to determine which patients may benefit from Notch inhibitors, which agents should be combined with them, and which biomarkers indicate Notch activity in vivo. We explored expression of Notch receptors and ligands in clinical specimens, as well as activity, regulation, and effectors of Notch signaling using cell lines and xenografts. Ductal and lobular carcinomas commonly expressed Notch-1, Notch-4, and Jagged-1 at variable levels. However, in breast cancer cell lines, Notch-induced transcriptional activity did not correlate with Notch receptor levels and was highest in estrogen receptor alpha-negative (ERalpha(-)), Her2/Neu nonoverexpressing cells. In ERalpha(+) cells, estradiol inhibited Notch activity and Notch-1(IC) nuclear levels and affected Notch-1 cellular distribution. Tamoxifen and raloxifene blocked this effect, reactivating Notch. Notch-1 induced Notch-4. Notch-4 expression in clinical specimens correlated with proliferation (Ki67). In MDA-MB231 (ERalpha(-)) cells, Notch-1 knockdown or gamma-secretase inhibition decreased cyclins A and B1, causing G(2) arrest, p53-independent induction of NOXA, and death. In T47D:A18 (ERalpha(+)) cells, the same targets were affected, and Notch inhibition potentiated the effects of tamoxifen. In vivo, gamma-secretase inhibitor treatment arrested the growth of MDA-MB231 tumors and, in combination with tamoxifen, caused regression of T47D:A18 tumors. Our data indicate that combinations of antiestrogens and Notch inhibitors may be effective in ERalpha(+) breast cancers and that Notch signaling is a potential therapeutic target in ERalpha(-) breast cancers.

Notch-1 activates estrogen receptor-α-dependent transcription via IKKα in breast cancer cells

Approximately 80% of breast cancers express the estrogen receptor-α (ERα) and are treated with anti-estrogens. Resistance to these agents is a major cause of mortality. We have shown that estrogen inhibits Notch, whereas anti-estrogens or estrogen withdrawal activate Notch signaling. Combined inhibition of Notch and estrogen signaling has synergistic effects in ERα-positive breast cancer models. However, the mechanisms whereby Notch-1 promotes the growth of ERα-positive breast cancer cells are unknown. Here, we demonstrate that Notch-1 increases the transcription of ERα-responsive genes in the presence or absence of estrogen via a novel chromatin crosstalk mechanism. Our data support a model in which Notch-1 can activate the transcription of ERα-target genes via IKKα-dependent cooperative chromatin recruitment of Notch–CSL–MAML1 transcriptional complexes (NTC) and ERα, which promotes the recruitment of p300. CSL binding elements frequently occur in close proximity to estrogen-responsive elements (EREs) in the human and mouse genomes. Our observations suggest that a hitherto unknown Notch-1/ERα chromatin crosstalk mediates Notch signaling effects in ERα-positive breast cancer cells and contributes to regulate the transcriptional functions of ERα itself.

Small Ubiquitin-like Modifier Modification of Arrestin-3 Regulates Receptor Trafficking

Nonvisual arrestins are regulated by direct post-translational modifications, such as phosphorylation, ubiquitination, and nitrosylation. However, whether arrestins are regulated by other post-translational modifications remains unknown. Here we show that nonvisual arrestins are modified by small ubiquitin-like modifier 1 (SUMO-1) upon activation of β2-adrenergic receptor (β2AR). Lysine residues 295 and 400 in arrestin-3 fall within canonical SUMO consensus sites, and mutagenic analysis reveals that Lys-400 represents the main SUMOylation site. Depletion of the SUMO E2 modifying enzyme Ubc9 blocks arrestin-3 SUMOylation and attenuates β2AR internalization, suggesting that arrestin SUMOylation mediates G protein-coupled receptor endocytosis. Consistent with this, expression of a SUMO-deficient arrestin mutant failed to promote β2AR internalization as compared with wild-type arrestin-3. Our data reveal an unprecedented role for SUMOylation in mediating GPCR endocytosis and provide novel mechanistic insight into arrestin function and regulation.

Preclinical study of a Kv11.1 potassium channel activator as antineoplastic approach for breast cancer

Potassium ion (K+) channels have been recently found to play a critical role in cancer biology. Despite that pharmacologic manipulation of ion channels is recognized as an important therapeutic approach, very little is known about the effects of targeting of K+ channels in cancer. In this study, we demonstrate that use of the Kv11.1 K+ channel activator NS1643 inhibits tumor growth in an in vivo model of breast cancer. Tumors exposed to NS1643 had reduced levels of proliferation markers, high expression levels of senescence markers, increased production of ROS and DNA damage compared to tumors of untreated mice. Importantly, mice treated with NS1643 did not exhibit significant cardiac dysfunction. In conclusion, pharmacological stimulation of Kv11.1 activity produced arrested TNBC-derived tumor growth by generating DNA damage and senescence without significant side effects. We propose that use of Kv11.1 channels activators could be considered as a possible pharmacological strategy against breast tumors.

Notch-1-PTEN-ERK1/2 signaling axis promotes HER2+ breast cancer cell proliferation and stem cell survival

Trastuzumab targets the HER2 receptor on breast cancer cells to attenuate HER2-driven tumor growth. However, resistance to trastuzumab-based therapy remains a major clinical problem for women with HER2+ breast cancer. Breast cancer stem cells (BCSCs) are suggested to be responsible for drug resistance and tumor recurrence. Notch signaling has been shown to promote BCSC survival and self-renewal. Trastuzumab-resistant cells have increased Notch-1 expression. Notch signaling drives cell proliferation in vitro and is required for tumor recurrence in vivo. We demonstrate herein a mechanism by which Notch-1 is required for trastuzumab resistance by repressing PTEN expression to contribute to activation of ERK1/2 signaling. Furthermore, Notch-1-mediated inhibition of PTEN is necessary for BCSC survival in vitro and in vivo. Inhibition of MEK1/2-ERK1/2 signaling in trastuzumab-resistant breast cancer cells mimics effects of Notch-1 knockdown on bulk cell proliferation and BCSC survival. These findings suggest that Notch-1 contributes to trastuzumab resistance by repressing PTEN and this may lead to hyperactivation of ERK1/2 signaling. Furthermore, high Notch-1 and low PTEN mRNA expression may predict poorer overall survival in women with breast cancer. Notch-1 protein expression predicts poorer survival in women with HER2+ breast cancer. These results support a potential future clinical trial combining anti-Notch-1 and anti-MEK/ERK therapy for trastuzumab-resistant breast cancer.

Inhibition of HER2 Increases JAGGED1-dependent Breast Cancer Stem Cells: Role for Membrane JAGGED1

Purpose: HER2-positive breast cancer is driven by cells possessing stem-like properties of self-renewal and differentiation, referred to as cancer stem cells (CSC). CSCs are implicated in radiotherapy, chemotherapy resistance, and tumor recurrence. NOTCH promotes breast CSC survival and self-renewal, and overexpression of NOTCH1 and the NOTCH ligand JAGGED1 predict poor outcome. Resistance to anti-HER2 therapy in HER2+ breast cancer requires NOTCH1, and that combination of trastuzumab and a gamma secretase inhibitor (GSI) prevents tumor relapse in xenograft models. Experimental Design: The current study investigates mechanisms by which HER2 tyrosine kinase activity regulates NOTCH-dependent CSC survival and tumor initiation. Results: Lapatinib-mediated HER2 inhibition shifts the population of HER2+ breast cancer cells from low membrane JAGGED1 expression to higher levels, independent of sensitivity to anti-HER2 treatment within the bulk cell population. This increase in membrane JAGGED1 is associated with higher NOTCH receptor expression, activation, and enrichment of CSCs in vitro and in vivo. Importantly, lapatinib treatment results in growth arrest and cell death of JAGGED1 low-expressing cells while the JAGGED1 high-expressing cells continue to cycle. High membrane JAGGED1 protein expression predicts poor overall cumulative survival in women with HER2+ breast cancer. Conclusions: These results indicate that higher membrane JAGGED1 expression may be used to either predict response to anti-HER2 therapy or for detection of NOTCH-sensitive CSCs posttherapy. Sequential blockade of HER2 followed by JAGGED1 or NOTCH could be more effective than simultaneous blockade to prevent drug resistance and tumor progression. Clin Cancer Res; 24(18); 4566–78. ©2018 AACR.

Abstract 4769: Targeting HER2 enriches Jagged1 high cancer stem cells in breast cancer

The human epidermal growth factor receptor 2 (HER2) positive subtype of breast cancer is characterized by gene amplification and/or protein overexpression of HER2. It is driven by a subpopulation of cells possessing stem cell properties of self-renewal and differentiation, known as Cancer Stem cells (CSCs). CSCs are implicated in tumor growth as well as radiotherapy and chemotherapy associated resistance. Notch receptors promote breast CSCs survival and self-renewal, and overexpression of a Notch ligand Jagged1 mRNA predicts poor prognosis in women with breast cancer. Our lab has published that Jagged1 or Notch1 is a critical target in trastuzumab/lapatinib (LAP) resistant HER2+ breast cancer. The study aimed to determine whether anti-HER2 therapy selects for Jagged-1/Notch-dependent CSCs that are responsible for tumor initiation. Surface expression of Notch1 and Jagged1 upon HER2 blockade using LAP was measured in HER2+ breast cancer cell lines (MDA-MB-453 and HCC1954) using flow cytometry. LAP treatment increased the Jagged1-positive subpopulation compared to vehicle. Cells were sorted based on Jagged1 cell surface expression and assessed for CSC-like properties (i.e. mammosphere forming efficiency, Aldefluor activity, expression of CD44high /CD24low, Notch target transcripts expression, and limiting dilution tumor initiating potential in athymic, nude mice). In addition, immunohistochemistry was performed on 145 HER2+ breast tumor microarray to detect cytoplasmic, membrane, or nuclear Jagged1 protein expression. Kaplan-Meier analysis was performed to determine overall survival. The results showed that upon HER2 inhibition, Jagged1 cell surface expression increased and Notch1 cell surface expression was unchanged. The Jagged1high subpopulation of cells showed elevated levels of Aldehyde dehydrogenase activity, Notch target gene transcripts, and mammosphere formation efficiency compared to vehicle treated cells. The MRK-003 γ-secretase inhibitor (GSI) prevented mammosphere formation in the Jagged1high cells indicating that Notch activation drives Jagged1high CSC survival. Also, we confirmed that Jagged1 expression is required for the enrichment of CSCs using a Jagged1 siRNA. Combined knockdown of Notch1 and Notch3 receptors was necessary to reduce LAP-enriched mammospheres suggesting that targeting HER2 enriches for a Jagged1-Notch1+Notch3 driven CSC phenotype. Importantly, higher membrane expression of Jagged1 protein in 145 HER2+ breast tumor specimens correlated with significantly lower overall cumulative survival. These results reveal that HER2 blockade in breast cancer cells enriches for a Jagged1high subpopulation that has higher CSC potential and is resistant to HER2 inhibitors. The implications of this work are that dual blockade of Jagged-1/Notch1/Notch3 and HER2 could be more effective than either therapy alone to eliminate both HER2 and Jagged-1-dependent cancer cells. Citation Format: Deep S. Shah, Debra Wyatt, Andrew Baker, Andrew Green, Aleksandra Filipovic, Lucio Miele, Clodia Osipo. Targeting HER2 enriches Jagged1 high cancer stem cells in 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 4769. doi:10.1158/1538-7445.AM2017-4769

Abstract LB-121: DAXX is a novel Notch-1 gene target and biomarker of GSI-sensitivity in ER+ breast cancer

Breast cancer is a heterogeneous disease that is best treated based on the expressed biomarker profile. Currently, the major clinical challenges are drug resistance and metastatic spread. Cancer stem cells (CSCs) are believed to be responsible for drug resistance and disease progression despite therapy. We previously identified novel biomarkers that could potentially be used with novel therapeutic strategies that target CSCs. We demonstrated in a presurgical window biomarker study of 20 breast cancer patients with ER-positive disease that signaling through Notch receptors mediates expression of 18 genes. Twenty women with ER-positive tumors were treated with 14 days of ET (tamoxifen or letrozole), with the addition of the oral GSI MK-0752 on day 15 (3 days on, 4 days off, 3 days on). Definitive surgery was on day 25. Analysis of biomarkers using microarray and RT-PCR during ET combined with MK-0752 GSI revealed statistically significant modulation of 18 genes: pro-apoptotic DAXX and NOXA; a tumor suppressor LFNG; Notch signaling such as NOTCH1, NOTCH4, HEYL, HES1, and HEY2; as well as proliferation-associated transcripts MIK67, CCND1, CCNA2; stem cell markers RUNX1 and ALDH1; and novel genes such as RICTOR, RPTOR, MMP7, ADAM19, and PgR (Albain et al. Proc SABCS 2014). The goal of the current study was to identify whether Notch1 directly regulated the 18 identified genes. We measured binding of Notch1 to CBF-1 (CSL/RPBJκ) binding sites of the 18 genes using a Chromatin Immunoprecipitation assay (ChIP) to determine whether Notch1 directly regulates the 18 genes. We scanned and designed primers 5 kb upstream and downstream of transcription start sites of the 18 genes and demonstrated that Notch1 was recruited to CBF-1 sites of 16 out of 18 genes. Different distribution of Notch1 binding was observed across all of the genes. Classical Notch targets, HEY1 and HES1 were among the most responsive genes and used as positive controls. Notch1 was found to be highly enriched on CBF-1 regulatory elements for the DAXX and NOXA genes. Binding of Notch1 to DAXX promoter elements increased in response to estrogen deprivation and this increase was attenuated upon GSI treatment. The biological activity of most genes was measured using the mammosphere-forming assay as a surrogate for CSC survival. Estrogen deprivation increased mammosphere-forming efficiency of ER+ breast cancer cells more than 2 fold compared to estrogen treatment. GSI blocked mammosphere formation by 95%-98% in response to anti-estrogen treatment. DAXX expression, was found to be necessary for GSI-mediated blockade of mammosphere formation. ChIP and mammosphere forming data calculated by ANOVA were found to be statistically significant. These results demonstrated that Notch1 is a direct transcriptional regulator of 16 genes identified by the clinical trial and in particular, DAXX, a pro-apoptotic gene that could serve as a cancer stem cell biomarker for anti-Notch therapy in ER+ breast cancer. Supported by a grant from the Breast Cancer Research Foundation. Citation Format: Andrei Zlobin, Debra Wyatt, Jeffrey C. Bloodworth, Susan Hilsenbeck, Suzanne Fuqua, Lucio Miele, Kathy S. Albain, Clodia Osipo. DAXX is a novel Notch-1 gene target and biomarker of GSI-sensitivity in ER+ 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 LB-121.

Abstract 1823: Novel regulation of Jagged1 by ErbB2 in breast cancer: implications for anti-ErbB2 therapy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA We have demonstrated that Notch1 is required for trastuzumab resistance in ErbB2 positive breast cancer. This indicates that ErbB2 suppresses Notch1 in breast cancer and therapeutic intervention targeting ErbB2 might have an unintended consequence which is aberrant up regulation of Notch1 which is a breast oncogene. However, the mechanism of action by which ErbB2 restricts Notch1 activation is unknown. In this current study, we investigated the role of cis- and trans-activation of Notch signaling by Notch ligands which are developmentally conversed to tightly regulate Notch activation. To address this hypothesis, we performed co-culture studies using fibroblasts expressing no Notch ligands or over-expressing human Jagged1 or Deltalike1 and ErbB2 positive breast cancer cells. We performed flow cytometry to isolate breast cancer cells after co-culture and extracted RNA to measure expression of Notch gene targets as a measure of Notch activity. The results showed that trastuzumab, Lapatinib, or ErbB2 knockdown increased overall Notch activition. Similarly, Co-culture with Jagged1-expressing fibroblasts increased overall Notch activation. However, Knocked down of Jagged1 in the breast cancer cells had little effect on ligand-induced Notch activation relieving the possibility of cis-inhibition. In contrast, Jagged1 knocked down abrogated trastuzumab-induced Notch activation in the breast cancer cells. These results suggest that ErbB2 might restrict Notch activation by preventing Jagged1-mediated trans activation of Notch and not by promoting cis-inhibition. Confocal immunofluorescence showed that Jagged1 is localized with Notch1 when ErbB2 is hyperactive but is trafficked to the cell surface in response to trastuzumab. K44ADynamin abrogated Jagged1 expression on the cell surface as measured by IF and surface biotinylation studies. Furthermore, K44ADynamin expression abrogated trastuzumab-induced Notch1 activation. Importantly, we measured growth consequences of Jagged1-mediated Notch activation in response to trastuzumab and found that Jagged1 is necessary for survival of ErbB2 positive breast cancer cells and trastuzumab resistance as measured by cell cycle analysis and Annexin V staining. These results taken together indicate that ErbB2 restricts Notch by limiting Jagged1-mediated trans-activation. Citation Format: Clodia Osipo, Kinnari Pandya, Debra Wyatt. Novel regulation of Jagged1 by ErbB2 in breast cancer: implications for anti-ErbB2 therapy. [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 1823. doi:10.1158/1538-7445.AM2014-1823