Andrei Zlobin

Activation of Notch-1 signaling maintains the neoplastic phenotype in human Ras-transformed cells

Truncated Notch receptors have transforming activity in vitro and in vivo. However, the role of wild-type Notch signaling in neoplastic transformation remains unclear. Ras signaling is deregulated in a large fraction of human malignancies and is a major target for the development of novel cancer treatments. We show that oncogenic Ras activates Notch signaling and that wild-type Notch-1 is necessary to maintain the neoplastic phenotype in Ras-transformed human cells in vitro and in vivo. Oncogenic Ras increases levels and activity of the intracellular form of wild-type Notch-1, and upregulates Notch ligand Delta-1 and also presenilin-1, a protein involved in Notch processing, through a p38-mediated pathway. These observations place Notch signaling among key downstream effectors of oncogenic Ras and suggest that it might be a novel therapeutic target.

CBP-mediated acetylation of histone H3 lysine 27 antagonizes Drosophila Polycomb silencing

Trimethylation of histone H3 lysine 27 (H3K27me3) by Polycomb repressive complex 2 (PRC2) is essential for transcriptional silencing of Polycomb target genes, whereas acetylation of H3K27 (H3K27ac) has recently been shown to be associated with many active mammalian genes. The Trithorax protein (TRX), which associates with the histone acetyltransferase CBP, is required for maintenance of transcriptionally active states and antagonizes Polycomb silencing, although the mechanism underlying this antagonism is unknown. Here we show that H3K27 is specifically acetylated by Drosophila CBP and its deacetylation involves RPD3. H3K27ac is present at high levels in early embryos and declines after 4 hours as H3K27me3 increases. Knockdown of E(Z) decreases H3K27me3 and increases H3K27ac in bulk histones and at the promoter of the repressed Polycomb target gene abd-A, suggesting that these indeed constitute alternative modifications at some H3K27 sites. Moderate overexpression of CBP in vivo causes a global increase in H3K27ac and a decrease in H3K27me3, and strongly enhances Polycomb mutant phenotypes. We also show that TRX is required for H3K27 acetylation. TRX overexpression also causes an increase in H3K27ac and a concomitant decrease in H3K27me3 and leads to defects in Polycomb silencing. Chromatin immunoprecipitation coupled with DNA microarray (ChIP-chip) analysis reveals that H3K27ac and H3K27me3 are mutually exclusive and that H3K27ac and H3K4me3 signals coincide at most sites. We propose that TRX-dependent acetylation of H3K27 by CBP prevents H3K27me3 at Polycomb target genes and constitutes a key part of the molecular mechanism by which TRX antagonizes or prevents Polycomb silencing.

HPV16 E6 and E7 oncoproteins regulate Notch-1 expression and cooperate to induce transformation.

Notch receptor signaling has been implicated in cellular transformation. Notch‐1 receptor expression is increased during the progression from cervical intraepithelial lesions (CIN) to invasive cervical carcinoma. Moreover, the main cellular localization of Notch‐1 protein changes from cytoplasmic to nuclear with the transition from CIN III to microinvasive carcinoma. Since the E6 and E7 proteins encoded by human papilloma virus (HPV) are a causative agent of cervical carcinoma, this study determined whether E6 and E7 protein expression causes the observed upregulation in Notch‐1 expression. Mouse and human primary cell lines were transfected with HPV16 E6 and E7 and Notch‐1 expression and activity were analyzed. We show that Notch‐1 expression and activity are upregulated by E6 and E7 independently. This was due to both transcriptional and post‐transcriptional mechanisms. A protein involved in Notch processing, Presenilin‐1 (PS‐1), was also upregulated by E6 and E7. In the presence of E6 and E7, Notch‐1 protein expression is localized in the cytoplasm. Downregulation of Notch‐1 expression in a human cervical carcinoma cell line expressing E6/E7 caused striking inhibition of proliferation in vitro and tumorigenicity in vivo. These data suggest that E6‐ and E7‐mediated upregulation of Notch signaling may contribute to disruption of regular cell growth in cervical cancer.

Notch-1 regulates cell death independently of differentiation in murine erythroleukemia cells through multiple apoptosis and cell cycle pathways.

Notch signaling is a potential therapeutic target for various solid and hematopoietic malignancies. We have recently shown that downregulation of Notch‐1 expression has significant anti‐neoplastic activity in pre‐clinical models. However, the mechanisms through which Notch modulation may affect cell fate in cancer remain poorly understood. We had previously shown that Notch‐1 prevents apoptosis and is necessary for pharmacologically induced differentiation in murine erythroleukemia (MEL) cells. We investigated the mechanisms of these effects using three experimental strategies: (1) MEL cells stably transfected with antisense Notch‐1 or constitutively active Notch‐1, (2) activation of Notch‐1 by a cell‐associated ligand, and (d3) activation of Notch‐1 by a soluble peptide ligand. We show that: (1) downregulation of Notch‐1 sensitizes MEL cells to apoptosis induced by a Ca2+ influx or anti‐neoplastic drugs; (2) Notch‐1 downregulation induces phosphorylation of c‐Jun N‐terminal kinase (JNK) while constitutive activation of Notch‐1 or prolonged exposure to a soluble Notch ligand abolishes it; (3) Notch‐1 has dose‐ and time‐dependent effects on the levels of apoptotic inhibitor Bcl‐xL and cell cycle regulators p21cip1/waf1, p27kip1, and Rb; and (4) Notch‐1 activation by a cell‐associated ligand is accompanied by rapid and transient induction of NF‐κB DNA‐binding activity. The relative effects of Notch‐1 signaling on these pathways depend on the levels of Notch‐1 expression, the mechanism of activation, and the timing of activation. The relevance of these findings to the role of Notch signaling in differentiation and cancer are discussed.

Toward The Rational Design of Cell Fate Modifiers: Notch Signaling as a Target for Novel Biopharmaceuticals

Recent advances in our understanding of highly conserved mechanisms that control cell fate determination are paving the way towards rationally designed biologics that modulate specific cell fate decisions. Cell fate decisions leading to proliferation, differentiation or apoptosis are crucial elements in the pathogenesis of countless human diseases. Biopharmaceuticals designed to regulate such processes in specific cell types in vivo or ex vivo have vast potential applications in oncology, stem cell technology, immunomodulation and neuropathology. One of the most conserved mechanisms controlling cell fate determination is based upon Notch-ligand interactions and subsequent signaling events. Recent studies have shown that this mechanism regulates cell differentiation, proliferation and apoptosis in a wide variety of cell maturation processes and in neoplastic cells. These observations identify the Notch signaling network as a promising drug target for numerous indications. In this review, we describe: 1) potential drug targets in the Notch signaling network; 2) the Notch agonists and antagonists developed so far, including recombinant proteins, antibody-based agents, synthetic peptides, antisense oligonucleotides and gene therapy approaches, as well as possible strategies to design novel Notch-targeting biopharmaceuticals; 3) the possible clinical applications of such biopharmaceuticals and 4) a model strategy for the selection and developement of a Notch-targeting biopharmaceutical.

Gamma secretase inhibitors of Notch signaling

The numerous processes involved in the etiology of breast cancer such as cell survival, metabolism, proliferation, differentiation, and angiogenesis are currently being elucidated. However, underlying mechanisms that drive breast cancer progression and drug resistance are still poorly understood. As we discuss here in detail, the Notch signaling pathway is an important regulatory component of normal breast development, cell fate of normal breast stem cells, and proliferation and survival of breast cancer initiating cells. Notch exerts a wide range of critical effects through a canonical pathway where it is expressed as a type I membrane precursor heterodimer followed by at least two subsequent cleavages induced by ligand engagement to ultimately release an intracellular form to function as a transcriptional activator. Notch and its ligands are overexpressed in breast cancer, and one method of effectively blocking Notch activity is preventing its cleavage at the cell surface with γ-secretase inhibitors. In the context of Notch signaling, the application of clinically relevant anti-Notch drugs in treatment regimens may contribute to novel therapeutic interventions and promote more effective clinical response in women with breast cancer.

Notch-EGFR/HER2 Bidirectional Crosstalk in Breast Cancer

The Notch pathway is a well-established mediator of cell–cell communication that plays a critical role in stem cell survival, self-renewal, cell fate decisions, tumorigenesis, invasion, metastasis, and drug resistance in a variety of cancers. An interesting form of crosstalk exists between the Notch receptor and the Epidermal Growth Factor Receptor Tyrosine Kinase family, which consists of HER-1, -2, -3, and -4. Overexpression of HER and/or Notch occurs in several human cancers including brain, lung, breast, ovary, and skin making them potent oncogenes capable of advancing malignant disease. Continued assessment of interplay between these two critical signaling networks uncovers new insight into mechanisms used by HER-driven cancer cells to exploit Notch as a compensatory pathway. The compensatory Notch pathway maintains HER-induced downstream signals transmitted to pathways such as Mitogen Activated Protein Kinase and Phosphatidylinositol 3-Kinase (PI3K), thereby allowing cancer cells to survive molecular targeted therapies, undergo epithelial to mesenchymal transitioning, and increase cellular invasion. Uncovering the critical crosstalk between the HER and Notch pathways can lead to improved screening for the expression of these oncogenes enabling patients to optimize their personal treatment options and predict potential treatment resistance. This review will focus on the current state of crosstalk between the HER and Notch receptors and the effectiveness of current therapies targeting HER-driven cancers.

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 P6-05-25: Ratio of notch receptors is critical for response to notch inhibition by a gamma-secretase inhibitor in triple negative breast cancer cells

Background: Women with triple-negative breast cancer have the worst prognosis, and have few targeted therapy options. Notch receptor genes are potent breast oncogenes, overexpressed in triple-negative breast cancer, and critical for survival of breast cancer stem cells. However, the contribution of each Notch receptor and the significance of the ratio of receptors to growth and sensitivity to Notch inhibitors have not yet been fully explored. We hypothesized that each Notch receptor has distinct roles in the etiology of triple negative breast cancer and designed a study to explore these roles in a panel of three triple negative cell lines and primary, human triple negative breast cancer tissue. Methods: Human cell lines BT-549, MDA-MB-231, and MDA-MB-468 were used to measure endogenous mRNA transcript levels of Notch ligands, receptors, and gene targets important for canonical Notch signaling (i.e. Hes-1, Hes-5, Hey-1, and Deltex-1), apoptosis (Noxa), inflammation (MMP-9 and IL-8), and cancer stem cells (ALDH1) by means of real time PCR under conditions where all four receptors were inhibited by a pan-inhibitor, gamma-secretase inhibitor or each receptor was individually knocked down using RNA interference. Furthermore, both anchorage-dependent and –independent growth were measured by counting cells and methylcellulose colony forming assay, respectively. In addition, the RNA from formalin-fixed, paraffin-embedded specimen from women-diagnosed with TNBC who had undergone breast surgery was also analyzed for comparable gene expression for similar targets following Laser capture micro-dissection. Results: The results showed that only MDA-MB-468 cells were sensitive to growth inhibition by a GSI while MDA-MB-231 and BT549 were resistant. Transcripts of Notch-4, Deltex-1, Hes-1 and Hes-5, as well as IL-8 and MMP-9 were significantly increased in MDA-MB-468 as compared to BT549 or MDA-MB-231 cells that were resistant. Interestingly, the cancer stem marker, ALDH1 was significantly increased only in MDA-MB-468 while the apoptotic marker, Noxa was decreased compared to resistant cells: BT549 and MDA-MB-231. The PCR and growth results from the Notch-1 knocked down were similar to GSI inhibition. However, knocked down of Notch-2 resulted in resistance to GSI. Notch-3 knocked down showed an increase in Notch-1, Notch-2, and Notch-4 transcripts implying that Notch-3 might suppress the other Notch receptors. Notch-4 knocked down alone had little effect on growth of any of the three cell lines. The results from human specimens showed an inverse relationship between Notch-1 and Notch-2, and Notch-1 and Notch-4. For example, Notch-1, Jagged-1, and Hes-5 genes were up-regulated in triple negative tumor tissue (n = 20), while the Notch-4 gene was down-regulated as compared to normal control specimens from reduction mammoplasty (n = 4). Conclusions: The results from this study indicate that Notch-1 is probably the growth driver in certain triple negative cancer types.. Furthermore, the ratios of Notch receptors and their activity states could predict sensitivity to Notch inhibition by a GSI or other inhibitors. Lastly, these results suggest that the best predictor genes for Notch activation are: Notch-1, Hes-1, and ALDH1. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-05-25.

Abstract P4-08-06: Notch-dependent Regulation of Novel Genes Associated with Trastuzumab Resistance

Background: We have shown previously that Notch-1 is critical for the development and maintenance of trasuzumab or lapatinib resistance in HER2 positive breast cancer cell lines. Here we sought to identify the critical genes that are regulated by Notch to promote anti-HER2 targeted resistance. Methods: We measured expression of 84 known breast cancer-associated transcripts using a real-time PCR array in both trastuzumab sensitive and resistant BT474 cell lines. A 4 fold increase or decrease in transcripts levels were measured to be statisitically significant. To confirm the change in levels, we designed primers to each gene of interest and repeated the PCR at least three independent times. Furthermore, we specifically asked whether Notch-1 or it9s transcriptional mediator, CBF-1, directly regulated any of the identified genes in HER2 positive breast cancer cells using a genetic knocked down approach. An ANOVA for multiple comparisons was used to compute statistical significance. Results: We identified more than 4 fold increase in APC, ABCG2, ABCB1, Gata-3, Bcl-2, Id2, and HRG-2 transcripts in trastuzumab resistant versus sensitive cells. Conversley, Gli1 was downregulated in resistant versus sensitive cells. Interestingly, siRNA directed against Notch-1 or CBF-1 decreased ABCG2 and Gata-3, but conversely increased ABCB1 and HRG-2 in resistant cells. Conclusions: Our findings demonstrate APC, ABCG2, ABCB1, Gata-3, Bcl-2, Id2, and HRG-2 transcripts are signficantly increased in trastuzumab resistant breast cancer cells compared to sensitive cells. More importantly, Notch-1 or it9s direct transcriptional activator CBF-1 may contribute to traztuzumab resistance by directly regulating critical genes that contribute to multidrug resistance: ABCB1 and ABCG2 and/or genes associated with stem cell survival: Gata-3 and HRG-2. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-08-06.