David N. Boone

Nucleophosmin interacts directly with c-Myc and controls c-Myc-induced hyperproliferation and transformation

The transcription factor c-Myc is essential for cellular proliferation and is one of the most frequently activated oncogenes, but the molecular mechanism mediating its critical role in transformation is unclear. Like c-Myc, multifunctional nucleophosmin (NPM) is tightly regulated during proliferation and is overexpressed in several different types of cancer. Overexpression of NPM enhances proliferation and oncogene-mediated transformation, but the mechanism mediating these effects is unknown. We examined whether NPM stimulates proliferation and transformation by affecting c-Myc. Here, we show that NPM is essential for the activities of oncogenic c-Myc and that overexpressed NPM dramatically stimulates c-Myc-induced hyperproliferation and transformation. Endogenous and exogenous NPM directly interact with c-Myc and regulate the expression of endogenous c-Myc target genes at the promoter. Therefore, NPM is a key cofactor for the transforming activity of c-Myc and the interaction with c-Myc may mediate the enhancement of proliferation and transformation induced by NPM overexppression.

Egr1 mediates p53-independent c-Myc–induced apoptosis via a noncanonical ARF-dependent transcriptional mechanism

c-Myc is frequently deregulated in human cancers. Although deregulated c-Myc leads to tumor growth, it also triggers apoptosis in partnership with tumor suppressors such as ARF and p53. Apoptosis induced by c-Myc is a critical fail-safe mechanism for the cell to protect against unrestrained proliferation. Despite the plethora of information on c-Myc, the molecular mechanism of how c-Myc induces both transformation and apoptosis is unclear. Oncogenic c-Myc can indirectly induce the expression of the tumor suppressor ARF, which leads to apoptosis through the stabilization of p53, but both c-Myc and ARF have apoptotic activities that are independent of p53. In cells without p53, ARF directly binds to c-Myc protein and inhibits c-Myc–induced hyperproliferation and transformation with a concomitant inhibition of canonical c-Myc target gene induction. However, ARF is an essential cofactor for p53-independent c-Myc–induced apoptosis. Here we show that ARF is necessary for c-Myc to drive transcription of a unique noncanonical target gene, Egr1. In contrast, c-Myc induces another family member, Egr2, through a canonical mechanism that is inhibited by ARF. We further demonstrate that Egr1 is essential for p53-independent c-Myc–induced apoptosis, but not ARF-independent c-Myc–induced apoptosis. Therefore, ARF binding switches the inherent activity of c-Myc from a proliferative to apoptotic protein without p53 through a unique noncanonical transcriptional mechanism. These findings also provide evidence that cofactors can differentially regulate specific transcriptional programs of c-Myc leading to different biological outcomes.

Role of IGF1R in Breast Cancer Subtypes, Stemness, and Lineage Differentiation

Insulin-like growth factor (IGF) signaling is fundamental for growth and survival. A large body of evidence (laboratory, epidemiological, and clinical) implicates the exploitation of this pathway in cancer. Up to 50% of breast tumors express the activated form of the type 1 insulin-like growth factor receptor (IGF1R). Breast cancers are categorized into subtypes based upon hormone and ERRB2 receptor expression and/or gene expression profiling. Even though IGF1R influences tumorigenic phenotypes and drug resistance across all breast cancer subtypes, it has specific expression and function in each. In some subtypes, IGF1R levels correlate with a favorable prognosis, while in others it is associated with recurrence and poor prognosis, suggesting different actions based upon cellular and molecular contexts. In this review, we examine IGF1R expression and function as it relates to breast cancer subtype and therapy-acquired resistance. Additionally, we discuss the role of IGF1R in stem cell maintenance and lineage differentiation and how these cell fate influences may alter the differentiation potential and cellular composition of breast tumors.

Domain-specific c-Myc ubiquitylation controls c-Myc transcriptional and apoptotic activity

The oncogenic transcription factor c-Myc causes transformation and tumorigenesis, but it can also induce apoptotic cell death. Although tumor suppressors are necessary for c-Myc to induce apoptosis, the pathways and mechanisms are unclear. To further understand how c-Myc switches from an oncogenic protein to an apoptotic protein, we examined the mechanism of p53-independent c-Myc–induced apoptosis. We show that the tumor suppressor protein ARF mediates this switch by inhibiting ubiquitylation of the c-Myc transcriptional domain (TD). Whereas TD ubiquitylation is critical for c-Myc canonical transcriptional activity and transformation, inhibition of ubiquitylation leads to the induction of the noncanonical c-Myc target gene, Egr1, which is essential for efficient c-Myc–induced p53-independent apoptosis. ARF inhibits the interaction of c-Myc with the E3 ubiquitin ligase Skp2. Overexpression of Skp2, which occurs in many human tumors, inhibits the recruitment of ARF to the Egr1 promoter, leading to inhibition of c-Myc–induced apoptosis. Therapeutic strategies could be developed to activate this intrinsic apoptotic activity of c-Myc to inhibit tumorigenesis.

A Role for Histone H2B Variants in Endocrine-Resistant Breast Cancer

Acquired resistance to aromatase inhibitors (AIs) remains a major clinical problem in the treatment of estrogen receptor-positive (ER+) breast cancer. We and others have previously reported widespread changes in DNA methylation using breast cancer cell line models of endocrine resistance. Here, we show that the histone variant HIST1H2BE is hypomethylated in estrogen deprivation-resistant C4-12 and long-term estrogen-deprived (LTED) cells compared with parental MCF-7 cells. As expected, this hypomethylation associates with increased expression of HIST1H2BE in C4-12 and LTED cells. Both overexpression and downregulation of HIST1H2BE caused decreased proliferation in breast cancer cell lines suggesting the need for tightly controlled expression of this histone variant. Gene expression analysis showed varied expression of HIST1H2BE in a large panel of breast cancer cell lines, without restriction to specific molecular subtypes. Analysis of HIST1H2BE messenger RNA (mRNA) expression in ER+ AI-treated breast tumors showed significantly higher expression in resistant (n = 19) compared with sensitive (n = 37) tumors (p = 0.01). Using nanostring analysis, we measured expression of all 61 histone variants in endocrine-resistant and endocrine-sensitive tumors. We found significant overexpression of 22 variant histone genes in tumors resistant to AI therapy. In silico The Cancer Genome Atlas (TCGA) analysis showed frequent amplification of the HIST1 locus. In summary, our studies show, for the first time, that overexpression of histone variants might be important in endocrine response in ER+ breast cancer, and that overexpression is at least in part mediated via epigenetic mechanisms and amplifications. Future studies addressing endocrine response should include a potential role of these currently understudied histone variants.

Controlled dimerization of insulin-like growth factor-1 and insulin receptors reveals shared and distinct activities of holo and hybrid receptors

Breast cancer development and progression are influenced by insulin-like growth factor receptor 1 (IGF1R) and insulin receptor (InsR) signaling, which drive cancer phenotypes such as cell growth, proliferation, and migration. IGF1R and InsR form IGF1R/InsR hybrid receptors (HybRs) consisting of one molecule of IGF1R and one molecule of InsR. The specific signaling and functions of HybR are largely unknown, as HybR is activated by both IGF1 and insulin, and no cellular system expresses HybR in the absence of holo-IGF1R or holo-InsR. Here we studied the role of HybR by constructing inducible chimeric receptors and compared HybR signaling with that of holo-IGF1R and holo-InsR. We cloned chemically inducible chimeric IGF1R and InsR constructs consisting of the extracellular domains of the p75 nerve growth factor receptor fused to the intracellular β subunit of IGF1R or InsR and a dimerization domain. Dimerization with the drugs AP20187 or AP21967 allowed specific and independent activation of holo-IGF1R, holo-InsR, or HybR, resulting in activation of the PI3K pathway. Holo-IGF1R and HybR both promoted cell proliferation and glucose uptake, whereas holo-InsR only promoted glucose uptake, and only holo-IGF1R showed anti-apoptotic effects. We also found that the three receptors differentially regulated gene expression: holo-IGF1R and HybR up-regulated EGR3; holo-InsR specifically down-regulated JUN and BCL2L1; holo-InsR down-regulated but HybR up-regulated HK2; and HybR specifically up-regulated FHL2, ITGA6, and PCK2. Our findings suggest that, when expressed and activated in mammary epithelial cells, HybR acts in a manner similar to IGF1R and support further investigation of the role of HybR in breast cancer.

Computer science, biology and biomedical informatics academy: outcomes from 5 years of immersing high-school students into informatics research

The University of Pittsburghs Department of Biomedical Informatics and Division of Pathology Informatics created a Science, Technology, Engineering, and Mathematics (STEM) pipeline in 2011 dedicated to providing cutting-edge informatics research and career preparatory experiences to a diverse group of highly motivated high-school students. In this third editorial installment describing the program, we provide a brief overview of the pipeline, report on achievements of the past scholars, and present results from self-reported assessments by the 2015 cohort of scholars. The pipeline continues to expand with the 2015 addition of the innovation internship, and the introduction of a program in 2016 aimed at offering first-time research experiences to undergraduates who are underrepresented in pathology and biomedical informatics. Achievements of program scholars include authorship of journal articles, symposium and summit presentations, and attendance at top 25 universities. All of our alumni matriculated into higher education and 90% remain in STEM majors. The 2015 high-school program had ten participating scholars who self-reported gains in confidence in their research abilities and understanding of what it means to be a scientist.

Insights from Global Analyses of Long Noncoding RNAs in Breast Cancer

Purpose of ReviewThe goal of this review was to compare and contrast the results and implications from several recent transcriptomic studies that analyzed the expression of long noncoding RNAs (lncRNAs) in breast cancer. How many lncRNAs are dysregulated in breast cancer? Do dysregulated lncRNAs contribute to breast cancer etiology? Are lncRNAs viable biomarkers in breast cancer?Recent FindingsTranscriptomic profiling of breast cancer tissues, mostly from The Cancer Genome Atlas, identified thousands of long noncoding RNAs that are expressed and dysregulated in breast cancer. The expression of lncRNAs alone can divide patients into molecular subtypes. Subsequent functional studies demonstrated that several of these lncRNAs have important roles in breast cancer cell biology.SummaryThousands of lncRNAs are dysregulated in breast cancer that can be developed as biomarkers for prognostic or therapeutic purposes. The reviewed reports provide a roadmap to guide functional studies to discover lncRNAs with critical biological functions relating to breast cancer development and progression.

Loss of E-cadherin Enhances IGF1–IGF1R Pathway Activation and Sensitizes Breast Cancers to Anti-IGF1R/InsR Inhibitors

Purpose: Insulin-like growth factor 1 (IGF1) signaling regulates breast cancer initiation and progression and associated cancer phenotypes. We previously identified E-cadherin (CDH1) as a repressor of IGF1 signaling and in this study examined how loss of E-cadherin affects IGF1R signaling and response to anti-IGF1R/insulin receptor (InsR) therapies in breast cancer. Experimental Design: Breast cancer cell lines were used to assess how altered E-cadherin levels regulate IGF1R signaling and response to two anti-IGF1R/InsR therapies. In situ proximity ligation assay (PLA) was used to define interaction between IGF1R and E-cadherin. TCGA RNA-seq and RPPA data were used to compare IGF1R/InsR activation in estrogen receptor-positive (ER+) invasive lobular carcinoma (ILC) and invasive ductal carcinoma (IDC) tumors. ER+ ILC cell lines and xenograft tumor explant cultures were used to evaluate efficacy to IGF1R pathway inhibition in combination with endocrine therapy. Results: Diminished functional E-cadherin increased both activation of IGF1R signaling and efficacy to anti-IGF1R/InsR therapies. PLA demonstrated a direct endogenous interaction between IGF1R and E-cadherin at points of cell–cell contact. Increased expression of IGF1 ligand and levels of IGF1R/InsR phosphorylation were observed in E-cadherin–deficient ER+ ILC compared with IDC tumors. IGF1R pathway inhibitors were effective in inhibiting growth in ER+ ILC cell lines and synergized with endocrine therapy and similarly IGF1R/InsR inhibition reduced proliferation in ILC tumor explant culture. Conclusions: We provide evidence that loss of E-cadherin hyperactivates the IGF1R pathway and increases sensitivity to IGF1R/InsR targeted therapy, thus identifying the IGF1R pathway as a potential novel target in E-cadherin–deficient breast cancers. Clin Cancer Res; 24(20); 5165–77. ©2018 AACR.

Abstract 800: Simultaneous methylation and mutational analysis of breast cancer genomes using droplet-based targeted sequencing.

Targeted sequencing using droplet-based PCR provides an established method to selectively and uniformly amplify thousands of genomic regions of interest for deep, cost-effective next generation sequencing. Recently, this approach has been adapted for the analysis of bisulfite-treated DNA, enabling both quantitative epigenomic analysis (strand-specific determination of cytosine methylation status with single-base pair resolution) and genomic mutation detection. Simultaneous collection of both methylation and genomic aberration information with a single method makes efficient use of precious clinical material and allows both researchers and clinicians to use this information to sub-type cancers. Here we report on our sequencing results using a single breast cancer targeted panel for sequence specific analysis of both epigenomic methylation and genomic mutation in breast tumor samples. The Breast Cancer MethylSeq Panel includes PCR primer pairs designed to amplify 1000 targets, leaving room for an additional 3000 target loci of interest to be added by individual researchers in the future. This single panel provides information about both loci-specific CpG methylation and genomic mutations, enabling analysis of multiple content types including: A) cell subtype (CELL); B) genomic mutation (MUT); C) surrogate gene expression subtypes using promoter CpG islands or other methylation marks (EXP); D) copy-number variation (CNV); E) Loss of Heterozygosity (LOH); and F) methylation-mediated gene silencing and loss of imprinting (IMP). In addition, miRNA associated with breast cancer will be targeted for analysis (MIR). The Breast Cancer MethylSeq Panel was tested and validated with a set of 64 bisulfite-converted DNA samples that included breast cancer cell lines, a range of ER positive and negative tumors and TCGA samples, and a set of 8 metastatic tumors from a single individual. Overall Illumina HiSeq sequencing success metrics, concordance with metadata on the various target classes (A-F above) using validated controls, and results from the patient samples will be presented. The Breast Cancer MethylSeq Panel allows simultaneous analysis of epigenomic and genomic sequence alterations associated with breast tumor samples, enabling tumor sub-typing and correlation with clinical outcomes. This ‘core’ panel provides a unique (expandable) screening tool for the breast cancer research community, and a paradigm for efficient analysis of multiple endpoints from a single assay for other diseases. Citation Format: Michael L. Samuels, Matt Roth, Preethi Srinivasan, Kevin Riehle, Adam Brufsky, Ryan Hartmaier, David Boone, R. Alan Harris, Chia-Chin Wu, Steffi Oesterreich, Adrian Lee, Aleksandar Milosavljevic. Simultaneous methylation and mutational analysis of breast cancer genomes using droplet-based targeted sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 800. doi:10.1158/1538-7445.AM2013-800