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ErbB2 is overexpressed in approximately 25% of human breast cancers, associated with clinically aggressive disease. No soluble ligand has been identified and the receptor is regulated by heterodimerization with other ErbB family receptors, including EGFR, and other receptor tyrosine kinases including EphA2. EGFR is activated by seven different growth factors including EGF and Amphiregulin. Downstream signaling modules required for ErbB2 induced tumorigenesis in genetically engineered mouse models (GEMM) include the phosphatidylinositol 3-kinase/Akt (PKB) pathway, the Ras/Raf/MEK/ERK1/2 pathway and the phospholipase C (PLCγ) pathways. ErbB2-mediated tumorigenesis involves activation of receptor tyrosine kinases, induction of cyclin D1/CDK activity, and functional restraint by tumor suppressors. The receptor tyrosine kinase EPH receptor A2 (EphA2), a member of the Eph RTK family, is overexpressed in aggressive breast cancer and EphA2 forms a complex with ErbB2 thereby enhancing ErbB2-induced tumor onset and progression. The host immune system participates in the therapeutic response of HER2+ breast cancer. The tumor microenvironment (TME) is regulated by chemokines and their G protein coupled receptors binds several ligands, including Cxcl5 which binds Cxcr2, to augment the pro-tumor immune response, tumor growth and metastasis. Identifying genetic programs that participate in ErbB2-induced tumors may provide the rational basis for co-extinction therapeutic approaches. Peroxisome proliferator-activated receptor γ (PPARγ), which is expressed in a variety of malignancies, governs biological functions through transcriptional programs. Herein, genetic deletion of endogenous Pparγ1 restrained mammary tumor progression, lipogenesis, and induced local mammary tumor F4/80+ tumor-associated macrophage infiltration, without affecting other tissue hematopoietic stem cell pools. Pparγ1 induced peroxisomal target genes in the mammary tumors as evidenced by increased expression of PEX-11, together with PPARGC1 and ESRR induced regulator, muscle 1, Perm1 (PGC-1 and ERR-induced Regulator in Muscle 1). Peroxisomes induced by Pparγ1, activated Type1 interferons (IFNs) and IFN-stimulated gene expression, including Cxcl5. Endogenous Pparγ1 induced expression of both an EphA2-Amphiregulin and an inflammatory INFγ -Cxcl5 signaling module. Pparγ1 bound directly to growth promoting and proinflammatory target genes in the context of chromatin. We conclude Pparγ1 promotes ErbB2-induced tumor growth and inflammation and represents a relevant target for therapeutic coextinction. Citation Format: Richard G Pestell, Xuanmao Jiao, Andrew V Kossenkov, Adam Ertel, Wei Tong, Zhao Zhang, Peter A McCue. Pparg1 induces an EGF-EphA2 receptor tyrosine kinase module to promote ErbB2- mammary adenocarcinoma in mice [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-58.

The essential G1-cyclin, CCND1, is a collaborative nuclear oncogene that is frequently overexpressed in cancer. D-type cyclins bind and activate CDK4 and CDK6 thereby contributing to G1–S cell-cycle progression. In addition to the nucleus, herein cyclin D1 was also located in the cytoplasmic membrane. In contrast with the nuclear-localized form of cyclin D1 (cyclin D1NL), the cytoplasmic membrane-localized form of cyclin D1 (cyclin D1MEM) induced transwell migration and the velocity of cellular migration. The cyclin D1MEM was sufficient to induce G1–S cell-cycle progression, cellular proliferation, and colony formation. The cyclin D1MEM was sufficient to induce phosphorylation of the serine threonine kinase Akt (Ser473) and augmented extranuclear localized 17β-estradiol dendrimer conjugate (EDC)-mediated phosphorylation of Akt (Ser473). These studies suggest distinct subcellular compartments of cell cycle proteins may convey distinct functions.

Summary DACH1 abundance is reduced in human malignancies, including breast cancer. Herein DACH1 was detected among multipotent fetal mammary stem cells in the embryo, among mixed lineage precursors, and in adult basal cells and (ERα+) luminal progenitors. Dach1 gene deletion at 6 weeks in transgenic mice reduced ductal branching, reduced the proportion of mammary basal cells (Lin− CD24med CD29high) and reduced abundance of basal cytokeratin 5, whereas DACH1 overexpression induced ductal branching, increased Gata3 and Notch1, and expanded mammosphere formation in LA-7 breast cells. Mammary gland-transforming growth factor β (TGF-β) activity, known to reduce ductal branching and to reduce the basal cell population, increased upon Dach1 deletion, associated with increased SMAD phosphorylation. Association of the scaffold protein Smad anchor for receptor activation with Smad2/3, which facilitates TGF-β activation, was reduced by endogenous DACH1. DACH1 increases basal cells, enhances ductal formation and restrains TGF-β activity in vivo., Highlights • Dach1 is expressed in mammary gland fetal stem cells and adult luminal cells • Dach1 expands mammary gland basal/myoepithelial cells • Dach1 induces post-natal mammary gland ductal formation • Dach1 retrains TGF-β activity in the mammary gland in vivo, DACH1 abundance, is reduced in human malignancies, including breast cancer. In this article, Dr. Pestell and his colleagues showed that DACH1 is expressed in multipotent mammary gland fetal stem cells and both the adult basal and ERα+ luminal cells, promotes mammary gland stem cell and basal/myoepithelial cell expansion, promotes mammary gland ductal branching, and restrains TGF-β action in the pubertal mammary gland.

Genome-wide association studies (GWAS) for kidney function identified hundreds of risk regions; however, the causal variants, target genes, cell types, and disease mechanisms remain poorly understood. Here, we performed transcriptome-wide association studies (TWAS), summary Mendelian randomization, and MetaXcan to identify genes whose expression mediates the genotype effect on the phenotype. Our analyses identified Dachshund homolog 1 (DACH1), a cell-fate determination factor. GWAS risk variant was associated with lower DACH1 expression in human kidney tubules. Human and mouse kidney single-cell open chromatin data (snATAC-Seq) prioritized estimated glomerular filtration rate (eGFR) GWAS variants located on an intronic regulatory region in distal convoluted tubule cells. CRISPR-Cas9-mediated gene editing confirmed the role of risk variants in regulating DACH1 expression. Mice with tubule-specific Dach1 deletion developed more severe renal fibrosis both in folic acid and diabetic kidney injury models. Mice with tubule-specific Dach1 overexpression were protected from folic acid nephropathy. Single-cell RNA sequencing, chromatin immunoprecipitation, and functional analysis indicated that DACH1 controls the expression of cell cycle and myeloid chemotactic factors, contributing to macrophage infiltration and fibrosis development. In summary, integration of GWAS, TWAS, single-cell epigenome, expression analyses, gene editing, and functional validation in different mouse kidney disease models identified DACH1 as a kidney disease risk gene.

Simple Summary HER2, which is associated with clinically aggressive disease, is overexpressed in 15–20% of breast cancers (BC). Peroxisome proliferator-activated receptor γ (PPARγ), is expressed in a variety of malignancies. The aim of our study was to determine the function of endogenous Pparγ1 in the onset and progression of mammary tumors induced by ErbB2 in mice. Genetic deletion of Pparγ1 slowed the rate of tumor progression and death from ErbB2-induced mammary tumors. The deletion of Pparγ1 correlated with reduced pro-tumorigenic inflammation. We conclude ErbB2 collaborates with endogenous Pparγ1 in the onset and progression of mammary tumorigenesis. Abstract HER2, which is associated with clinically aggressive disease, is overexpressed in 15–20% of breast cancers (BC). The host immune system participates in the therapeutic response of HER2+ breast cancer. Identifying genetic programs that participate in ErbB2-induced tumors may provide the rational basis for co-extinction therapeutic approaches. Peroxisome proliferator-activated receptor γ (PPARγ), which is expressed in a variety of malignancies, governs biological functions through transcriptional programs. Herein, genetic deletion of endogenous Pparγ1 restrained mammary tumor progression, lipogenesis, and induced local mammary tumor macrophage infiltration, without affecting other tissue hematopoietic stem cell pools. Endogenous Pparγ1 induced expression of both an EphA2-Amphiregulin and an inflammatory INFγ and Cxcl5 signaling module, that was recapitulated in human breast cancer. Pparγ1 bound directly to growth promoting and proinflammatory target genes in the context of chromatin. We conclude Pparγ1 promotes ErbB2-induced tumor growth and inflammation and represents a relevant target for therapeutic coextinction. Herein, endogenous Pparγ1 promoted ErbB2-mediated mammary tumor onset and progression. PPARγ1 increased expression of an EGF-EphA2 receptor tyrosine kinase module and a cytokine/chemokine 1 transcriptional module. The induction of a pro-tumorigenic inflammatory state by Pparγ1 may provide the rationale for complementary coextinction programs in ErbB2 tumors.

Reprogramming of metabolic priorities promotes tumor progression. Our understanding of the Warburg effect, based on studies of cultured cancer cells, has evolved to a more complex understanding of tumor metabolism within an ecosystem that provides and catabolizes diverse nutrients provided by the local tumor microenvironment. Recent studies have illustrated that heterogeneous metabolic changes occur at the level of tumor type, tumor subtype, within the tumor itself, and within the tumor microenvironment. Thus, altered metabolism occurs in cancer cells and in the tumor microenvironment (fibroblasts, immune cells and fat cells). Herein we describe how these growth advantages are obtained through either “convergent” genetic changes, in which common metabolic properties are induced as a final common pathway induced by diverse oncogene factors, or “divergent” genetic changes, in which distinct factors lead to subtype-selective phenotypes and thereby tumor heterogeneity. Metabolic heterogeneity allows subtyping of cancers and further metabolic heterogeneity occurs within the same tumor mass thought of as “microenvironmental metabolic nesting”. Furthermore, recent findings show that mutations of metabolic genes arise in the majority of tumors providing an opportunity for the development of more robust metabolic models of an individual patient’s tumor. The focus of this review is on the mechanisms governing this metabolic heterogeneity in breast cancer.

The mechanisms governing therapeutic resistance of the most aggressive and lethal primary brain tumor in adults, glioblastoma, have increasingly focused on tumor stem cells. These cells, protected by the periarteriolar hypoxic GSC niche, contribute to the poor efficacy of standard of care treatment of glioblastoma. Integrated proteogenomic and metabolomic analyses of glioblastoma tissues and single cells have revealed insights into the complex heterogeneity of glioblastoma and stromal cells, comprising its tumor microenvironment (TME). An additional factor, which isdriving poor therapy response is the distinct genetic drivers in each patient’s tumor, providing the rationale for a more individualized or personalized approach to treatment. We recently reported that the G protein-coupled receptor CCR5, which contributes to stem cell expansion in other cancers, is overexpressed in glioblastoma cells. Overexpression of the CCR5 ligand CCL5 (RANTES) in glioblastoma completes a potential autocrine activation loop to promote tumor proliferation and invasion. CCL5 was not expressed in glioblastoma stem cells, suggesting a need for paracrine activation of CCR5 signaling by the stromal cells. TME-associated immune cells, such as resident microglia, infiltrating macrophages, T cells, and mesenchymal stem cells, possibly release CCR5 ligands, providing heterologous signaling between stromal and glioblastoma stem cells. Herein, we review current therapies for glioblastoma, the role of CCR5 in other cancers, and the potential role for CCR5 inhibitors in the treatment of glioblastoma.

Cancer stem cells (CSCs) are believed to be the main source of cancer relapse and metastasis. PIWI-interacting small non-coding RNAs (piRNAs) have been recently recognized to be relevant to cancer biology. Whether and how piRNAs regulate human CSCs remain unknown. Herein, upregulation of piR-823 was identified in tested luminal breast cancer cells, especially in the luminal subtype of breast CSCs. Enforced expression or targeted knockdown of piR-823 demonstrated its oncogenic function in regulating cell proliferation and colony formation in MCF-7 and T-47D breast cancer cells. In addition, piR-823 induced ALDH (+) breast CSC subpopulation promoted the expression of stem cell markers including OCT4, SOX2, KLF4, NANOG, and hTERT, and increased mammosphere formation. Tail vein injection of magnetic nanoparticles carrying anti-piR-823 into the mammary gland of tumor-burdened mice significantly inhibited tumor growth in vivo. DNA methyltransferases (DNMTs) including DNMT1, DNMT3A, and DNMT3B were demonstrated to be the downstream genes of piR-823, which regulate gene expression by maintaining DNA methylation. piR-823 increased the expression of DNMTs, promoted DNA methylation of gene adenomatous polyposis coli (APC), thereby activating Wnt signaling and inducing cancer cell stemness in the luminal subtype of breast cancer cells. The current study not only revealed a novel mechanism through which piRNAs contribute to tumorigenesis in breast cancer by regulating CSCs, but also provided a therapeutic strategy using non-coding genomes in the suppression of human breast cancer.

The chemokine CCL5/RANTES is a versatile inflammatory mediator, which interacts with the receptor CCR5, promoting cancer cell interactions within the tumor microenvironment. Glioblastoma is a highly invasive tumor, in which CCL5 expression correlates with shorter patient survival. Using immunohistochemistry, we identified CCL5 and CCR5 in a series of glioblastoma samples and cells, including glioblastoma stem cells. CCL5 and CCR5 gene expression were significantly higher in a cohort of 38 glioblastoma samples, compared to low-grade glioma and non-cancerous tissues. The in vitro invasion of patients-derived primary glioblastoma cells and glioblastoma stem cells was dependent on CCL5-induced CCR5 signaling and is strongly inhibited by the small molecule CCR5 antagonist maraviroc. Invasion of these cells, which was enhanced when co-cultured with mesenchymal stem cells (MSCs), was inhibited by maraviroc, suggesting that MSCs release CCR5 ligands. In support of this model, we detected CCL5 and CCR5 in MSC monocultures and glioblastoma-associated MSC in tissue sections. We also found CCR5 expressing macrophages were in close proximity to glioblastoma cells. In conclusion, autocrine and paracrine cross-talk in glioblastoma and, in particular, glioblastoma stem cells with its stromal microenvironment, involves CCR5 and CCL5, contributing to glioblastoma invasion, suggesting the CCL5/CCR5 axis as a potential therapeutic target that can be targeted with repositioned drug maraviroc.

The molecular mechanisms governing the secretion of the non-coding genome are poorly understood. We show herein that cyclin D1, the regulatory subunit of the cyclin-dependent kinase that drives cell-cycle progression, governs the secretion and relative proportion of secreted non-coding RNA subtypes (miRNA, rRNA, tRNA, CDBox, scRNA, HAcaBox. scaRNA, piRNA) in human breast cancer. Cyclin D1 induced the secretion of miRNA governing the tumor immune response and oncogenic miRNAs. miR-21 and miR-93, which bind Toll-Like Receptor 8 to trigger a pro-metastatic inflammatory response, represented >85% of the cyclin D1-induced secreted miRNA transcripts. Furthermore, cyclin D1 regulated secretion of the P-element Induced WImpy testis (PIWI)-interacting RNAs (piRNAs) including piR-016658 and piR-016975 that governed stem cell expansion, and increased the abundance of the PIWI member of the Argonaute family, piwil2 in ERα positive breast cancer. The cyclin D1-mediated secretion of pro-tumorigenic immuno-miRs and piRNAs may contribute to tumor initiation and progression.

Objective Menage a trois 1 (MAT1) is a targeting subunit of cyclin-dependent kinase-activating kinase and general transcription factor IIH kinase, which modulates cell cycle, transcription and DNA repair. Its dysregulation is responsible for diseases including cancers. To further explore the role of MAT1 in breast cancer, we investigated the pathways in which MAT1 might be involved, the association between MAT1 and molecular subtypes, and the role of MAT1 in clinical outcomes of breast cancer patients. Methods We conducted immunohistochemistry staining on tissue microarray and immunofluorescence staining on sections of MAT1 stable breast cancer cells. Also, we performed Kyoto Encyclopedia of Genes and Genomes pathway analysis, correlation analysis and prognosis analysis on public databases. Results MAT1 was involved in multiple pathways including normal physiology signaling and disease-related signaling. Furthermore, MAT1 positively correlated with the protein status of estrogen receptor and progesterone receptor, and was enriched in luminal-type and human epidermal growth factor receptor 2-enriched breast cancer in comparison with basal-like subtype at both mRNA and protein levels. Correlation analysis revealed significant association between MAT1 mRNA amount and epithelial markers, mesenchymal markers, cancer stem cell markers, apoptosis markers, transcription markers and oncogenes. Consistently, the results of immunofluorescence stain indicated that MAT1 overexpression enhanced the protein abundance of epidermal growth factor receptor, vimentin, sex determining region Y-box 2 and sine oculis homeobox homolog 1. Importantly, Kaplan-Meier Plotter analysis reflected that MAT1 could serve as a prognostic biomarker predicting worse relapse-free survival and metastasis-free survival. Conclusions MAT1 is correlated with molecular subtypes and is associated with unfavorable prognosis for breast cancer patients.

Cardiac toxicity from chemotherapy, which contributes substantially to morbidity and mortality in cancer survivors is dose-dependent, but sensitivity can vary greatly between patients. Doxorubicin (DOX) reduces ventricular ejection fraction to 1.5 million women develop breast cancer annually, it is estimated that >50,000 women/year will develop severe cardiotoxicity. With cancer survivors estimated at 19 million in the USA by 2025, DOX-induced cardiotoxicity is considered part of the “cardio-oncology epidemic”. Two cellular activities: DNA damage, causing double-strand breaks (DSBs) following poisoning of topoisomerase II (Topo II), and chromatin damage, mediated through histone eviction, are required for cardiotoxicity. The only FDA approved preventive, dexrazoxane reduces the anti-cancer efficacy of the DOX-based chemotherapeutic agents has the concerning side effects of myelotoxicity and a possible increased prevalence of secondary malignancies. We previously showed that the G-protein coupled receptor CCR5 is expressed on both immune and epithelial cells of ~50% of human breast cancers (BCa), thereby inducing cancer “stemness”, cell survival and DNA repair and a pro-metastatic phenotype. Moreover, CCR5 expression is enhanced in DOX-resistant BCa cells and CCR5 inhibitors (CCR5i) enhance DOX-induced cell death of BCa cells. Whilst expression of CCR5 and its ligands (CCL3 and CCL5) were induced by DOX-treatment in cardiac myocytes in both a murine model of DOX-cardiac toxicity and in the hearts of patients undergoing cardiac transplantation for DOX-induced cardiomyopathy. Unlike in BCa cells (where the enhanced CCR5 expression is transcriptionaly regulated) the increased expression in the myocardium occurs in the absence of altered mRNA levels suggesting epigenetic control over CCR5 expression during DOX-induced cardiotoxicity. In the myocardium CCR5 signaling promotes cardiac remodeling and heart failure, opposite that in BCa where CCR5 signaling promotes survival. Not surprisingly, CCR5i protected human iPSC-derived cardiomyocytes and isolated canine cardiomyocytes from DOX-induced cell death using nuclear fragmentation (hypodiploid peak) and mitochondrial function (MitoGlo reagent) as endpoints. Further, in a murine model of DOX cardiotoxicity (3 mg/kgx8 doses over 2 weeks) CCR5i substantially reduced (>90%) DOX-induced cardiac dysfunction and mortality in mice. CCR5 inhibitors (CCR5i) are “dual function” compounds that provide both cardiac protection and enhanced breast cancer cell killing in the presence of DNA damaging chemotherapy agents. Our studies may have a broad impact by identifying a novel approach to both enhancing therapeutic efficacy and providing cardioprotection from DNA damaging agents that are widely used in cancer treatment. Citation Format: Anthony W. Ashton, Xuanmao Jiao, Zhiping Li, Joseph C. Wu, Cristobal dos Remedios, Sean Lal, Richard Kitsis, Richard G. Pestell. CCR5 inhibitors enhance doxorubicin-induced breast cancer cell killing while reducing cardiotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1334.

Human coronavirus, hCoV-19 (SARS-CoV-2) is associated with pneumonia, severe systemic disease and, in fatal cases, a high prevalence of cardiac dysfunction. Cellular entry requires a cell-surface receptor, angiotensin-converting enzyme 2 (ACE2), and activation of the spike protein by host proteases, including TMPRSS2 and cathepsins. Cardiac toxicity is a serious cause of morbidity and mortality from SARS-CoV-2 with chronic sequelae reducing cardiac function in many survivors. We determined mRNA expression for genes related to SARS-Cov-2 infection in iPSC-derived cardiac myocytes treated with the widely used chemotherapeutic, Doxorubicin (DOX). DOX induced expression of TMPSS2, but not Ace2 or Furin, 4- to 5-fold. Further, DOX treatment enhanced expression of cathepsins A, B and F between 1.2-1.5 fold. Overall these changes promote an environment of enhanced SARS-CoV-2 susceptibility in the myocardium placing cancer patients on DOX therapy at increased risk of cardiac damage. To further characterize the effects of SARS-CoV-2 on the myocardium we examined the local spatial gene expression response in human post-mortem cardiac samples. A genome wide (1,864 genes) and matching proteome analysis on SARS-CoV-2 infected myocardium was conducted using Digital spatial profiling (DSP) (NanoString GeoMxTM). RNAscopeTM, a sensitive form of fluorescence in situ hybridization (FISH), identified SARS-CoV-2 spike RNA in cells enriched for ACE2 and TMPRSS2. Immunohistochemistry on serial sections identified the SARS-CoV-2 spike protein in both myocyte (desmin+) and monocyte (CD45+) populations suggesting multiple targets for viral infection in the myocardium. SCANscopeTM was used to determine the effects of SARS-CoV-2 on cardiac gene. Probes directed to the spike protein were used to identify regions of interest (ROI) for SARS-CoV-2-infection. Unsupervised principal component analysis (PCA) of the differentially expressed gene transcripts separated gene expression of SARS-CoV-2 ROI from control ROI areas in the same patient and was distinct from the PCA of normal myocardium control ROI. PCA of the SARS-CoV-2-associated gene expression of the ROI from male vs. female myocardium identified distinct gender-related gene expression patterns. Further, PCA based on SARS-Cov-2 spike RNA abundance correlated with induction of innate and acquired immunity signaling in the myocardial cells. Moreover, gene ontology (Reactome) analysis of SARS-CoV-2 infected regions of the myocardium displayed characteristic signatures indicating enhanced apoptosis and autophagy, chromatin remodeling and reduced DNA repair, and reduced oxidoreductase activity in regions of SARS-CoV-2 infection compared to uninfected myocardium. These data probe the underlying molecular mechanisms that enhance the risk of death from SARS-CoV-2 for cancer patients and the cardiac remodeling that results in death for many patients with COVID-19. Citation Format: Anthony W. Ashton, Liang Zhang, Yan Liang, Prajan Divakar, Carolos Cordon-Cardo, Richard G. Pestell. SARS-CoV-2 infection of the human heart governs intracardiac innate immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 705.

miRNAs are a class of endogenous noncoding small RNAs that participate in diverse biological processes through base-pairing to the complementary sequence in 3' untranslated region (3' UTR) of mRNA Specific miRNA have been associated with the initiation, progression and therapy resistance and regulate immunological processes including the activation and ageing of innate and adaptive immune cells. Immuno-miRs, including. miR-21 and miR-93, regulate immune function in the tumor microenvironment by binding to the 3′-UTR region of target mRNAs, or by binding to- and activating Toll-Like Receptor 8 (TLR8) in surrounding cells of the immune system. The cyclin D1 (CCND1) gene, which encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the Rb protein, is overexpressed in up to 50% of human breast cancers. Cyclin D1, governs the expression, processing, the secretion and the relative proportion of secreted non-coding RNA subtypes (miRNA, rRNA, tRNA, CDBox, scRNA, HAcaBox. scaRNA, piRNA) in human breast cancer and the secretion of piRNA. Exosomes, which consist of a lipid bilayer membrane surrounding a small cytosol, contain constituents of their cell of origin, including mRNA and miRNA. Exosomes in the tumor microenvironment, participate in therapy resistance, cancer metastasis and the metastatic niche. Herein, knock down of cyclin D1 in MCF-7 cells with tet-inducible cyclin D1 shRNA decreased MCF-7 cell proliferation and mammosphere formation. Exosomes isolated from wild-type and cyclin D1 knock-down MCF-7 cell culture media, showed that exosomes from cyclin D1 knock down cells dramatically decreased proliferation of both wild-type and cyclin D1 knock-down cells, whereas the wild-type exosomes partially reversed the cyclin D1 knock down effects. Cyclin D1 knock-down exosomes decreased mammosphere formation of wild-type MCF-7 cells and wild-type exosomes increased mammosphere formation of cyclin D1 knock-down cells. miRNA profiles within the exosomes, showed that cyclin D1 regulated the abundance of miRNA within exosomes. The pattern of miRNA within exosomes regulated by cyclin D1 were enriched for miRNA governing mesenchymal epithelial transition and the induction of stem cell function. The most enriched miRNA in cyclin D1 wild-type exosomes was hsa-mir-21 which was recognized tumor promoting function, whereas the most enriched miRNAs in cyclin D1 knock-down exosomes were has-mir-16 and has-mir-92a, both of which were shown as tumor suppressor in breast cancer cells. The immune-miR miR-21 represented >85% of the cyclin D1-induced exosome miRNA transcripts, The cyclin D1-mediated miRNA exosome content may contribute to tumor initiation and progression through heterotypic expansion of cancer stem cells. Citation Format: Xuanmao Jiao, Chongwen Xu, Lifeng Tian, Zhao Zhang, Anthony W. Ashton, Zhiping Li, Richard G. Pestell. Cyclin D1 mediated exosomes are enriched for pro-oncogenic miRNAs and promote cancer stem cell expansion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2356.

Molecular analysis of all cancer types in the cancer genome atlas (TCGA) demonstrated that multiple pathways may be disrupted within a given tumor including PCa. Prostate cancer (PCa), the second leading cause of death in American men, includes distinct genetic subtypes with distinct therapeutic vulnerabilities. Substratification of PCa into genetic subtypes, forms the basis of rational therapy for PCa. Known genetic drivers to PCa include deletions of target genes (PTEN and NKX3.1 deletions), rearrangements/fusions of multiple genes (including TMPRSS2 and the oncogenic ETS transcription factor, ERG) and genetic predisposing factors (including germline DNA-repair gene mutations). An increasingly common finding in PCa are defects in DNA repair. The Drosophila Dac gene was initially cloned as dominant inhibitor of Elipse. The human homologue, DACH1, encodes a winged helix/Forkhead DNA-binding protein that binds in chromatin to FOXM1 sites. Herein, reduced expression of DACH1 is shown in PCa, correlating with DACH1 promoter methylation. Homozygous deletion of DACH1 occurred in up to 18% of human PCa, associated with increased AR activity, increased somatic copy-number alteration and poor prognosis. DACH1 increased DNA repair, as shown in comet assays, enhancing nonhomologous end joining (NHEJ) while reducing homology directed repair (HDR) in DNA repair reporter assays. Laser wounding showed DACH1 was recruited to sites of DNA damage, augmenting the recruitment of Ku70/Ku80. In Dach1 deletion cells reduced expression of Dach1 was associated with resistance to inhibitors of Poly-(ADP)-ribose polymerase (PARP) and enhanced sensitivity to inhibitors of WEE1 kinase and DNA-PK. The abundance of DACH1 in prostate cancer may define a subclass of prostate cancer warranting selective therapies. Citation Format: Zhiping Li, Xuanmao Jiao, A. Gordon Robertson, Anthony W. Ashton, Andrew Kossenkov, Hao Chen, Lai Yee Phoon, Li Lan, Richard G. Pestell. The Forkhead-like protein Dach1 governs sensitivity to WEE1 kinase and PARP inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2500.

Cancer immunotherapy is the most exciting advancement in cancer therapy. Similar to immune checkpoint blockade and chimeric antigen receptor T cell (CAR-T), bispecific antibody (BsAb) is attracting more and more attention as a novel strategy of antitumor immunotherapy. BsAb not only offers an effective linkage between therapeutics (e.g., immune effector cells, radionuclides) and targets (e.g., tumor cells) but also simultaneously blocks two different oncogenic mediators. In recent decades, a variety of BsAb formats have been generated. According to the structure of Fc domain, BsAb can be classified into two types: IgG-like format and Fc-free format. Among these formats, bispecific T cell engagers (BiTEs) and triomabs are commonly investigated. BsAb has achieved an exciting breakthrough in hematological malignancies and promising outcome in solid tumor as showed in various clinical trials. In this review, we focus on the preclinical experiments and clinical studies of epithelial cell adhesion molecule (EpCAM), human epidermal growth factor receptor (HER) family, carcinoembryonic antigen (CEA), and prostate-specific membrane antigen (PSMA) related BsAbs in solid tumors, as well as discuss the challenges and corresponding approaches in clinical application.

Autophagy activated after DNA damage or other stresses mitigates cellular damage by removing damaged proteins, lipids, and organelles. Activation of the master metabolic kinase AMPK enhances autophagy. Here we report that cyclin D1 restrains autophagy by modulating the activation of AMPK. In cell models of human breast cancer or in a cyclin D1–deficient model, we observed a cyclin D1–mediated reduction in AMPK activation. Mechanistic investigations showed that cyclin D1 inhibited mitochondrial function, promoted glycolysis, and reduced activation of AMPK (pT172), possibly through a mechanism that involves cyclin D1-Cdk4/Cdk6 phosphorylation of LKB1. Our findings suggest how AMPK activation by cyclin D1 may couple cell proliferation to energy homeostasis. Cancer Res; 77(13); 3391–405. ©2017 AACR.

Dachshund homolog 1 (DACH1), a key cell fate determination factor, contributes to tumorigenesis, invasion, metastasis of human breast neoplasm. However, the exact molecular mechanisms for the anti-tumor roles of DACH1 in breast carcinoma are still lack of extensive understanding. Herein, we utilized immunohistochemistry (IHC) staining and public microarray data analysis showing that DACH1 was higher in normal breast, low-grade and luminal-type cancer in comparison with breast carcinoma, high-grade and basal-like tumors respectively. Additionally, both correlation analysis of public databases of human breast carcinoma and IHC analysis of mice xenograft tumors demonstrated that DACH1 inversely related to cancer stem cells (CSCs) markers, epithelial-mesenchymal transition (EMT) inducers and basal-enriched molecules, while cluster of differentiation 44 (CD44) behaved in an opposite manner. Furthermore, mice transplanted tumor model indicated that breast cancer cells Met-1 with up-regulation of DACH1 were endowed with remarkably reduced potential of tumorigenesis. Importantly, meta-analysis of 19 Gene Expression Omnibus (GEO) databases of breast cancer implicated that patients with higher DACH1 expression had prolonged time to death, recurrence and metastasis, while CD44 was a promising biomarker predicting worse overall survival (OS) and metastasis-free survival (MFS). Collectively, our study indicated that CD44 might be a novel target of DACH1 in breast carcinoma.

// Alison Shupp 1 , Mathew C. Casimiro 2 and Richard G. Pestell 2 1 Departments of Cancer Biology, Medical Oncology, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA 2 Pennsylvania Cancer and Regenerative Medicine Research Center, Baruch S Blumberg Institute, Doylestown, PA, USA Correspondence to: Richard G. Pestell, email: // Keywords : CDK5, cancer Received : October 22, 2016 Accepted : December 17, 2016 Published : January 06, 2017 Abstract Cyclin dependent kinases are proline-directed serine/threonine protein kinases that are traditionally activated upon association with a regulatory subunit. For most CDKs, activation by a cyclin occurs through association and phosphorylation of the CDK’s T-loop. CDK5 is unusual because it is not typically activated upon binding with a cyclin and does not require T-loop phosphorylation for activation, even though it has high amino acid sequence homology with other CDKs. While it was previously thought that CDK5 only interacted with p35 or p39 and their cleaved counterparts, Recent evidence suggests that CDK5 can interact with certain cylins, amongst other proteins, which modulate CDK5 activity levels. This review discusses recent findings of molecular interactions that regulate CDK5 activity and CDK5 associated pathways that are implicated in various diseases. Also covered herein is the growing body of evidence for CDK5 in contributing to the onset and progression of tumorigenesis.

MicroRNAs (miRNAs) loss-of-function phenotypes are mainly induced by chemically modified antisense oligonucleotides. Here we develop an alternative inhibitor for miRNAs, termed ‘small RNA zipper'. It is designed to connect miRNA molecules end to end, forming a DNA–RNA duplex through a complementary interaction with high affinity, high specificity and high stability. Two miRNAs, miR-221 and miR-17, are tested in human breast cancer cell lines, demonstrating the 70∼90% knockdown of miRNA levels by 30–50 nM small RNA zippers. The miR-221 zipper shows capability in rescuing the expression of target genes of miR-221 and reversing the oncogenic function of miR-221 in breast cancer cells. In addition, we demonstrate that the miR-221 zipper attenuates doxorubicin resistance with higher efficiency than anti-miR-221 in human breast cancer cells. Taken together, small RNA zippers are a miRNA inhibitor, which can be used to induce miRNA loss-of-function phenotypes and validate miRNA target genes., MicroRNAs regulate a wide range of biological processes and being able to inhibit their function could allow the development of therapeutic options. Here the authors describe a ‘small RNA zipper' that sequesters miRNAs by forming a chain of DNA:RNA duplexes.

Introduction: Collaborative interactions between several diverse biological processes govern the onset and progression of breast cancer. These processes include alterations in cellular metabolism, anti-tumor immune responses, DNA damage repair, proliferation, anti-apoptotic signals, autophagy, epithelial-mesenchymal transition, components of the non-coding genome or onco-mIRs, cancer stem cells and cellular invasiveness. The last two decades have revealed that each of these processes are also directly regulated by a component of the cell cycle apparatus, cyclin D1. Area covered: The current review is provided to update recent developments in the clinical application of cyclin/CDK inhibitors to breast cancer with a focus on the anti-tumor immune response. Expert opinion: The cyclin D1 gene encodes the regulatory subunit of a proline-directed serine-threonine kinase that phosphorylates several substrates. CDKs possess phosphorylation site selectivity, with the phosphate-acceptor residue preceding a proline. Several important proteins are substrates including all three retinoblastoma proteins, NRF1, GCN5, and FOXM1. Over 280 cyclin D3/CDK6 substrates have b\een identified. Given the diversity of substrates for cyclin/CDKs, and the altered thresholds for substrate phosphorylation that occurs during the cell cycle, it is exciting that small molecular inhibitors targeting cyclin D/CDK activity have encouraging results in specific tumors.

Lysine methylation of histones and non-histone substrates by the SET domain containing protein lysine methyltransferase (KMT) G9a/EHMT2 governs transcription contributing to apoptosis, aberrant cell growth, and pluripotency. The positioning of chromosomes within the nuclear three-dimensional space involves interactions between nuclear lamina (NL) and the lamina-associated domains (LAD). Contact of individual LADs with the NL are dependent upon H3K9me2 introduced by G9a. The mechanisms governing the recruitment of G9a to distinct subcellular sites, into chromatin or to LAD, is not known. The cyclin D1 gene product encodes the regulatory subunit of the holoenzyme that phosphorylates pRB and NRF1 thereby governing cell-cycle progression and mitochondrial metabolism. Herein, we show that cyclin D1 enhanced H3K9 dimethylation though direct association with G9a. Endogenous cyclin D1 was required for the recruitment of G9a to target genes in chromatin, for G9a-induced H3K9me2 of histones, and for NL-LAD interaction. The finding that cyclin D1 is required for recruitment of G9a to target genes in chromatin and for H3K9 dimethylation, identifies a novel mechanism coordinating protein methylation. Published version This work was supported in part by R01CA70896, R01CA75503, and R01CA86072 (RGP). This project was funded in part from the Dr. Ralph and Marian C. Falk Medical Research Trust (RGP), a grant from the Breast Cancer Research Foundation (RGP), and a grant from the Pennsylvania Department of Health (RGP).

Background: The androgen receptor (AR) plays a key role in normal prostate homeostasis and in prostate cancer (PCa) development, while the role of aromatase (Cyp19a1) is still unclear. We evaluated the effects of a treatment with Tadalafil (TAD) on both these proteins. Methods: Androgen-sensitive human PCa cell line (LnCAP) was incubated with/without TAD (10&minus, 6 M) and bicalutamide (BCT) (10&minus, 4 M) to evaluate a potential modulation on cell proliferation, protein and mRNA expression of Cyp19a, AR and estrogen receptor-&beta, (ER&beta, ), respectively. Results: TAD increased early AR nuclear translocation (p &lt, 0.05, after 15 min of exposure), and increased AR transcriptional activity (p &lt, 0.05) and protein expression (p &lt, 0.05) after 24 h. Moreover, after 24 h this treatment upregulated Cyp19a1 and ER&beta, mRNA (p &lt, 0.05 and p &lt, 0.005 respectively) and led to an increase in protein expression of both after 48 h (p &lt, 0.05). Interestingly, TAD counteracted Cyp19a1 stimulation induced by BCT (p &lt, 0.05) but did not alter the effect induced by BCT on the AR protein expression. Conclusion: We demonstrate for the first time that TAD can significantly modulate AR expression and activity, Cyp19a1 and ER&beta, expression in PCa cells, suggesting a specific effect of these proteins. In addition, TAD potentiates the antiproliferative activity of BCT, opening a new clinical scenario in the treatment of PCa.

Proteomic analysis of castration-resistant prostate cancer demonstrated the enrichment of Src tyrosine kinase activity in approximately 90% of patients. Src is known to induce cyclin D1, and a cyclin D1–regulated gene expression module predicts poor outcome in human prostate cancer. The tumor-associated calcium signal transducer 2 (TACSTD2/Trop2/M1S1) is enriched in the prostate, promoting prostate stem cell self-renewal upon proteolytic activation via a γ-secretase cleavage complex (PS1, PS2) and TACE (ADAM17), which releases the Trop2 intracellular domain (Trop2 ICD). Herein, v-Src transformation of primary murine prostate epithelial cells increased the proportion of prostate cancer stem cells as characterized by gene expression, epitope characteristics, and prostatosphere formation. Cyclin D1 was induced by v-Src, and Src kinase induction of Trop2 ICD nuclear accumulation required cyclin D1. Cyclin D1 induced abundance of the Trop2 proteolytic cleavage activation components (PS2, TACE) and restrained expression of the inhibitory component of the Trop2 proteolytic complex (Numb). Patients with prostate cancer with increased nuclear Trop2 ICD and cyclin D1, and reduced Numb, had reduced recurrence-free survival probability (HR = 4.35). Cyclin D1, therefore, serves as a transducer of v-Src–mediated induction of Trop2 ICD by enhancing abundance of the Trop2 proteolytic activation complex. Cancer Res; 76(22); 6723–34. ©2016 AACR.

Therapy resistance and poor outcome in prostate cancer is associated with increased expression of cyclin D1. Androgens promote DNA double-strand break repair to reduce DNA damage, and cyclin D1 was also shown to enhance DNA damage repair (DDR). In this study, we investigated the significance of cyclin D1 in androgen-induced DDR using established prostate cancer cells and prostate tissues from cyclin D1 knockout mice. We demonstrate that endogenous cyclin D1 further diminished the dihydrotestosterone (DHT)-dependent reduction of γH2AX foci in vitro. We also show that cyclin D1 was required for the androgen-dependent DNA damage response both in vitro and in vivo. Furthermore, cyclin D1 was required for androgen-enhanced DDR and radioresistance of prostate cancer cells. Moreover, microarray analysis of primary prostate epithelial cells from cyclin D1-deficient and wild-type mice demonstrated that most of the DHT-dependent gene expression changes are also cyclin D1 dependent. Collectively, our findings suggest that the hormone-mediated recruitment of cyclin D1 to sites of DDR may facilitate the resistance of prostate cancer cells to DNA damage therapies and highlight the need to explore other therapeutic approaches in prostate cancer to prevent or overcome drug resistance. Cancer Res; 76(2); 329–38. ©2015 AACR.

Background C-X-C motif ligand 8 (CXCL8), known as a proinflammatory chemokine, exerts multiple effects on the proliferation, invasion, and migration of tumor cells via the autocrine or paracrine manner. Conversely, the human Dachshund homologue 1 (DACH1) is recognized as a tumor suppressor which retards the progression of various cancers. In prostate cancer, it has been demonstrated that DACH1 was negatively correlated with the expression of CXCL8 and able to antagonize the effects of CXCL8 on cellular migration. Herein, we explored the mechanisms by which DACH1 regulated the CXCL8 in non-small cell lung cancer (NSCLC). Methods Public microarray and Kaplan-Meier plotter datasets were analyzed. Blood serum samples from lung adenocarcinoma (ADC) patients were collected for enzyme-linked immunosorbent assay (ELISA) analysis. Immunohistochemical staining was conducted on tissue microarray. Cell lines with stable expression of DACH1 were established, and relative gene expression was measured by Western blot, ELISA, real-time PCR, and human cytokine array. Correspondingly, cell lines transfected with shDACH1 were established, and relative gene expression was measured by real-time PCR and immunofluorescence array. Functional studies were performed by transwell and xenograft mice models. Luciferase reporter gene assay was applied to measure the regulation of DACH1 on CXCL8. Results Our study indicated that CXCL8 both at the mRNA and protein level was associated with the high tumor burden of ADC. Correlational analyses in ADC cell lines and ADC tissues showed that DACH1 was inversely correlated with CXCL8. Meanwhile, patients with high DACH1 expression and low CXCL8 expression had prolonged time to death and recurrence. Moreover, we verified the inhibitory effects of DACH1 on CXCL8 both in vitro and in vivo. Mechanism studies proved that DACH1 transcriptionally repressed CXCL8 promoter activity through activator protein-1 (AP-1) and nuclear transcription factor-kappa B (NF-κB) sites. Conclusions Our study proved that CXCL8 acted as an unfavorable factor promoting to tumor progression and poor prognosis of ADC, while DACH1 antagonized CXCL8 to provide a favorable survival of ADC patients. Double detection of DACH1 and CXCL8 may provide a precise information for further evaluating the prognosis of ADC patients.

Rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in childhood, rarely affects adults, preferring male. RMS expresses the receptor for androgen (AR) and responds to androgen; however, the molecular action of androgens on RMS is unknown. Herein, testosterone (T) effects were tested in embryonal (ERMS) and alveolar (ARMS) RMS cell lines, by performing luciferase reporter assay, RT-PCR, and western blotting experiments. RNA interference experiments or bicalutamide treatment was performed to assess the specific role of AR. Radiation treatment was delivered to characterise the effects of T treatment on RMS intrinsic radioresistance. Our study showed that RMS cells respond to sub-physiological levels of T stimulation, finally promoting AR-dependent genomic and non-genomic effects, such as the transcriptional regulation of several oncogenes, the phosphorylation-mediated post-transductional modifications of AR and the activation of ERK, p38 and AKT signal transduction pathway mediators that, by physically complexing or not with AR, participate in regulating its transcriptional activity and the expression of T-targeted genes. T chronic daily treatment, performed as for the hormone circadian rhythm, did not significantly affect RMS cell growth, but improved RMS clonogenic and radioresistant potential and increased AR mRNA both in ERMS and ARMS. AR protein accumulation was evident in ERMS, this further developing an intrinsic T-independent AR activity. Our results suggest that androgens sustain and improve RMS transformed and radioresistant phenotype, and therefore, their therapeutic application should be avoided in RMS post puberal patients.

The functional significance of the chemokine receptor CCR5 in human breast cancer epithelial cells is poorly understood. Here, we report that CCR5 expression in human breast cancer correlates with poor outcome. CCR5+ breast cancer epithelial cells formed mammospheres and initiated tumors with >60-fold greater efficiency in mice. Reintroduction of CCR5 expression into CCR5-negative breast cancer cells promoted tumor metastases and induced DNA repair gene expression and activity. CCR5 antagonists Maraviroc and Vicriviroc dramatically enhanced cell killing mediated by DNA-damaging chemotherapeutic agents. Single-cell analysis revealed CCR5 governs PI3K/Akt, ribosomal biogenesis, and cell survival signaling. As CCR5 augments DNA repair and is reexpressed selectively on cancerous, but not normal breast epithelial cells, CCR5 inhibitors may enhance the tumor-specific activities of DNA damage response–based treatments, allowing a dose reduction of standard chemotherapy and radiation. Significance: This study offers a preclinical rationale to reposition CCR5 inhibitors to improve the treatment of breast cancer, based on their ability to enhance the tumor-specific activities of DNA-damaging chemotherapies administered in that disease. Cancer Res; 78(7); 1657–71. ©2018 AACR.

Background: Genetic classification of breast cancer based on the coding mRNA suggests the evolution of distinct subtypes. Whether the non-coding genome is altered concordantly with the coding genome and the mechanism by which the cell cycle directly controls the non-coding genome is poorly understood. Methods: Herein, the miRNA signature maintained by endogenous cyclin D1 in human breast cancer cells was defined. In order to determine the clinical significance of the cyclin D1-mediated miRNA signature, we defined a miRNA expression superset from 459 breast cancer samples. We compared the coding and non-coding genome of breast cancer subtypes. Results: Hierarchical clustering of human breast cancers defined four distinct miRNA clusters (G1-G4) associated with distinguishable relapse-free survival by Kaplan-Meier analysis. The cyclin D1-regulated miRNA signature included several oncomirs, was conserved in multiple breast cancer cell lines, was associated with the G2 tumor miRNA cluster, ERα+ status, better outcome and activation of the Wnt pathway. The coding and non-coding genome were discordant within breast cancer subtypes. Seed elements for cyclin D1-regulated miRNA were identified in 63 genes of the Wnt signaling pathway including DKK. Cyclin D1 restrained DKK1 via the 3'UTR. In vivo studies using inducible transgenics confirmed cyclin D1 induces Wnt-dependent gene expression. Conclusion: The non-coding genome defines breast cancer subtypes that are discordant with their coding genome subtype suggesting distinct evolutionary drivers within the tumors. Cyclin D1 orchestrates expression of a miRNA signature that induces Wnt/β-catenin signaling, therefore cyclin D1 serves both upstream and downstream of Wnt/β-catenin signaling. Published version

The EglN2/PHD1 prolyl hydroxylase is an important oxygen sensor contributing to breast tumorigenesis. Emerging studies suggest that there is functional cross talk between oxygen sensing and mitochondrial function, both of which play an essential role for sustained tumor growth. However, the potential link between EglN2 and mitochondrial function remains largely undefined. Here, we show that EglN2 depletion decreases mitochondrial respiration in breast cancer under normoxia and hypoxia, which correlates with decreased mitochondrial DNA in a HIF1/2α‐independent manner. Integrative analyses of gene expression profile and genomewide binding of EglN2 under hypoxic conditions reveal nuclear respiratory factor 1 (NRF1) motif enrichment in EglN2‐activated genes, suggesting NRF1 as an EglN2 binding partner. Mechanistically, by forming an activator complex with PGC1α and NRF1 on chromatin, EglN2 promotes the transcription of ferridoxin reductase (FDXR) and maintains mitochondrial function. In addition, FDXR, as one of effectors for EglN2, contributes to breast tumorigenesis in vitro and in vivo. Our findings suggest that EglN2 regulates mitochondrial function in ERα‐positive breast cancer.

Ovarian cancer (OC) is a leading cause of cancer mortality, but aside from a few well-studied mutations, very little is known about its underlying causes. As such, we performed survival analysis on ovarian copy number amplifications and gene expression datasets presented by The Cancer Genome Atlas in order to identify potential drivers and markers of aggressive OC. Additionally, two independent datasets from the Gene Expression Omnibus web platform were used to validate the identified markers. Based on our analysis, we identified FXYD5, a glycoprotein known to reduce cell adhesion, as a potential driver of metastasis and a significant predictor of mortality in OC. As a marker of poor outcome, the protein has effective antibodies against it for use in tissue arrays. FXYD5 bridges together a wide variety of cancers, including ovarian, breast cancer stage II, thyroid, colorectal, pancreatic, and head and neck cancers for metastasis studies.

In this study, we show that the transmembrane glycoprotein Trop-2 is up-regulated in human prostate cancer (PCa) with extracapsular extension (stages pT3/pT4) as compared to organ-confined (stage pT2) PCa. Consistent with this evidence, Trop-2 expression is found to be increased in metastatic prostate tumors of Transgenic Adenocarcinoma of Mouse Prostate mice and to strongly correlate with α5β1 integrin levels. Using PCa cells, we show that Trop-2 specifically associates with the α5 integrin subunit, as binding to α3 is not observed, and that Trop-2 displaces focal adhesion kinase from focal contacts. In support of the role of Trop-2 as a promoter of PCa metastatic phenotype, we observe high expression of this molecule in exosomes purified from Trop-2-positive PCa cells. These vesicles are then found to promote migration of Trop-2-negative PCa cells on fibronectin, an α5β1 integrin/focal adhesion kinase substrate, thus suggesting that the biological function of Trop-2 may be propagated to recipient cells. In summary, our findings show that Trop-2 promotes an α5β1 integrin-dependent pro-metastatic signaling pathway in PCa cells and that the altered expression of Trop-2 may be utilized for early identification of capsule-invading PCa.

Cyclin D1 is an important molecular driver of human breast cancer but better understanding of its oncogenic mechanisms is needed, especially to enhance efforts in targeted therapeutics. Currently, pharmaceutical initiatives to inhibit cyclin D1 are focused on the catalytic component since the transforming capacity is thought to reside in the cyclin D1/CDK activity. We initiated the following study to directly test the oncogenic potential of catalytically inactive cyclin D1 in an in vivo mouse model that is relevant to breast cancer. Herein, transduction of cyclin D1(-/-) mouse embryonic fibroblasts (MEFs) with the kinase dead KE mutant of cyclin D1 led to aneuploidy, abnormalities in mitotic spindle formation, autosome amplification, and chromosomal instability (CIN) by gene expression profiling. Acute transgenic expression of either cyclin D1(WT) or cyclin D1(KE) in the mammary gland was sufficient to induce a high CIN score within 7 days. Sustained expression of cyclin D1(KE) induced mammary adenocarcinoma with similar kinetics to that of the wild-type cyclin D1. ChIP-Seq studies demonstrated recruitment of cyclin D1(WT) and cyclin D1(KE) to the genes governing CIN. We conclude that the CDK-activating function of cyclin D1 is not necessary to induce either chromosomal instability or mammary tumorigenesis.

We have previously demonstrated that the anti-apoptotic protein BAD is expressed in normal human breast tissue and shown that BAD inhibits expression of cyclin D1 to delay cell-cycle progression in breast cancer cells. Herein, expression of proteins in breast tissues was studied by immunohistochemistry and results were analyzed statistically to obtain semi-quantitative data. Biochemical and functional changes in BAD-overexpressing MCF7 breast cancer cells were evaluated using PCR, reporter assays, western blotting, ELISA and extracellular matrix invasion assays. Compared to normal tissues, Grade II breast cancers expressed low total/phosphorylated forms of BAD in both cytoplasmic and nuclear compartments. BAD overexpression decreased the expression of β-catenin, Sp1, and phosphorylation of STATs. BAD inhibited Ras/MEK/ERK and JNK signaling pathways, without affecting the p38 signaling pathway. Expression of the metastasis-related proteins, MMP10, VEGF, SNAIL, CXCR4, E-cadherin and TlMP2 was regulated by BAD with concomitant inhibition of extracellular matrix invasion. Inhibition of BAD by siRNA increased invasion and Akt/p-Akt levels. Clinical data and the results herein suggest that in addition to the effect on apoptosis, BAD conveys anti-metastatic effects and is a valuable prognostic marker in breast cancer.

D-type cyclins contribute the regulatory subunits to the holoenzymes that phosphorylate distinct substrates and regulate diverse biological processes, including cellular proliferation and differentiation. A growing body of evidence has demonstrated that the D-type cyclins are located in distinct subcellular pools with distinct functions. These subcellular locations include the cell membrane, the cytoplasm, the nuclear lamina, the nucleus, and DNA binding sites. The distribution of D-type cyclins in each one of these compartments is regulated by distinct signaling pathways and contributes to a vast array of biological processes. Importantly, D-type cyclins can also conduct transcriptional functions. Initially shown to regulate the activity of transcription factors in gene reporter assays, subsequent studies have shown that D-type cyclins can affect gene expression through the regulation of coactivators, the modulation of transcription factor binding in the context of chromatin, and their ability to serve as molecular scaffolds that facilitate the interactions between chromatin-modifying enzymes (histone and DNA methylases [Suv39, HP1α, G9a, DNMT1, PRMT1], histone acetylases [SRC1, p300, P/CAF], and histone deacetylases [HDAC1,3]). Genome-wide studies have shown that cyclin D1 associates with the regulatory regions of more than 2840 genes. The importance of the transcriptional functions of D-type cyclins has been demonstrated in vivo, as hormone signaling in prostate and mammary gland tissues is critically dependent upon the presence of cyclin D1. Furthermore, it is the cyclin D1-regulated gene expression signature, not the abundance of the protein, which strongly predicts poor outcome in prostate cancer. Together, these findings are consistent with the evolving realization that the D-type cyclins play an important biological role in governing gene transcription.

The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that drives cell autonomous cell cycle progression and proliferation. Herein we show cyclin D1 abundance is increased >30-fold in the stromal fibroblasts of patients with invasive breast cancer, associated with poor outcome. Cyclin D1 transformed hTERT human fibroblast to a cancer-associated fibroblast phenotype. Stromal fibroblast expression of cyclin D1 (cyclin D1Stroma) in vivo, enhanced breast epithelial cancer tumor growth, restrained apoptosis, and increased autophagy. Cyclin D1Stroma had profound effects on the breast tumor microenvironment increasing the recruitment of F4/80+ and CD11b+ macrophages and increasing angiogenesis. Cyclin D1Stroma induced secretion of factors that promoted expansion of stem cells (breast stem-like cells, embryonic stem cells and bone marrow derived stem cells). Cyclin D1Stroma resulted in increased secretion of proinflammatory cytokines (CCL2, CCL7, CCL11, CXCL1, CXCL5, CXCL9, CXCL12), CSF (CSF1, GM-CSF1) and osteopontin (OPN) (30-fold). OPN was induced by cyclin D1 in fibroblasts, breast epithelial cells and in the murine transgenic mammary gland and OPN was sufficient to induce stem cell expansion. These results demonstrate that cyclin D1Stroma drives tumor microenvironment heterocellular signaling, promoting several key hallmarks of cancer. Published version

Uncontrolled cell division is the hallmark of cancers. Full understanding of cell cycle regulation would contribute to promising cancer therapies. In particular, cyclin-dependent kinases 4/6 (CDK4/6), which are pivotal drivers of cell proliferation by combination with cyclin D, draw more and more attention. Subsequently, extensive studies were carried out to explore drugs inhibiting CDK4/6 and assess the efficacy and safety of these drugs in cancer, especially breast cancer. Due to the insuperable adverse events and the less activity observed in vivo, the drug development of the initial pan-CDK inhibitor flavopiridol was consequently discontinued, and then highly specific inhibitors were extensively researched and developed, including palbociclib (PD0332991), ribociclib (LEE011), and abemaciclib (LY2835219). Food and Drug Administration has approved palbociclib and ribociclib for the treatment of hormone receptor-positive, human epidermal growth factor receptor 2-negative advanced or metastatic breast cancer, and recent clinical trial data suggest that palbociclib significantly improved clinical outcome when combined with letrozole or fulvestrant. Besides, the favorable effects of abemaciclib on prolonging survival of breast cancer patients have also been observed in clinical trials both for single-agent and combination strategy. In this review, we outline the preclinical and clinical advancement of these three orally bioavailable and highly selective CDK4/6 inhibitors in breast cancer.

Awareness that the metabolic phenotype of cells within tumours is heterogeneous - and distinct from that of their normal counterparts - is growing. In general, tumour cells metabolize glucose, lactate, pyruvate, hydroxybutyrate, acetate, glutamine, and fatty acids at much higher rates than their nontumour equivalents; however, the metabolic ecology of tumours is complex because they contain multiple metabolic compartments, which are linked by the transfer of these catabolites. This metabolic variability and flexibility enables tumour cells to generate ATP as an energy source, while maintaining the reduction-oxidation (redox) balance and committing resources to biosynthesis - processes that are essential for cell survival, growth, and proliferation. Importantly, experimental evidence indicates that metabolic coupling between cell populations with different, complementary metabolic profiles can induce cancer progression. Thus, targeting the metabolic differences between tumour and normal cells holds promise as a novel anticancer strategy. In this Review, we discuss how cancer cells reprogramme their metabolism and that of other cells within the tumour microenvironment in order to survive and propagate, thus driving disease progression; in particular, we highlight potential metabolic vulnerabilities that might be targeted therapeutically.

Naturally occurring extracellular vesicles (EVs) play essential roles in intracellular communication and delivery of bioactive molecules. Therefore it has been suggested that EVs could be used for delivery of therapeutics. However, to date the therapeutic application of EVs has been limited by number of factors, including limited yield and full understanding of their biological activities. To address these issues, we analyzed the morphology, molecular composition, fusion capacity and biological activity of Cytochalasin B-induced membrane vesicles (CIMVs). The size of these vesicles was comparable to that of naturally occurring EVs. In addition, we have shown that CIMVs from human SH-SY5Y cells contain elevated levels of VEGF as compared to the parental cells, and stimulate angiogenesis in vitro and in vivo. Published version

// Gabriele Di Sante 1 , Agnese Di Rocco 1 , Claudia Pupo 1 , Mathew C. Casimiro 1 and Richard G. Pestell 1,2 1 Pennsylvania Cancer and Regenerative Medicine Research Center, Baruch S. Blumberg Institute, Pennsylvania Biotechnology Center, PA, USA 2 Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore Correspondence to: Richard G. Pestell, email: // Keywords : cyclin D1, DNA damage, hormones, genomic instability, DNA repair Received : December 21, 2016 Accepted : June 26, 2017 Published : July 20, 2017 Abstract Cell cycle control proteins govern events that leads to the production of two identical daughter cells. Distinct sequential temporal phases, Gap 1 (G 1 ), Gap 0 (G 0 ), Synthesis (S), Gap 2 (G 2 ) and Mitosis (M) are negotiated through a series of check points during which the favorability of the local cellular environment is assessed, prior to replicating DNA [ 1 ]. Cyclin D1 has been characterized as a key regulatory subunit of the holoenzyme that promotes the G 1 /S-phase transition through phosphorylating the pRB protein. Cyclin D1 overexpression is considered a driving force in several types of cancers and cdk inhibitors are being used effectively in the clinic for treatment of ERα + breast cancer [ 1 , 2 ]. Genomic DNA is assaulted by damaging ionizing radiation, chemical carcinogens, and reactive oxygen species (ROS) which are generated by cellular metabolism. Furthermore, specific hormones including estrogens [ 3 , 4 ] and androgens [ 5 ] govern pathways that damage DNA. Defects in the DNA Damage Response (DDR) pathway can lead to genomic instability and cancer. Evidence is emerging that cyclin D1 bind proteins involved in DNA repair including BRCA1 [ 6 ], RAD51 [ 7 ], BRCA2 [ 8 ] and is involved in the DNA damage and DNA repair processes [ 7 , 8 ]. Because the repair of damaged DNA appears to be an important and unexpected role for cyclin D1, and inhibitors of cyclin D1-dependent kinase activity are being used in the clinic, the latest findings on the role of cyclin D1 in mediating the DDR including the DDR induced by the hormones estrogen [ 9 ] and androgen [ 10 , 11 ] is reviewed.

Src family kinases (SFK) integrate signal transduction for multiple receptors, regulating cellular proliferation, invasion, and metastasis in human cancer. Although Src is rarely mutated in human prostate cancer, SFK activity is increased in the majority of human prostate cancers. To determine the molecular mechanisms governing prostate cancer bone metastasis, FVB murine prostate epithelium was transduced with oncogenic v-Src. The prostate cancer cell lines metastasized in FVB mice to brain and bone. Gene expression profiling of the tumors identified activation of a CCR5 signaling module when the prostate epithelial cell lines were grown in vivo versus tissue cultures. The whole body, bone, and brain metastatic prostate cancer burden was reduced by oral CCR5 antagonist. Clinical trials of CCR5 inhibitors may warrant consideration in patients with CCR5 activation in their tumors. Cancer Res; 74(23); 7103–14. ©2014 AACR.

// Lymor Ringer 1,* , Paul Sirajuddin 1,* , Lucas Tricoli 1,* , Sarah Waye 1 , Muhammad Umer Choudhry 1 , Erika Parasido 1 , Angiela Sivakumar 1 , Mary Heckler 1 , Aisha Naeem 1 , Iman Abdelgawad 1,6 , Xuefeng Liu 2 , Adam S. Feldman 3 , Richard J. Lee 3 , Chin-Lee Wu 3 , Venkata Yenugonda 1 , Bhaskar Kallakury 2 , Anatoly Dritschilo 4 , John Lynch 4 , Richard Schlegel 1,2 , Olga Rodriguez 1 , Richard G. Pestell 5 , Maria Laura Avantaggiati 1,* and Chris Albanese 1,2,* 1 Department of Oncology and Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA 2 Department of Pathology, Georgetown University Medical Center, Washington, DC, USA 3 Massachusetts General Hospital, Boston, USA 4 Georgetown University Hospital, Washington, DC, USA 5 Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA 6 National Cancer Institute of Egypt, Cairo, Egypt * These authors contributed equally to this work Correspondence: Chris Albanese, email: // Keywords : p53, apoptosis, autophagy, primary cells, prostate Received : July 14, 2014 Accepted : September 25, 2014 Published : September 26, 2014 Abstract The p53 tumor suppressor protein plays a crucial role in influencing cell fate decisions in response to cellular stress. As p53 elicits cell cycle arrest, senescence or apoptosis, the integrity of the p53 pathway is considered a key determinant of anti-tumor responses. p53 can also promote autophagy, however the role of p53-dependent autophagy in chemosensitivity is poorly understood. VMY-1-103 (VMY), a dansylated analog of purvalanol B, displays rapid and potent anti-tumor activities, however the pathways by which VMY works are not fully defined. Using established prostate cancer cell lines and novel conditionally reprogrammed cells (CRCs) derived from prostate cancer patients; we have defined the mechanisms of VMY-induced prostate cancer cell death. Herein, we show that the cytotoxic effects of VMY required a p53-dependent induction of autophagy, and that inhibition of autophagy abrogated VMY-induced cell death. Cancer cell lines harboring p53 missense mutations evaded VMY toxicity and treatment with a small molecule compound that restores p53 activity re-established VMY-induced cell death. The elucidation of the molecular mechanisms governing VMY-dependent cell death in cell lines, and importantly in CRCs, provides the rationale for clinical studies of VMY, alone or in combination with p53 reactivating compounds, in human prostate cancer.

// Lifeng Tian 1 , Chenguang Wang 1 , Fred K. Hagen 2 , Michael Gormley 1 , Sankar Addya 1 , Raymond Soccio 3 , Mathew C. Casimiro 1 , Jie Zhou 1 , Michael J. Powell 1 , Ping Xu 4 , Haiteng Deng 5 , Anthony A. Sauve 4 and Richard G. Pestell 1 1 Department of Cancer Biology, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA 2 Department of Biochemistry and Biophysics, University of Rochester, Rochester, NY, USA 3 Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Department of Genetics, and The Institute for Diabetes, Obesity, and Metabolism, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA 4 Department of Pharmacology, Weill Medical College of Cornell University, York Avenue LC216, New York, NY, USA 5 Proteomics Resource Center, Rockefeller University, New York, NY, USA Correspondence: Richard G. Pestell, email: // Chenguang Wang, email: // Keywords : acetylation; peroxisome proliferator-activated receptor gamma (Pparγ); nuclear receptors; silent mating type information regulation 2 homolog 1 (SIRT1); lipogenesis. Received : JULY 18, 2014 Accepted : August 18, 2014 Published : August 19, 2014 Abstract In our prior publications we characterized a conserved acetylation motif (K(R)xxKK) of evolutionarily related nuclear receptors. Recent reports showed that peroxisome proliferator activated receptor gamma (PPARγ) deacetylation by SIRT1 is involved in delaying cellular senescence and maintaining the brown remodeling of white adipose tissue. However, it still remains unknown whether lysyl residues 154 and 155 (K154/155) of the conserved acetylation motif (RIHKK) in Pparγ1 are acetylated. Herein, we demonstrate that Pparγ1 is acetylated and regulated by both endogenous TSA-sensitive and NAD-dependent deacetylases. Acetylation of lysine 154 was identified by mass spectrometry (MS) while deacetylation of lysine 155 by SIRT1 was confirmed by in vitro deacetylation assay. An in vivo labeling assay revealed K154/K155 as bona fide acetylation sites. The conserved acetylation sites of Pparγ1 and the catalytic domain of SIRT1 are both required for the interaction between Pparγ1 and SIRT1. Sirt1 and Pparγ1 converge to govern lipid metabolism in vivo. Acetylation-defective mutants of Pparγ1 were associated with reduced lipid synthesis in ErbB2 overexpressing breast cancer cells. Together, these results suggest that the conserved lysyl residues K154/K155 of Pparγ1 are acetylated and play an important role in lipid synthesis in ErbB2-positive breast cancer cells.

The miR-221/222 cluster has been demonstrated to function as oncomiR in human cancers. miR-221/222 promotes epithelial-to-mesenchymal transition (EMT) and confers tamoxifen resistance in breast cancer. However, the effects and mechanisms by which miR-221/222 regulates breast cancer aggressiveness remain unclear. Here we detected a much higher expression of miR-221/222 in highly invasive basal-like breast cancer (BLBC) cells than that in non-invasive luminal cells. A microRNA dataset from breast cancer patients indicated an elevated expression of miR-221/222 in BLBC subtype. S-phase entry of the cell cycle was associated with the induction of miR-221/222 expression. miRNA inhibitors specially targeting miR-221 or miR-222 both significantly suppressed cellular migration, invasion and G1/S transition of the cell cycle in BLBC cell types. Proteomic analysis demonstrated the down-regulation of two tumor suppressor genes, suppressor of cytokine signaling 1 (SOCS1) and cyclin-dependent kinase inhibit 1B (CDKN1B), by miR-221/222. This is the first report to reveal miR-221/222 regulation of G1/S transition of the cell cycle. These findings demonstrate that miR-221/222 contribute to the aggressiveness in control of BLBC.

// Zuoren Yu 1,2 , Zengguang Xu 1 , Gabriele DiSante 2 , Jennifer Wright 2 , Min Wang 2 , Yuan Li 1 , Qian Zhao 1 , Tao Ren 1 , Xiaoming Ju 2 , Ellen Gutman 2 , Guangxue Wang 1 , Sankar Addya 2 , Tieyan Li 1 , Zhendong Xiang 3 , Chenguang Wang 2 , Xiongfei yang 3 , Xiaolai Yang 3 , Richard Pestell 2 1 Research Center for Translational Medicine, Key Laboratory of Arrhythmias of the Ministry of Education, East Hospital, Tongji University School of Medicine, Shanghai, China, 2 Departments of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia PA 3 People’s Hospital of Gansu Province, Lanzhou, China Correspondence: Richard G. Pestell, email: // Zuoren Yu, email: // Keywords : miR-17/20, breast cancer, AKT, apoptosis Received : January 29, 2014 Accepted : March 2, 2014 Published : March 4, 2014 Abstract The serine threonine kinase Akt1 has been implicated in the control of cellular metabolism, survival and growth. Herein, disruption of the ubiquitously expressed member of the Akt family of genes, Akt1, in the mouse, demonstrates a requirement for Akt1 in miRNA-mediated cellular apoptosis. The miR-17/20 cluster is known to inhibit breast cancer cellular proliferation through G1/S cell cycle arrest via binding to the cyclin D1 3’UTR. Here we show that miR-17/20 overexpression sensitizes cells to apoptosis induced by either Doxorubicin or UV irradiation in MCF-7 cells via Akt1. miR-17/20 mediates apoptosis via increased p53 expression which promotes Akt degradation. Akt1 -/- mammary epithelial cells which express Akt2 and Akt3 demonstrated increased apoptosis to DNA damaging agents. Akt1 deficiency abolished the miR-17/20-mediated apoptosis. These results demonstrated a novel pathway through which miR17/20 regulate p53 and Akt controlling breast cancer cell apoptosis.

CAPER is an estrogen receptor (ER) co-activator that was recently shown to be involved in human breast cancer pathogenesis. Indeed, we reported increased expression of CAPER in human breast cancer specimens. We demonstrated that CAPER was undetectable or expressed at relatively low levels in normal breast tissue and assumed a cytoplasmic distribution. In contrast, CAPER was expressed at higher levels in ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) specimens, where it assumed a predominantly nuclear distribution. However, the functional role of CAPER in human breast cancer initiation and progression remained unknown. Here, we used a lentiviral-mediated gene silencing approach to reduce the expression of CAPER in the ER-positive human breast cancer cell line MCF-7. The proliferation and tumorigenicity of MCF-7 cells stably expressing control or human CAPER shRNAs was then determined via both in vitro and in vivo experiments. Knockdown of CAPER expression significantly reduced the proliferation of MCF-7 cells in vitro. Importantly, nude mice injected with MCF-7 cells harboring CAPER shRNAs developed smaller tumors than mice injected with MCF-7 cells harboring control shRNAs. Mechanistically, tumors derived from mice injected with MCF-7 cells harboring CAPER shRNAs displayed reduced expression of the cell cycle regulators PCNA, MCM7, and cyclin D1, and the protein synthesis marker 4EBP1. In conclusion, knockdown of CAPER expression markedly reduced human breast cancer cell proliferation in both in vitro and in vivo settings. Mechanistically, knockdown of CAPER abrogated the activity of proliferative and protein synthesis pathways.

Cancer pathogenesis has been attributed to the minor and major disruptions in the cell cycle, with a key role being played by several of the recently discovered epigenetic factors. Lately, UHRF1 (Ubiquitin-like with containing PHD and RING Finger domains 1), an epigenetic regulator has been shown to be evidently over expressed in numerous malignancies through an in-depth review of literature. Molecular docking studies have found that existing drugs such as propranolol, naphthazarin and thymoquinone have favourable interactions with specific domains of UHRF1. However, these findings would need large scale clinical trials to confirm their potency and safety during chemotherapy. UHRF1 (Ubiquitin-like with containing PHD and RING Finger domains 1), an epigenetic factor, plays a crucial role as an important checkpoint in the cell machinery. Basic science continues to unravel multiple facets of this five domain protein which includes a detailed elucidation of its roles and mechanisms of interaction with various enzymes during DNA replication. The gene has recently begun to be also termed as the “Universal Oncogene” in response to the results of research conducted in heterogenous populations and in over 17 cancers displaying heightened mRNA and protein expression in breast, liver, lung, head and neck cancers and many more. This gene could therefore, be a potential biomarker for diagnosis and for the prediction of the prognosis and survival of the diseased. A scientifically established solution in the form of targeted treatment must follow such a discovery and therefore, several natural and synthetic compounds such as thymoquinone and the well-known antihypertensive, propranolol have been docked and reported to have favourable interactions with the SRA (Set and Ring Associated) domain of UHRF1 in this review. This comprehensive review is thus, a brief synopsis of details regarding the structure and heightened levels of UHRF1 in several malignancies. Furthermore, pharmacogenomic research revolving around this oncogene is a potential sphere for clinical studies to be conducted in much larger and heterogenous populations to not only validate these therapeutic docking results but to also to bring personalised medicine to the bedside for the benefit of the patients.

Peroxisome proliferator-activated receptorg (Pparγ) is a member of the nuclear receptor (NR) superfamily, which regulates diverse biological functions including lipogenesis and differentiation, anti-inflammation, insulin sensitivity, cellular proliferation, and autophagy. Independent lines of evidence support a role for Pparγ as either a collaborative oncogene or as a tumor suppressor. Heterozygous mutations of Pparγ have been detected in 4/55 patients with colon cancer and a chromosomal translocation between PAX8 and Pparγ in follicular thyroid cancer appeared to serve as a dominant inhibitor of endogenous Pparγ expression. Pparγ agonists reduced tumorigenesis in several in vivo models. In contrast, several studies suggest Pparγ may enhance tumor growth. Pparγ ligands increased polyp numbers in the Apc mouse model of familial adenomatosis. Pparγ and its ligands inhibit breast tumor growth; however, constitutively active Pparγ collaborated in mammary oncogenesis with polyoma middle T antigen or oncogenic ErbB2. Pparγ activation involves post-translational modifications including phosphorylation and sumoylation upon growth factor or ligand stimulus. Mutation of the Pparγ1 sumoylation site at K77 and K365 demonstrated that K77 may either reduce Pparγ-dependent gene induction and enhance repression or reduce repression, depending upon the synthetic reporter gene used. Lysine residues of nuclear receptors also serve as substrates for acetylation and Pparγ binds co-activators and co-repressors with intrinsic or associated histone acetylase or deacetylase activity including NCoR, SMRT, SIRT1, and p300. Initially characterized for the ERα, AR and, subsequently, the orphan nuclear receptor steroidogenic factor 1 (SF-1), acetylation occurs at a conserved lysine motif shared amongst evolutionarily related nuclear receptors. Several nuclear receptors and co-integrators involved in lipid metabolism are regulated by acetylation including p300, PGC1α, FXR, LXR and RAR. Both TSA- and NAD-sensitive HDACs (e.g. SIRT1) regulate Pparγ function and SIRT1 inhibits Pparγ-dependent adipocyte differentiation. Whether Pparγ is acetylated in cancer cells and how Pparγ exerts it's crucial, though controversial, function in tumorigenesis have not been established. Pparγ induces gene transcription through binding specific NR half-sites and through non-canonical binding sequences (such as CREB/AP-1 sites). Transcriptional repression involves Pparγ sumoylation at lysine 77 (K77). Herein, Pparγ was shown to be acetylated at nine distinct lysine residues. SIRT1 bound and deacetylated Pparγ at K154/155. ChIP-Seq analysis for genome-wide DNA binding demonstrated the acetylation site was required for binding NR half-sites, but was not required for non-canonical site binding. Breast tumor growth, de novo lipid synthesis, induction of autophagy and evasion of apoptosis was promoted by K154/155 and inhibited by K77 in vivo. Pparγ acetylation induced a gene signature that was increased in breast cancer, associated with a reduction in SIRT1 abundance and poor outcome. The Pparγ acetylation site determines binding to autophagy and apoptosis signaling to regulate breast tumor lipid metabolism and growth. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-06-02.

MicroRNAs (miRNAs), a class of non-coding small RNA, regulate gene expression through base-pairing binding to the complementary sequence in the 3’ untranslated region (3’ UTR) of mRNA. miRNAs contribute to the timing of development, apoptosis, cell cycle progression, cellular proliferation, stem cell self-renewal, cancer initiation and metastasis. The expression of miRNA is regulated during cell-cycle transition and cellular contact in part via active degradation. Aberrant expression of miRNAs or mutations of miRNA genes have been described in many types of tumors, including mammary tumors. The RNase III endoribonuclease Dicer cleaves long double-stranded RNA (dsRNA) or stem-loop-stem structured pre-miRNA to form mature miRNAs. RNAi-mediated knock-down of Dicer in human cells led to defects in both miRNA production and shRNA-mediated RNAi. Initially cloned as a breakpoint rearrangement in parathyroid adenoma, the cyclin D1 gene encodes the regulatory subunit of the holoenzyme that phosphorylates and inactivates both the pRb tumor suppressor and the key inducer of mitochondrial biogenesis NRF-1. In addition, a DNA bound form of cyclin D1 regulates gene expression. Cyclin D1 expression is induced during mammary gland and retinal differentiation, and deletion of the murine cyclin D1 gene resulted in failed terminal alveolar breast bud development and retinal degeneration. Diverse biological functions regulated by cyclin D1 include the induction of cellular proliferation, angiogenesis, cellular migration, DNA damage repair, mitochondrial biogenesis, stem cell maintenance, and miRNA expression. Cyclin D1 was shown to regulate the miR-17/20 locus and found to bind the miR-17/20 regulatory region. In order to determine further the mechanism by which cyclin D1 regulates non-coding RNA, we conducted studies of miRNA processing. We established cyclin D1-/- mouse embryonic fibroblasts cells (MEFs) and cyclin D1 knockdown (KD) MCF-7 human breast cancer cells. miRNA analysis indicated an induction of mature miRNA expression in cyclin D1 overexpressing cells. Analysis of the miRNA processing regulators demonstrated the selective induction of Dicer expression by cyclin D1. In cyclin D1-/- cells the reduction of Dicer abundance was accompanied by impairment of pre-miRNA to mature miRNA processing, which was restored with cyclin D1 rescue. Transient transgenic expression of cyclin D1 in the mouse mammary gland, or sustained transgenic expression of cyclin D1 induced mouse mammary gland tumors, recapitulated the induction of Dicer expression. Cyclin D1 and Dicer expression were correlated in luminal A and basal-like human breast cancer. Cyclin D1 regulation of cellular proliferation and migration was dependent upon Dicer. By demonstrating cyclin D1 induced Dicer abundance and function in tissue culture and in vivo, we provide evidence for novel crosstalk between the cell-cycle and non-coding miRNA biogenesis. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-07-05.

The epithelial-mesenchymal transition (EMT) enhances cellular invasiveness and confers tumor cells with cancer stem cell like characteristics, through transcriptional and translational mechanisms. The mechanisms maintaining transcriptional and translational repression of EMT and cellular invasion are poorly understood. The Drosophila homologue of DACH1, the Dac gene is a key member of the retinal determination gene network that specifies organismal development. The dachshund (dac), eya1, eyes-absent (eya), twin of eyeless (toy), teashirt (tsh) and sinoculues (so) are expressed in progenitor cells, contributing to development of the eye and genitalia. Loss of DACH1 expression contributes to the expansion of neural progenitors, muscle satellite cell differentiation and breast cancer stem cells. In recent studies Dachshund repressed breast cancer stem cell expansion. DACH1 expression is reduced in a variety of human cancers including prostate, ovarian and human breast cancer. Herein, the cell fate-determination factor Dachshund (DACH1), suppressed EMT via repression of cytoplasmic translational induction of Snail by inactivating the Y box-binding protein (YB-1). In the nucleus, DACH1 antagonized YB-1-mediated oncogenic transcriptional modules governing cell invasion. DACH1 blocked YB-1-induced mammary tumor growth and EMT in mice. In basal-like breast cancer (BLBC) the reduced expression of DACH1 and increased YB-1, correlated with poor metastasis free survival. The loss of DACH1 suppression of both cytoplasmic translational and nuclear transcriptional events governing EMT and tumor invasion may contribute to poor prognosis in BLBC. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-07-05.